Search PubMed⌕ Search

Biomedical subjects

A W Murray

Publications and source records attributed to A W Murray.

At least 55 records · Page 3Linked to original sources
PubMed1998

Microinjection of antibody to Mad2 protein into mammalian cells in mitosis induces premature anaphase.

In yeast, the Mad2 protein is required for the M phase arrest induced by microtubule inhibitors, but the protein is not essential under normal culture conditions. We tested whether the Mad2 protein participates in regulating the timing of anaphase onset in mammalian cells in the absence of microtubule drugs. When microinjected into living prophase or prometaphase PtK1 cells, anti-Mad2 antibody induced the onset of anaphase prematurely during prometaphase, before the chromosomes had assembled at the metaphase plate. Anti-Mad2 antibody-injected cells completed all aspects of anaphase including chromatid movement to the spindle poles and pole-pole separation. Identical results were obtained when primary human keratinocytes were injected with anti-Mad2 antibody. These studies suggest that Mad2 protein function is essential for the timing of anaphase onset in somatic cells at each mitosis. Thus, in mammalian somatic cells, the spindle checkpoint appears to be a component of the timing mechanism for normal mitosis, blocking anaphase onset until all chromosomes are aligned at the metaphase plate.

Anaphase↗
PubMed1998

Pro- and anti-apoptotic effects of K+ in HeLa cells.

The present study determined the effects of osmotic stress induced by sorbitol and KCl on apoptosis. Sorbitol induced apoptosis, activated the mitogen-activated protein kinase (MAPK) family and stimulated accumulation of cytosolic cytochrome c and procaspase-3 cleavage. KCl (0.2 M) also activated the MAPKs and induced cytosolic cytochrome c accumulation but did not induce procaspase-3 cleavage or apoptosis and was protective against sorbitol-induced apoptosis. However, when cells were exposed to KCl for 1 h, washed and returned to isotonic medium, caspase-3 was rapidly cleaved and apoptosis induced. We conclude that hyperosmotic KCl initiates early events in apoptosis but blocks caspase-3 activation by preventing the loss of intracellular K+.

Apoptosis↗
PubMed1998

Budding yeast Cdc20: a target of the spindle checkpoint.

The spindle checkpoint regulates the cell division cycle by keeping cells with defective spindles from leaving mitosis. In the two-hybrid system, three proteins that are components of the checkpoint, Mad1, Mad2, and Mad3, were shown to interact with Cdc20, a protein required for exit from mitosis. Mad2 and Mad3 coprecipitated with Cdc20 at all stages of the cell cycle. The binding of Mad2 depended on Mad1 and that of Mad3 on Mad1 and Mad2. Overexpression of Cdc20 allowed cells with a depolymerized spindle or damaged DNA to leave mitosis but did not overcome the arrest caused by unreplicated DNA. Mutants in Cdc20 that were resistant to the spindle checkpoint no longer bound Mad proteins, suggesting that Cdc20 is the target of the spindle checkpoint.

Amino Acid Sequence↗
PubMed1998

Sister chromatid cohesion in mitosis.

Sister chromatid cohesion is essential for accurate chromosome segregation during the cell cycle. Newly identified structural proteins are required for sister chromatid cohesion and there may be a link in some organisms between the processes of cohesion and condensation. Proteins that induce and regulate the separation of sister chromatids have also been recently identified.

Animals↗
PubMed1998

Neurotrophic factors prevent ceramide-induced apoptosis downstream of c-Jun N-terminal kinase activation in PC12 cells.

Neurotrophic factors prevent apoptosis of PC12 cells in serum-free medium. The present study determines whether neurotrophic factors can prevent ceramide-induced apoptosis in PC12 cells and investigates the role that c-Jun N-terminal kinase (JNK) activation may play in this system. Ceramide-induced apoptosis was inhibited by nerve growth factor, basic fibroblast growth factor, pituitary adenylyl cyclase-activating peptide, 4-(8-chlorophenylthio)cyclic AMP, and the caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp fluoromethyl ketone (zVAD-FMK). It was surprising that inhibition of extracellular signal-regulated kinase and/or phosphatidylinositol 3-kinase did not markedly block the protective effects exerted by neurotrophic factors against ceramide-induced apoptosis, suggesting that neurotrophic factors can promote survival independently of these signaling pathways. Treatment of PC12 cells with ceramide resulted in a time-dependent increase in JNK activity. However, neither neurotrophic factors nor zVAD-FMK attenuated ceramide-stimulated JNK activation. Further experiments indicated that ceramide-induced apoptosis in PC12 cells requires new protein synthesis, and that nerve growth factor and zVAD-FMK can prevent apoptosis after JNK activity has been detected. These results indicate that ceramide-induced JNK activation is an early event and may be required for the expression of essential components of the apoptotic machinery. It is anticipated that neurotrophic factors inhibit ceramide-induced apoptosis by affecting signaling events downstream of JNK activation.

