Search PubMed⌕ Search

Biomedical subjects

A W Murray

Publications and source records attributed to A W Murray.

At least 37 records · Page 2Linked to original sources
PubMed2000

NO protects alveolar type II cells from stretch-induced apoptosis. A novel role for macrophages in the lung.

We have previously shown that mechanical distortion or stretch of alveolar type II (ATII) cells induces both surfactant release and the induction of apoptosis. We hypothesize that nitric oxide (NO) secreted from alveolar macrophages (AMs) prevents cyclic stretch-induced apoptosis. We show that S-nitroso-N-acetyl-D, L-penicillamine (SNAP), a chemical donor of NO, protects cells against nuclear condensation and DNA fragmentation induced by stretch (30% at 60 cycles/min) as well as by sorbitol. SNAP depleted of NO had no protective effect, and the NO scavenger 2-phenyl-4,4,5, 5-tetramethylimidazoline-1-oxyl 3-oxide blocked the antiapoptotic effect of SNAP. We also show that AMs isolated from rat lung lavage fluid actively synthesize and secrete NO. Using a novel technique in which AMs were cocultured with ATII cells while adhered to floating membrane rafts, we found that NO released from AMs was effective in protecting ATII cells from undergoing apoptosis. We therefore propose that NO secreted by AMs may function as part of a physiological antiapoptotic mechanism that prevents ATII cells from undergoing stretch-induced cell death in the lung.

Animals↗
PubMed1999

From molecular to modular cell biology.

Cellular functions, such as signal transmission, are carried out by 'modules' made up of many species of interacting molecules. Understanding how modules work has depended on combining phenomenological analysis with molecular studies. General principles that govern the structure and behaviour of modules may be discovered with help from synthetic sciences such as engineering and computer science, from stronger interactions between experiment and theory in cell biology, and from an appreciation of evolutionary constraints.

Action Potentials↗
PubMed1999

Genetic selection of peptide inhibitors of biological pathways.

Genetic selections were used to find peptides that inhibit biological pathways in budding yeast. The peptides were presented inside cells as peptamers, surface loops on a highly expressed and biologically inert carrier protein, a catalytically inactive derivative of staphylococcal nuclease. Peptamers that inhibited the pheromone signaling pathway, transcriptional silencing, and the spindle checkpoint were isolated. Putative targets for the inhibitors were identified by a combination of two-hybrid analysis and genetic dissection of the target pathways. This analysis identified Ydr517w as a component of the spindle checkpoint and reinforced earlier indications that Ste50 has both positive and negative roles in pheromone signaling. Analysis of transcript arrays showed that the peptamers were highly specific in their effects, which suggests that they may be useful reagents in organisms that lack sophisticated genetics as well as for identifying components of existing biological pathways that are potential targets for drug discovery.

Amino Acid Sequence↗
PubMed1999

Mad2 binding by phosphorylated kinetochores links error detection and checkpoint action in mitosis.

The spindle checkpoint must detect the presence of unattached or improperly attached kinetochores and must then inhibit progression through the cell cycle until the offending condition is resolved. Detection probably involves attachment-sensitive kinetochore phosphorylation (reviewed in [1,2]). A key player in the checkpoint's response is the Mad2 protein, which prevents activation of the anaphase-promoting complex (APC) by the Cdc20 protein [3-8]. Microinjection of Mad2 antibodies results in premature anaphase onset [9,10], and excess Mad2 protein causes arrest in mitosis [5,11]. We have previously shown that Mad2 localizes to unattached kinetochores in vertebrate cells, and that this localization ceases as kinetochores accumulate microtubules [10,12,13]. But how is Mad2 binding limited to unattached kinetochores? Here, we used lysed PtK1 cells to study kinetochore phosphorylation and Mad2 binding. We found that Mad2 binds to phosphorylated kinetochores, but not to unphosphorylated ones. Our data suggest that it is kinetochore protein phosphorylation that promotes Mad2 binding to unattached kinetochores. Thus, we have identified a probable molecular link between attachment-sensitive kinetochore phosphorylation and the inhibition of anaphase. The complete pathway for error control in mitosis can now be outlined.

Animals↗
PubMed1999

Cyclic stretch induces both apoptosis and secretion in rat alveolar type II cells.

