The persistence of value-driven attention capture is task-dependent.

Visual features previously associated with reward can capture attention even when task-irrelevant, a phenomenon known as value-driven attention capture (VDAC). VDAC persists without reinforcement, unlike other forms of learning, where removing reinforcement typically leads to extinction. In five experiments, factors common to many studies were manipulated to examine their impact on VDAC and its extinction. All experiments included learning and test phases. During learning, participants completed a visual search task during which one of two target colors was associated with a reward, and the other with no reward. During test, 1 week later, participants completed another visual search task in which the reward association was not reinforced. When a rewarded feature remained task-relevant (Experiment 1), VDAC was observed. When the rewarded feature was made task-irrelevant (Experiments 2-5) there was no evidence of a VDAC effect, except when the target feature was physically salient and there was a reduction in the frequency of exposure to the reward-associated feature (Experiment 5). We failed to find evidence of VDAC in Experiments 2-4, suggesting that VDAC may depend on the demands of the task resulting in vulnerability to VDAC. When VDAC was observed, extinction was also observed. This indicates that VDAC is subject to extinction as would be expected from an effect driven by reinforcement learning.

Higher physical fitness levels are associated with less language decline in healthy ageing.

Healthy ageing is associated with decline in cognitive abilities such as language. Aerobic fitness has been shown to ameliorate decline in some cognitive domains, but the potential benefits for language have not been examined. In a cross-sectional sample, we investigated the relationship between aerobic fitness and tip-of-the-tongue states. These are among the most frequent cognitive failures in healthy older adults and occur when a speaker knows a word but is unable to produce it. We found that healthy older adults indeed experience more tip-of-the-tongue states than young adults. Importantly, higher aerobic fitness levels decrease the probability of experiencing tip-of-the-tongue states in healthy older adults. Fitness-related differences in word finding abilities are observed over and above effects of age. This is the first demonstration of a link between aerobic fitness and language functioning in healthy older adults.

bcl-2 modulation of apoptosis induced by anticancer drugs: resistance to thymidylate stress is independent of classical resistance pathways.

The hypothesis was tested that expression of bcl-2 could provide protection against apoptosis induced by cytotoxic drugs via a mechanism which was different from the classical determinants of drug resistance. Sensitivity and resistance to inhibitors of thymidylate synthase (EC 2.1.1. 45) were chosen for study since these drugs have a well-defined and quantifiable locus of action with similarly well defined biochemical sequelae resulting from enzyme inhibition. Human lymphoma cells transfected with the vector alone readily underwent apoptosis after a 36-h exposure to various drugs. For example, 5-fluorodeoxyuridine (0.1 microM) induced 67% apoptosis in vector control cells 24 h after removal of the drug. In contrast, cells treated under identical conditions, but which expressed the bcl-2 protein, showed only basal levels of apoptosis (8%), with no significant fall in viability. Similar results were obtained using two quinazoline-based inhibitors of thymidylate synthase, N10-propargyl-5,8-dideazafolic acid (CB3717) and ICI M247496. Determinants of resistance to these three drugs were investigated. Analysis of the cell cycle, thymidylate synthase levels, and activity showed these to be unchanged by expression of bcl-2. Addition of the drugs brought about equivalent inhibition of proliferation in the presence or absence of bcl-2 expression. 5-Fluorodeoxyuridine treatment reduced TTP synthesis, induced strand breaks in nascent DNA, measured by alkaline elution, and increased the synthesis of thymidylate synthase; these changes preceded the onset of apoptosis and were identical in the vector controls and bcl-2 transfectants. Resistance to thymidylate stress in bcl-2-expressing cells therefore occurred by a mechanism different from those which classically define resistance to this type of cytotoxic drug.

Apoptosis in Burkitt lymphoma cells is driven by c-myc.

Chromosomal translocation and subsequent de-regulation of the c-myc proto-oncogene are considered to be critical events in the multi-stage evolution of Burkitt lymphoma (BL). It is widely accepted that Myc protein functions as a competence factor for proliferation. However, recent studies indicate that it can also act in some cell types as a regulator of apoptosis. BL cell populations display a high frequency of apoptosis in vivo, a property which is also readily demonstrable in vitro in group I BL cell lines. Such lines are known to retain the cell surface marker characteristics of the parental tumour cells and, in the case of Epstein-Barr virus-positive tumours, their restricted viral protein expression. We have shown previously that apoptosis in a group I BL cell line is inhibited by interferon (IFN)-alpha. Here we show that IFN-alpha-mediated suppression of apoptosis in group I BL cells corresponds temporally with inhibition of Myc protein levels. Furthermore, inhibition of Myc expression following treatment with c-myc anti-sense oligonucleotides markedly enhanced survival of group I BL cells. These results indicate that, whilst c-myc may facilitate cycling of tumour cells in which it is de-regulated, it also stimulates their apoptosis.