Amino Acid Chloromethyl Ketones↗
PubMed1998

Osmotic stress induces both secretion and apoptosis in rat alveolar type II cells.

The aim of this study was to analyze the effects of osmotic shock and secretagogues such as ATP and 12-O-tetradecanoylphorbol 13-acetate (TPA) on various intracellular signaling pathways in primary cultures of alveolar type II cells and examine their potential role in regulating events such as secretion and apoptosis in these cells. Sorbitol-induced osmotic stress caused the sustained release of [3H]phosphatidylcholine ([3H]PC) from primary cultures of rat alveolar type II cells prelabeled with [3H]choline chloride. This release was not dependent on protein kinase C because downregulation of the major protein kinase C isoforms (alpha, betaII, delta, and eta) expressed in alveolar type II cells had no effect on [3H]PC secretion. Sorbitol, as well as the known secretagogues TPA and ATP, activated extracellular signal-regulated kinase. Although an inhibitor of the extracellular signal-regulated kinase cascade, PD-98059, blocked this activation, it had no effect on the release of [3H]PC. Sorbitol and ultraviolet C radiation, but not TPA or ATP, were also found to activate both p38 and stress-activated protein kinase/c-Jun NH2-terminal kinase. Furthermore, both sorbitol and ultraviolet C radiation induced apoptosis in alveolar type II cells as demonstrated by Hoechst 33258 staining of the condensed nuclei, the generation of DNA ladders, and the activation of caspases. The data indicate that multiple signaling pathways are activated by traditional secretagogues such as TPA and ATP and by cellular stresses such as osmotic shock and that these may be involved in regulating secretory and apoptotic events in alveolar type II cells.

Adenosine Triphosphate↗
PubMed1997

Interphase chromosomes undergo constrained diffusional motion in living cells.

BACKGROUND: Structural studies of fixed cells have revealed that interphase chromosomes are highly organized into specific arrangements in the nucleus, and have led to a picture of the nucleus as a static structure with immobile chromosomes held in fixed positions, an impression apparently confirmed by recent photobleaching studies. Functional studies of chromosome behavior, however, suggest that many essential processes, such as recombination, require interphase chromosomes to move around within the nucleus. RESULTS: To reconcile these contradictory views, we exploited methods for tagging specific chromosome sites in living cells of Saccharomyces cerevisiae with green fluorescent protein and in Drosophila melanogaster with fluorescently labeled topoisomerase ll. Combining these techniques with submicrometer single-particle tracking, we directly measured the motion of interphase chromatin, at high resolution and in three dimensions. We found that chromatin does indeed undergo significant diffusive motion within the nucleus, but this motion is constrained such that a given chromatin segment is free to move within only a limited subregion of the nucleus. Chromatin diffusion was found to be insensitive to metabolic inhibitors, suggesting that it results from classical Brownian motion rather than from active motility. Nocodazole greatly reduced chromatin confinement, suggesting a role for the cytoskeleton in the maintenance of nuclear architecture. CONCLUSIONS: We conclude that chromatin is free to undergo substantial Brownian motion, but that a given chromatin segment is confined to a subregion of the nucleus. This constrained diffusion is consistent with a highly defined nuclear architecture, but also allows enough motion for processes requiring chromosome motility to take place. These results lead to a model for the regulation of chromosome interactions by nuclear architecture.

Animals↗
PubMed1997

Integrating genetic approaches into the discovery of anticancer drugs.

The discovery of anticancer drugs is now driven by the numerous molecular alterations identified in tumor cells over the past decade. To exploit these alterations, it is necessary to understand how they define a molecular context that allows increased sensitivity to particular compounds. Traditional genetic approaches together with the new wealth of genomic information for both human and model organisms open up strategies by which drugs can be profiled for their ability to selectively kill cells in a molecular context that matches those found in tumors. Similarly, it may be possible to identify and validate new targets for drugs that would selectively kill tumor cells with a particular molecular context. This article outlines some of the ways that yeast genetics can be used to streamline anticancer drug discovery.

Animals↗
PubMed1997

Chromosome and low copy plasmid segregation in E. coli: visual evidence for distinct mechanisms.