We examined the effects of short-term cyclic stretch on both phosphatidylcholine (PC) secretion and apoptosis in primary cultures of rat alveolar type II cells. A 22% cyclic stretch (3 cycles/min) was applied to type II cells cultured on silastic membranes using a Flexercell strain unit. This induced, after a lag period of about 1 h, a small, but significant release of [3H]PC from prelabelled cells. In addition, stretch increased nuclear condensation, the generation of oligosomal DNA fragments and the activation of caspases. Similar responses were triggered by sorbitol-induced osmotic shock, but not by the secretagogue ATP. We conclude that stretch can induce both apoptosis and PC secretion in alveolar type II cells and propose that these diverse responses occur within the lung as a consequence of normal respiratory distortion of the alveolar epithelium.

Animals↗
PubMed1999

The conserved protein kinase Ipl1 regulates microtubule binding to kinetochores in budding yeast.

Chromosome segregation depends on kinetochores, the structures that mediate chromosome attachment to the mitotic spindle. We isolated mutants in IPL1, which encodes a protein kinase, in a screen for budding yeast mutants that have defects in sister chromatid separation and segregation. Cytological tests show that ipl1 mutants can separate sister chromatids but are defective in chromosome segregation. Kinetochores assembled in extracts from ipl1 mutants show altered binding to microtubules. Ipl1p phosphorylates the kinetochore component Ndc10p in vitro and we propose that Ipl1p regulates kinetochore function via Ndc10p phosphorylation. Ipl1p localizes to the mitotic spindle and its levels are regulated during the cell cycle. This pattern of localization and regulation is similar to that of Ipl1p homologs in higher eukaryotes, such as the human aurora2 protein. Because aurora2 has been implicated in oncogenesis, defects in kinetochore function may contribute to genetic instability in human tumors.

Aurora Kinases↗
PubMed1999

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. (This review is an updated version of one that was published in Current Opinion in Cell Biology 1998, 10:769-775.)

Animals↗
PubMed1999

The spindle checkpoint of budding yeast depends on a tight complex between the Mad1 and Mad2 proteins.

The spindle checkpoint arrests the cell cycle at metaphase in the presence of defects in the mitotic spindle or in the attachment of chromosomes to the spindle. When spindle assembly is disrupted, the budding yeast mad and bub mutants fail to arrest and rapidly lose viability. We have cloned the MAD2 gene, which encodes a protein of 196 amino acids that remains at a constant level during the cell cycle. Gel filtration and co-immunoprecipitation analyses reveal that Mad2p tightly associates with another spindle checkpoint component, Mad1p. This association is independent of cell cycle stage and the presence or absence of other known checkpoint proteins. In addition, Mad2p binds to all of the different phosphorylated isoforms of Mad1p that can be resolved on SDS-PAGE. Deletion and mutational analysis of both proteins indicate that association of Mad2p with Mad1p is critical for checkpoint function and for hyperphosphorylation of Mad1p.

Benomyl↗
PubMed1999

Lesions in many different spindle components activate the spindle checkpoint in the budding yeast Saccharomyces cerevisiae.

The spindle checkpoint arrests cells in mitosis in response to defects in the assembly of the mitotic spindle or errors in chromosome alignment. We determined which spindle defects the checkpoint can detect by examining the interaction of mutations that compromise the checkpoint (mad1, mad2, and mad3) with those that damage various structural components of the spindle. Defects in microtubule polymerization, spindle pole body duplication, microtubule motors, and kinetochore components all activate the MAD-dependent checkpoint. In contrast, the cell cycle arrest caused by mutations that induce DNA damage (cdc13), inactivate the cyclin proteolysis machinery (cdc16 and cdc23), or arrest cells in anaphase (cdc15) is independent of the spindle checkpoint.

Cell Division↗
PubMed1999

Role of the extracellular signal-regulated protein kinase cascade in human neutrophil killing of Staphylococcus aureus and Candida albicans and in migration.