Repression of apoptosis in human B-lymphoma cells by CD40-ligand and Bcl-2: relationship to the cell-cycle and role of the retinoblastoma protein.

Using a Burkitt lymphoma cell line to model human B-cell apoptosis in vitro, we observed that crosslinking, by antibody, of cell surface immunoglobulin induced G1 growth-arrest followed by apoptosis. By contrast, cells treated with the Ca(2+)-ionophore, ionomycin, generated apoptotic signals in G2/M as well as in G1. Both ionomycin and anti-immunoglobulin treatment induced rapid dephosphorylation of Rb prior to apoptosis. Apoptosis was repressed following exposure to CD40-ligand and was accompanied by hyperphosphorylation of Rb and cell-cycle progression but not Bcl-2 expression. Expression of Bcl-2 protein in stable bcl-2-transfectants, also resulted in repression of apoptosis and anti-immunoglobulin-treated cells no longer underwent growth-arrest. In Bcl-2-expressing cells in which apoptosis was repressed, Rb remained hyperphosphorylated, even during G1-arrest induced by ionomycin. TGF beta treatment of Bcl-2-expressing cells induced G1-arrest, de-phosphorylation of Rb and apoptosis. These results suggest that the functional activity of Bcl-2 in B-lymphoma cells is dependent upon, or leads to, sustained hyperphosphorylation of Rb and that Rb hyperphosphorylation can be uncoupled from cell-cycle progression.

Differential effects of BCL-2 on survival and proliferation of human B-lymphoma cells following gamma-irradiation.

Bcl-2 can inhibit apoptosis induced by a variety of stimuli, including radiation and its presence in tumour cells would be expected to indicate poor prognosis. Bcl-2-expressing tumours, however, are often low-grade and highly responsive to therapy. To investigate this apparent paradox, we analysed in vitro the responses of Burkitt lymphoma (BL) cells to gamma-irradiation in the presence and absence of Bcl-2. High-level expression of Bcl-2 was shown to promote BL cell survival following irradiation. However, a significant proportion of Bcl-2-rescued cells subsequently underwent apoptosis after an extended period in culture. In addition, in different BL lines, Bcl-2 was found either to promote or to inhibit long-term proliferative activity following gamma-irradiation. This differential regulation of proliferation correlated both with differential effects of Bcl-2 on the cell cycle and with differences in p53 status. Thus, by one week after irradiation, BL cells expressing only wild-type p53 (wt/wt) had arrested in G1, whereas those with a mutant allele (wt/mu) were arrested in all phases of the cell cycle. The proportion of Bcl-2-rescued cells that subsequently underwent apoptosis was reduced by ligation of CD40 at the time of irradiation in wt/wt BL cells, but not in wt/mu cells. CD40-ligation reduced both G1-arrest and apoptosis in parallel. These results indicate that, whilst Bcl-2 can delay apoptosis in BL cells following gamma-irradiation, the protein can also cause growth-arrest and thereby promote apoptosis. Long-term survival following Bcl-2-mediated rescue of gamma-irradiated cells may depend on p53 status and require additional death-repressing or growth-promoting signals.

Adenovirus early region 1A protein binds to mammalian SUG1-a regulatory component of the proteasome.