We have investigated DNA segregation in E. coli by inserting multiple lac operator sequences into the chromosome near the origin of replication (oriC), in the hisC gene, a terminus marker, and into plasmids P1 and F. Expression of a GFP-LacI fusion protein allowed visualization of lac operator localization. oriC was shown to be specifically localized at or near the cell poles, and when duplicated, one copy moved to the site of new pole formation near the site of cell division. In contrast, P1 and F localized to the cell center and on duplication appeared to move rapidly to the quarter positions in the cell. Our analysis suggests that different active processes are involved in movement and localization of the chromosome and of the two plasmids during segregation.

Cell Cycle↗
PubMed1997

Mitosis in living budding yeast: anaphase A but no metaphase plate.

Chromosome movements and spindle dynamics were visualized in living cells of the budding yeast Saccharomyces cerevisiae. Individual chromosomal loci were detected by expression of a protein fusion between green fluorescent protein (GFP) and the Lac repressor, which bound to an array of Lac operator binding sites integrated into the chromosome. Spindle microtubules were detected by expression of a protein fusion between GFP and Tub1, the major alpha tubulin. Spindle elongation and chromosome separation exhibited biphasic kinetics, and centromeres separated before telomeres. Budding yeast did not exhibit a conventional metaphase chromosome alignment but did show anaphase A, movement of the chromosomes to the poles.

Anaphase↗
PubMed1997

N-6 and n-3 polyunsaturated fatty acids stimulate translocation of protein kinase Calpha, -betaI, -betaII and -epsilon and enhance agonist-induced NADPH oxidase in macrophages.

The polyunsaturated fatty acids (PUFA), arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were poor inducers of oxygen-dependent respiratory activity (chemiluminescence) in human monocytes and macrophages, but markedly enhanced the response to the tripeptide, N-formylmethionyl-leucyl-phenylalanine. The effects of these fatty acids were seen at concentrations of 1 microg/ml. A similar enhancement was seen with PMA, a stimulus that acts on protein kinase C (PKC), or calcium ionophore (A23187), which increases intracellular calcium, suggesting that the effect of the fatty acids was post-surface receptor binding. HL-60 cells, differentiated to macrophage-like cells by culture in the presence of vitamin D3, were similarly affected by the fatty acids. In experiments in which the time of pre-exposure of the monocytes to PUFA was varied, it was found that the priming effect induced by AA, EPA and DHA was maximal at 5 min. The ability of these fatty acids to synergize with other agonists was completely lost if the fatty acids were either methylated or oxidized to the hydro and hydroperoxy derivatives. Saturated fatty acids were inactive. Western blot analysis demonstrated that the PUFA induced the translocation of PKCalpha, -betaI, -betaII and -epsilon isoenzymes to a particulate fraction. The synergistic response between fatty acids and A23187 was completely inhibited by pretreating the cells with a PKC inhibitor, GF-109203X, or by pretreatment of monocytes with PMA for 18 h, to deplete PKC levels. From these investigations it is evident that PUFA prime macrophages, causing increased/synergistic oxidative respiratory burst activity to other stimuli and that this priming is dependent on PKC translocation and activation.

Calcimycin↗
PubMed1997

Ceramide induces apoptosis in PC12 cells.

The novel lipid second messenger, ceramide, induced apoptosis in PC12 cells as determined morphologically by nuclear appearance and internucleosomal DNA fragmentation. Apoptosis was induced by exogenous C2-ceramide in a dose- and time-dependent manner. Natural ceramide and C6-ceramide had a similar effect. This response was specific since the structural analog C2-dihydroceramide and other related lipids failed to initiate apoptosis. The apoptotic effect of ceramide also depends critically on cell plating density. Furthermore, the peptide inhibitor of interleukin-1beta converting enzyme (ICE)-like proteases, Z-VAD.FMK, completely prevented the nuclear changes induced by ceramide, implicating the involvement of ICE-like protease activation in ceramide-induced apoptosis in PC12 cells.

Amino Acid Chloromethyl Ketones↗
PubMed1997

A novel yeast screen for mitotic arrest mutants identifies DOC1, a new gene involved in cyclin proteolysis.