Killing of Staphylococcus aureus and Candida albicans by neutrophils involves adherence of the microorganisms, phagocytosis, and a collaborative action of oxygen reactive species and components of the granules. While a number of intracellular signalling pathways have been proposed to regulate neutrophil responses, the extent to which each pathway contributes to the killing of S. aureus and C. albicans has not been clearly defined. We have therefore examined the effect of blocking one such pathway, the extracellular signal-regulated protein kinase (ERK) cascade, using the specific inhibitor of the mitogen-activated protein kinase/ERK kinase, PD98059, on the ability of human neutrophils to kill S. aureus and C. albicans. Our data demonstrate the presence of ERK2 and a 43-kDa form of ERK but not ERK1 in human neutrophils. Upon stimulation with formyl methionyl leucyl phenylalanine (fMLP), the activities of both ERK2 and the 43-kDa form were stimulated. Despite abrogating the activity of both ERK forms, PD98059 only slightly reduced the ability of neutrophils to kill S. aureus or C. albicans. This is consistent with our finding that PD98059 had no effect on neutrophil adherence or degranulation, although pretreatment of neutrophils with PD98059 inhibited fMLP-stimulated superoxide production by 50%, suggesting that a change in superoxide production per se is not strictly correlated with microbicidal activity. However, fMLP-stimulated chemokinesis was markedly inhibited, while random migration and fMLP-stimulated chemotaxis were partially inhibited, by PD98059. These data demonstrate, for the first time, that the ERK cascade plays only a minor role in the microbicidal activity of neutrophils and that the ERK cascade is involved primarily in regulating neutrophil migration in response to fMLP.

Blood Bactericidal Activity↗
PubMed1998

Time-lapse microscopy reveals unique roles for kinesins during anaphase in budding yeast.

The mitotic spindle is a complex and dynamic structure. Genetic analysis in budding yeast has identified two sets of kinesin-like motors, Cin8p and Kip1p, and Kar3p and Kip3p, that have overlapping functions in mitosis. We have studied the role of three of these motors by video microscopy of motor mutants whose microtubules and centromeres were marked with green fluorescent protein. Despite their functional overlap, each motor mutant has a specific defect in mitosis: cin8Delta mutants lack the rapid phase of anaphase B, kip1Delta mutants show defects in the slow phase of anaphase B, and kip3Delta mutants prolong the duration of anaphase to the point at which the spindle becomes longer than the cell. The kip3Delta and kip1Delta mutants affect the duration of anaphase, but cin8Delta does not.

Anaphase↗
PubMed1998

Spindle checkpoint protein Xmad1 recruits Xmad2 to unattached kinetochores.

The spindle checkpoint prevents the metaphase to anaphase transition in cells containing defects in the mitotic spindle or in chromosome attachment to the spindle. When the checkpoint protein Xmad2 is depleted from Xenopus egg extracts, adding Xmad2 to its endogenous concentration fails to restore the checkpoint, suggesting that other checkpoint component(s) were depleted from the extract through their association with Xmad2. Mass spectrometry provided peptide sequences from an 85-kD protein that coimmunoprecipitates with Xmad2 from egg extracts. This information was used to clone XMAD1, which encodes a homologue of the budding yeast (Saccharomyces cerevisiae) checkpoint protein Mad1. Xmad1 is essential for establishing and maintaining the spindle checkpoint in egg extracts. Like Xmad2, Xmad1 localizes to the nuclear envelope and the nucleus during interphase, and to those kinetochores that are not bound to spindle microtubules during mitosis. Adding an anti-Xmad1 antibody to egg extracts inactivates the checkpoint and prevents Xmad2 from localizing to unbound kinetochores. In the presence of excess Xmad2, neither chromosomes nor Xmad1 are required to activate the spindle checkpoint, suggesting that the physiological role of Xmad1 is to recruit Xmad2 to kinetochores that have not bound microtubules.

Animals↗
PubMed1998

How to compact DNA.

Explore the source record for details and available documents.

Adenosine Triphosphatases↗
PubMed1998

Evidence that protein kinase Cepsilon mediates phorbol ester inhibition of calphostin C- and tumor necrosis factor-alpha-induced apoptosis in U937 histiocytic lymphoma cells.

Protein kinase C (PKC) activators, such as the tumor-promoting phorbol esters, have been reported to protect several cell lines from apoptosis induced by a variety of agents. Recent evidence suggests that PKCepsilon is involved in protection of cardiac myocytes from hypoxia-induced cell death (Gray, M. O., Karliner, J. S., and Mochly-Rosen, D. (1997) J. Biol. Chem. 272, 30945-30951). We investigated the protective effects of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on U937 histiocytic lymphoma cells induced to undergo apoptosis by tumor necrosis factor-alpha (TNF-alpha) or by the specific PKC inhibitor calphostin C. U937 cells were transiently permeabilized with a peptide (epsilonV1-2) derived from the V1 region of PKCepsilon that has been reported to specifically block translocation of PKCepsilon. The epsilonV1-2 peptide blocked the inhibitory effect of TPA on both TNF-alpha- and calphostin C-induced apoptosis. A scrambled version of epsilonV1-2 and a peptide reported to inhibit PKCbeta translocation (betaC2-4) had no effect on the ability of TPA to inhibit apoptosis. These results suggest that PKCepsilon is required for the protective effect of TPA in TNF-alpha- and calphostin C-induced apoptosis. Furthermore, calphostin C reduced membrane-associated PKCepsilon activity and immunoreactivity, suggesting that PKCepsilon may play an important role in leukemic cell survival.