Adenovirus early region 1A (Ad E1A) is a multifunctional protein which is essential for adenovirus-mediated transformation and oncogenesis. Whilst E1A is generally considered to exert its influence on recipient cells through regulation of transcription it also increases the level of cellular p53 by increasing the protein half-life. With this in view, we have investigated the relationship of Ad E1A to the proteasome, which is normally responsible for degradation of p53. Here we have shown that both Ad5 and Ad12 E1A 12S and 13S proteins can be co-immunoprecipitated with proteasomes and that the larger Ad12 E1A protein binds strongly to at least three components of the 26S but not 20S proteasome. One of these interacting species has been identified as mammalian SUGI, a proteasome regulatory component which also plays a role in the cell as a mediator of transcription. In vitro assays have demonstrated a direct interaction between Ad12 E1A 13S protein and mouse SUGI. Following infection of human cells with Ad5 wt and Ad5 mutants with lesions in the E1A gene it has been shown that human SUG1 can be co-immunoprecipitated with full-length E1A and with E1A carrying a deletion in conserved region 1 which is the region considered to be responsible for increased expression of p53. We have concluded therefore that Ad EIA binds strongly to SUGI but that this interaction is not responsible for inhibition of proteasome activity. This is consistent with the observation that purified Ad12 E1A inhibits the activity of the purified 20S but not 26S proteasomes. We have also demonstrated that SUGI can be co-immunoprecipitated with SV40 T and therefore we suggest that this may represent a common interaction of transforming proteins of DNA tumour viruses.

Analysis and discrimination of necrosis and apoptosis (programmed cell death) by multiparameter flow cytometry.

Necrosis and apoptosis are two distinct modes of cell death which differ in morphology, mechanism and incidence. Membrane disruptants, respiratory poisons and hypoxia cause ATP depletion, metabolic collapse, cell swelling and rupture leading to inflammation. These are typical features of necrosis. Apoptosis plays a crucial role in embryogenesis and development and is also prevalent in tumours. It is characterised by cell shrinkage, chromatin condensation and systematic DNA cleavage. Apoptotic cells are rapidly engulfed by phagocytes, thus preventing inflammatory reaction to degradative cell contents. In vivo, apoptosis is almost impossible to quantify due to problems of heterogeneity and the short half-life of an apoptotic cell. In vitro, mechanistic studies are further complicated by a late phase of apoptosis where the cell membrane becomes permeable to vital dyes and which occurs in the absence of phagocytes. Here we describe a novel and rapid multiparameter flow cytometric assay which discriminates and quantifies viable, apoptotic and necrotic cells via measurement of forward and side light scatter (proportional to cell diameter and internal granularity, respectively) and the DNA-binding fluorophores Hoechst 33342 and propidium. It is anticipated that mechanistic studies of apoptosis in a variety of cell types will greatly benefit from this mode of analysis.

Induction of apoptosis by chemotherapeutic drugs: the role of FADD in activation of caspase-8 and synergy with death receptor ligands in ovarian carcinoma cells.

Although ovarian tumours initially respond to chemotherapy, they gradually acquire drug resistance. The aims of this study were to identify how chemotherapeutic drugs with diverse cellular targets activate apoptotic pathways and to investigate the mechanism by which exposure to a combination of drugs plus death receptor ligands can increase tumour cell kill. The results show that drugs with distinct cellular targets differentially up-regulate TRAIL and TNF as well CD95L, but do not require interaction of these ligands with their receptor partners to induce cell death. Factors that were critical in drug-induced apoptosis were activation of caspases, with caspase-8 being activated by diverse drugs in a FADD-independent manner. Certain drugs also demonstrated some dependence on FADD in the induction of cell death. Caspase-9 was activated more selectively by chemotherapeutic agents. Combining ligation of death receptors with exposure to drugs increased tumour cell kill in both drug resistant cell lines and primary ovarian carcinoma cells, even though these cells were not sensitive to death receptor ligation alone. CD95L was more consistent at combining with drugs than TRAIL or TNF. Investigation of the mechanism by which a combination of drugs plus CD95 ligation can increase cell death showed that caspase-8 was activated in cells exposed to a combination of cisplatin and anti-CD95, but not in cells exposed to either agent alone.

Homology between a human apoptosis specific protein and the product of APG5, a gene involved in autophagy in yeast.

Apoptosis specific proteins (ASP) are expressed in the cytoplasm of cultured mammalian cells of various lineages following induction of apoptosis. The cDNA encoding ASP has been cloned from a human expression library and has significant homology to the Saccharomyces cerevisiae APG5 gene which is essential for yeast autophagy. The ASP gene, known as hAPG5, can be transcribed to give mRNAs of 3.3 kbp, 2.5 kbp and 1.8 kbp which are present at comparable levels in viable and apoptotic cells, demonstrating that protein expression must be regulated at the translational level. These data indicate a possible relationship between apoptosis and autophagy and suggest evolutionary conservation in mammalian apoptosis of a degradative process present in yeast.

Cellular radiosensitivity in V79 cells is linked to alterations in chromatin structure.