B-type cyclins are rapidly degraded at the transition between metaphase and anaphase and their ubiquitin-mediated proteolysis is required for cells to exit mitosis. We used a novel enrichment to isolate new budding mutants that arrest the cell cycle in mitosis. Most of these mutants lie in the CDC16, CDC23, and CDC27 genes, which have already been shown to play a role in cyclin proteolysis and encode components of a 20S complex (called the cyclosome or anaphase promoting complex) that ubiquitinates mitotic cyclins. We show that mutations in CDC26 and a novel gene, DOC1, also prevent mitotic cyclin proteolysis. Mutants in either gene arrest as large budded cells with high levels of the major mitotic cyclin (Clb2) protein at 37 degrees C and cannot degrade Clb2 in G1-arrested cells. Cdc26 associates in vivo with Doc1, Cdc16, Cdc23, and Cdc27. In addition, the majority of Doc1 cosediments at 20S with Cdc27 in a sucrose gradient, indicating that Cdc26 and Doc1 are components of the anaphase promoting complex.

Amino Acid Sequence↗
PubMed1996

GFP tagging of budding yeast chromosomes reveals that protein-protein interactions can mediate sister chromatid cohesion.

BACKGROUND: Precise control of sister chromatid separation is essential for the accurate transmission of genetic information. Sister chromatids must remain linked to each other from the time of DNA replication until the onset of chromosome segregation, when the linkage must be promptly dissolved. Recent studies suggest that the machinery that is responsible for the destruction of mitotic cyclins also degrades proteins that play a role in maintaining sister chromatid linkage, and that this machinery is regulated by the spindle-assembly checkpoint. Studies on these problems in budding yeast are hampered by the inability to resolve its chromosomes by light or electron microscopy. RESULTS: We have developed a novel method for visualizing specific DNA sequences in fixed and living budding yeast cells. A tandem array of 256 copies of the Lac operator is integrated at the desired site in the genome and detected by the binding of a green fluorescent protein (GFP)-Lac repressor fusion expressed from the HIS3 promoter. Using this method, we show that sister chromatid segregation precedes the destruction of cyclin B. In mad or bub cells, which lack the spindle-assembly checkpoint, sister chromatid separation can occur in the absence of microtubules. The expression of a tetramerizing form of the GFP-Lac repressor, which can bind Lac operators on two different DNA molecules, can hold sister chromatids together under conditions in which they would normally separate. CONCLUSIONS: We conclude that sister chromatid separation in budding yeast can occur in the absence of microtubule-dependent forces, and that protein complexes that can bind two different DNA molecules are capable of holding sister chromatids together.

Bacterial Proteins↗
PubMed1996

Protein phosphatase 2A regulates MPF activity and sister chromatid cohesion in budding yeast.

BACKGROUND: Mitosis is regulated by MPF (maturation promoting factor), the active form of Cdc2/28-cyclin B complexes. Increasing levels of cyclin B abundance and the loss of inhibitory phosphates from Cdc2/28 drives cells into mitosis, whereas cyclin B destruction inactivates MPF and drives cells out of mitosis. Cells with defective spindles are arrested in mitosis by the spindle-assembly checkpoint, which prevents the destruction of mitotic cyclins and the inactivation of MPF. We have investigated the relationship between the spindle-assembly checkpoint, cyclin destruction, inhibitory phosphorylation of Cdc2/28, and exit from mitosis. RESULTS: The previously characterized budding yeast mad mutants lack the spindle-assembly checkpoint. Spindle depolymerization does not arrest them in mitosis because they cannot stabilize cyclin B. In contrast, a newly isolated mutant in the budding yeast CDC55 gene, which encodes a protein phosphatase 2A (PP2A) regulatory subunit, shows a different checkpoint defect. In the presence of a defective spindle, these cells separate their sister chromatids and leave mitosis without inducing cyclin B destruction. Despite the persistence of B-type cyclins, cdc55 mutant cells inactivate MPF. Two experiments show that this inactivation is due to inhibitory phosphorylation on Cdc28: phosphotyrosine accumulates on Cdc28 in cdc55 delta cells whose spindles have been depolymerized, and a cdc28 mutant that lacks inhibitory phosphorylation sites on Cdc28 allows spindle defects to arrest cdc55 mutants in mitosis with active MPF and unseparated sister chromatids. CONCLUSIONS: We conclude that perturbations of protein phosphatase activity allow MPF to be inactivated by inhibitory phosphorylation instead of by cyclin destruction. Under these conditions, sister chromatid separation appears to be regulated by MPF activity rather than by protein degradation. We discuss the role of PP2A and Cdc28 phosphorylation in cell-cycle control, and the possibility that the novel mitotic exit pathway plays a role in adaptation to prolonged activation of the spindle-assembly checkpoint.

CDC28 Protein Kinase, S cerevisiae↗