Apoptosis↗
PubMed1998

Stimulation of p38 phosphorylation and activity by arachidonic acid in HeLa cells, HL60 promyelocytic leukemic cells, and human neutrophils. Evidence for cell type-specific activation of mitogen-activated protein kinases.

Although it is well appreciated that arachidonic acid, a second messenger molecule that is released by ligand-stimulated phospholipase A2, stimulates a wide range of cell types, the mechanisms that mediate the actions of arachidonic acid are still poorly understood. We now report that arachidonic acid stimulated the appearance of dual-phosphorylated (active) p38 mitogen-activated protein kinase as detected by Western blotting in HeLa cells, HL60 cells, human neutrophils, and human umbilical vein endothelial cells but not Jurkat cells. An increase in p38 kinase activity caused by arachidonic acid was also observed. Further studies with neutrophils show that the stimulation of p38 dual phosphorylation by arachidonic acid was transient, peaking at 5 min, and was concentration-dependent. The effect of arachidonic acid was not affected by either nordihydroguaiaretic acid, an inhibitor of the 5-, 12-, and 15-lipoxygenases or by indomethacin, an inhibitor of cyclooxygenase. Arachidonic acid also stimulated the phosphorylation and/or activity of the extracellular signal-regulated protein kinase and of c-jun N-terminal kinase in a cell-type-specific manner. An examination of the mechanisms through which arachidonic acid stimulated the phosphorylation/activity of p38 and extracellular signal-regulated protein kinase in neutrophils revealed an involvement of protein kinase C. Thus, arachidonic acid stimulated the translocation of protein kinase C alpha, betaI, and betaII to a particulate fraction, and the effects of arachidonic acid on mitogen-activated protein kinase phosphorylation/activity were partially inhibited by GF109203X, an inhibitor of protein kinase C. This study is the first to demonstrate that a polyunsaturated fatty acid causes the dual phosphorylation and activation of p38.

Animals↗
PubMed1998

Localization of Mad2 to kinetochores depends on microtubule attachment, not tension.

A single unattached kinetochore can delay anaphase onset in mitotic tissue culture cells (Rieder, C.L., A. Schultz, R. Cole, G. Sluder. 1994. J. Cell Biol. 127:1301-1310). Kinetochores in vertebrate cells contain multiple binding sites, and tension is generated at kinetochores after attachment to the plus ends of spindle microtubules. Checkpoint component Mad2 localizes selectively to unattached kinetochores (Chen, R.-H., J.C. Waters, E.D. Salmon, and A.W. Murray. 1996. Science. 274:242-246; Li, Y., and R. Benezra. Science. 274: 246-248) and disappears from kinetochores by late metaphase, when chromosomes are properly attached to the spindle. Here we show that Mad2 is lost from PtK1 cell kinetochores as they accumulate microtubules and re-binds previously attached kinetochores after microtubules are depolymerized with nocodazole. We also show that when kinetochore microtubules in metaphase cells are stabilized with taxol, tension at kinetochores is lost. The phosphoepitope 3f3/2, which has been shown to become dephosphorylated in response to tension at the kinetochore (Nicklas, R.B., S.C. Ward, and G.J. Gorbsky. 1995. J. Cell Biol. 130:929-939), is phosphorylated on all 22 kinetochores after tension is reduced with taxol. In contrast, Mad2 only localized to an average of 2.6 out of the 22 kinetochores in taxol-treated PtK1 cells. Therefore, loss of tension at kinetochores occupied by microtubules is insufficient to induce Mad2 to accumulate on kinetochores, whereas unattached kinetochores consistently bind Mad2. We also found that microinjecting antibodies against Mad2 caused cells arrested with taxol to exit mitosis after approximately 12 min, while uninjected cells remained in mitosis for at least 6 h, demonstrating that Mad2 is necessary for maintenance of the taxol-induced mitotic arrest. We conclude that kinetochore microtubule attachment stops the Mad2 interactions at kinetochores which are important for inhibiting anaphase onset.

Animals↗