V79 cells grown as spheroids are more radioresistant than those grown as monolayers. Viable cells from spheroid culture contain restraints to ethidium bromide driven rewinding of DNA supercoils that are absent in monolayer cells. Spheroid cells also contain a DNA-protein matrix that is more resistant to detergent-induced degradation. The increase in structural integrity may be related to a 55-60 kD protein in the nucleoids of spheroid, but not monolayer cells. Spheroid cell radioresistance may therefore be related to a more stable chromatin platform for high fidelity repair of DNA damage.

Measurement of DNA damage in mammalian cells using flow cytometry.

A technique for the detection of DNA damage induced by radiation insult has been developed. Cells were lysed with a buffer containing 2 M sodium chloride to release the DNA in a supercoiled form, the nucleoid. These were stained with the DNA intercalating dye, ethidium bromide, and exposed to laser light within a flow cytometer. Scattered and fluorescent light was analyzed from the laser/nucleoid interaction following irradiation of viable cells with gamma rays. The addition of ethidium bromide to prepared nucleoids caused a reduction in scattered light due to condensation of the nucleoid. Irradiation of cells prior to nucleoid production and ethidium bromide treatment restricted this condensation and produced a dose-dependent increase in laser scatter. Nucleoids derived from human lymphocytes showed enhanced light scatter from 5 Gy, compared to Chinese hamster ovary (CHO) fibroblasts where doses above 10 Gy were required. Up to 30 Gy CHO nucleoids showed a dose-dependent reduction in the ethidium bromide fluorescence. This technique allows detection of altered light scattering and fluorescent behavior of nucleoids after cellular irradiation; these may be related to structural changes within the nucleus induced by the radiation. The use of flow cytometry compared to other methods allows a rapid analysis of nuclear damage within individual cells.

The increase in radioresistance of Chinese hamster cells cultured as spheroids is correlated to changes in nuclear morphology.

Chinese hamster V79 cells grown as spheroids in roller culture are more radioresistant than those grown as monolayers. The supercoiled structure of chromatin, as salt-extracted nucleoids, has been examined using flow cytometry. Irradiated viable cells from spheroid culture contain restraints to supercoil relaxation that are absent in monolayer cells. Further analysis of the chromatin organization from each growth form shows that the radioresistant spheroid cells contain a DNA-protein matrix that is more resistant to detergent-induced degradation. The increase in structural integrity may be due to the retention of a 55-60 kDa protein that is apparent in the nucleoids of spheroid, but not monolayer cells. The increase in structural integrity of the spheroid cells may explain their greater radioresistance by providing a more stable platform for high-fidelity DNA damage repair.

Detection of antithrombin III microheterogeneity.

Antithrombin III microheterogeneity was investigated by isoelectric focusing and immunofixation in healthy individuals and in patients with clinical conditions in which antithrombin III is known to vary (liver disease, nephrotic syndrome, after surgery and anticoagulant therapy). In normal plasma microheterogeneity was present with ten bands of varying intensity being visible in a pI range from 5.0-5.7. One variant was observed which was not associated with a clinical disorder. Low concentrations of antithrombin III were detected in some patients with liver disease, nephrotic syndrome and those on anticoagulant therapy and these demonstrated a decrease of intensity in all bands. Alterations in microheterogeneity were seen in patients tested after surgery and those with nephrotic syndrome. This indicates that changes in the subpopulations of antithrombin III can occur and may be relevant to clinical abnormalities.

A correlation between DNA-nuclear matrix binding and relative radiosensitivity in two human squamous cell carcinoma cell lines.

Three aspects of DNA topology were examined in two human squamous cell carcinoma lines of differing radiosensitivity (SQ-9G, D0 = 1.46 Gy; and SQ-20B, D0 = 2.36 Gy). High-salt-extracted nuclei (nucleoids) were taken from gamma-irradiated cells, stained with ethidium bromide and examined by flow cytometry. After 5 Gy, nucleoids from SQ-9G cells became 30% less efficient at adopting positive DNA supercoils than were unirradiated controls. In contrast, only a 4% difference was found with the radioresistant SQ-20B line. Both lines produced positive supercoils more efficiently after irradiation if first exposed to the topoisomerase II inhibitor VP16. Ethidium bromide titration of nucleoids was consistent with each containing similar numbers and sizes of DNA loops. In each line approximately 30-35% of DNA was accessible to trioxsalen, as shown by inter-strand crosslinking after UV photo-activation. Exhaustive digestion of nuclear DNA by DNase I removed more DNA from the radiosensitive than from the radioresistant cell line (12% vs 28% remaining). This difference was thought to be due to the increased accessibility of SQ-9G DNA in vitro. We suggest that a looser association of SQ-9G DNA with the nuclear matrix both promotes DNase I digestion and affects the ability of SQ-9G nucleoids to maintain positive DNA supercoils after irradiation. These data implicate the DNA matrix attachment region in the expression of radiation sensitivity in the cell lines studied.

Isolation of methicillin-resistant Staphylococcus aureus from non-sterile sites: evaluation of a new selective medium.

Methicillin-resistant Staphylococcus aureus (MRSA) are relatively common nosocomial isolates, causing problems for health-care professionals worldwide. Therefore, early detection of the organisms by the laboratory is essential. A new selective medium for MRSA is described, comprising a DNA-containing base and a combination of stains that permit direct visualisation of DNase activity around colonies. The medium is made partially selective by adding a number of antibiotics (aztreonam, polymyxin B, nystatin and oxacillin). When compared to other media used for the isolation of MRSA, it was found that the new medium allowed earlier detection of colonies and provided a good direct method of identification, reducing the need for time-consuming replating of colonies, and, therefore, the turnaround time for specimens entering the laboratory for MRSA screening.

Regulation of cell survival in Burkitt lymphoma: implications from studies of apoptosis following cold-shock treatment.

Burkitt lymphoma (BL) tumour-cell populations are known to display high rates both of proliferation and of apoptosis in vivo, but the mechanisms which determine whether a BL cell continues to cycle or engages its cell-death programme are not understood. Group-I BL-derived cell lines, which retain in vitro the proliferative and apoptotic capacities of the parental cells, selectively entered apoptosis when returned to 37 degrees C after a brief period at low temperature (1 degree C). The induction of apoptosis by cold treatment, as determined by morphological characteristics and DNA fragmentation, was readily detectable within the first 1 to 2 hr of re-incubation at 37 degrees C, reaching a maximum at 4 to 6 hours. Commitment to enter apoptosis occurred after as little as 20 to 30 min at 1 degree C. Significant cell death at 1 degree C occurred only during prolonged incubation in the cold and displayed the characteristics of necrosis. Both bcl-2-dependent and -independent survival pathways were found to provide protection from cold-induced apoptosis, but only if engaged before cold-shock treatment. These results indicate that continued cycling of group-I BL cells is dependent upon their capacity to inhibit or circumvent their normally constitutively active apoptotic programme, and are consistent with the notion that the synthesis of one or more critical "survival" proteins of short half-life is necessary to guarantee successful passage through the cell cycle. Notably, high levels of apoptosis were also inducible in group-I BL cells by inhibitors of RNA and protein synthesis.

Effects of interferon-alpha on human B cells: repression of apoptosis and prevention of cell growth are independent responses of Burkitt lymphoma lines.

We have previously shown that interferon-alpha (IFN-alpha) can repress apoptosis in Burkitt lymphoma (BL) cells. In this study, we have compared this protective response with a further, well-established effect of IFN-alpha on BL cells, that of growth arrest. Of a panel of BL lines comprising (i) EBV-positive and -negative lines that retain the phenotype of the parental tumour cells and (ii) the prototype IFN-alpha-growth-inhibited line, Daudi, only Daudi cells were found to undergo substantial growth inhibition in response to the cytokine. By contrast, all lines, with the notable exception of Daudi, were protected by IFN-alpha from high-rate apoptosis initiated by the Ca2+ ionophore ionomycin. Ionomycin failed to elicit an IFN-alpha-repressible apoptotic response in either wild-type Daudi cells or IFN-resistant sublines that were refractory to the growth-arresting effects of the cytokine. Analysis of c-myc protein levels confirmed previous observations that repression of apoptosis in IFN-alpha-rescuable BL cells was associated with an early inhibition of myc that was followed by a return to high-level expression. Significantly, ionomycin alone induced a comparable transient inhibition of myc protein in Daudi cells. In Daudi cells, but not in IFN-alpha-rescuable BL cells, renewed expression of myc observed after the early, transient down-regulation was followed by sustained down-regulation of the protein, which paralleled growth arrest. Our results indicate that long-term growth arrest and repression of apoptosis in BL are distinct cellular responses to IFN-alpha.