Mesh-free modeling of liquid crystals using modified smoothed particle hydrodynamics.

We present a generalization of the modified smooth particle hydrodynamics simulation technique capable of simulating static and dynamic liquid crystalline behavior. This generalization is then implemented in the context of the Qian-Sheng description of nematodynamics. To test the method, we first use it to simulate switching in both a Fréedericksz setup and a chiral hybrid aligned nematic cell. In both cases, the results obtained give excellent agreement with previously published results. We then apply the technique in a three-dimensional simulation of the switching dynamics of the post aligned bistable nematic device.

Fluvastatin reduces oxidative damage in human vascular endothelial cells by upregulating Bcl-2.

BACKGROUND: 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have been widely used in clinical practise and their efficacy in reducing cardiovascular risk has been well described. OBJECTIVES: To investigate the effect of low doses of fluvastatin (nanomolar) on H(2)O(2)-induced cell damage and the underlying mechanism. METHODS AND RESULTS: Primary cultures of human umbilical vein endothelial cells were used, and the effects of fluvastatin on H(2)O(2)-induced apoptosis, necrosis, and proliferation were observed. H(2)O(2) at a concentration of 100 mum significantly induced apoptotic cell death after 24-h cell culture. Fluvastatin at low concentrations (10-100 nm) prevented H(2)O(2)-induced apoptosis, as determined by a DNA fragmentation assay and by cell counting with trypan blue and Hoechst 33342 nuclei staining. The protective effect of fluvastatin was mediated by the upregulation of Bcl-2 expression as probed by real-time polymerase chain reaction and Western blotting. Using siRNA to knock down the expression of Bcl-2, the protective effect of fluvastatin was abolished. Fluvastatin had no direct effect on the H(2)O(2)-sensitive TRPM2 calcium channel. CONCLUSIONS: These results suggest that fluvastatin has a potent protective effect against H(2)O(2)-induced apoptosis via upregulation of Bcl-2 expression. The findings provide a new insight into the mechanism by which fluvastatin is able to modulate the influence of oxidative stress on vascular endothelial cells.

Topology and bistability in liquid crystal devices.

We study nematic liquid crystal configurations in a prototype bistable device -- the post aligned bistable nematic (PABN) cell. Working within the Oseen-Frank continuum model, we describe the liquid crystal configuration by a unit-vector field n , in a model version of the PABN cell. First, we identify four distinct topologies in this geometry. We explicitly construct trial configurations with these topologies which are used as initial conditions for a numerical solver, based on the finite-element method. The morphologies and energetics of the corresponding numerical solutions qualitatively agree with experimental observations and suggest a topological mechanism for bistability in the PABN cell geometry.

Fluvastatin induces apoptosis of vascular endothelial cells: blockade by glucocorticoids.

Statins block de novo synthesis of cholesterol by inhibiting the enzyme, HMG CoA reductase. The product of this reaction, mevalonic acid, is also a precursor of isoprenoids, molecules required for the activation of signaling G-proteins, such as Ras. Signal transduction pathways involving Ras are important for cell survival and this may be why statins induce apoptotic death of several cell types. Given that statins are used to treat vascular disease, surprisingly no studies have been conducted on vascular endothelial cells. Here we show that fluvastatin (FS), at concentrations from 1-2 microM, blocks growth and induces apoptosis of the endothelial cell line, EA.hy 926. Considerable redundancy is known to exist in cell signaling and in vivo toxicity of FS might be prevented by other signaling pathways, like those activated by adrenal or sex steroids. RT-PCR analysis revealed the expression of the androgen and glucocorticoid receptor in EA.hy 926 cells. Although the androgen, dihydrotestesterone (DHT) had no effect, the glucocorticoid, dexamethasone (Dex), blocked FS-induced apoptosis. Cell cycle analysis revealed that 24 h exposure to FS prevented cells from leaving G(1) and 24-48 h later a marked sub-G(1) peak was observed. Dex was able to reduce the sub-G(1) peak, but it failed to block accumulation of cells in G(1), indicating that it's effect was specific for blockade of apoptosis, and not specific to an effect on FS alone. This study strongly suggests that glucocorticoids have a role to play in preventing vascular injury and they may provide the reason why statins are not inherently toxic to vascular endothelial cells, in vivo.

Statin-induced apoptosis of vascular endothelial cells is blocked by dexamethasone.

Statins block de novo synthesis of cholesterol by inhibiting the enzyme, HMG CoA reductase. The product of this reaction, mevalonic acid, is also a precursor of isoprenoids, molecules required for the activation of signalling G-proteins, such as Ras. Signal transduction pathways involving Ras are important for cell survival and this may be why statins induce apoptotic death of several cell types. Given that statins are used to treat vascular disease, it is surprising that no studies have been conducted on vascular endothelial cells. For this reason, we have tested the effect of fluvastatin (FS) on the endothelial cell line EA.hy 926. Here we show that FS, at concentrations from 1 to 2 microM, blocks growth and induces apoptosis of the endothelial cell line, EA.hy 926. As considerable redundancy exists in cell signalling pathways for cell survival, toxicity of FS under more physiological conditions might be prevented by pathways that do not require Ras, such as those activated by adrenal or sex steroids. To test this hypothesis, first RT-PCR analysis was performed for nuclear receptor mRNA expression. This revealed the presence of mRNA for the androgen receptor (AR) and glucocorticoid receptor (GR). The effect of the AR agonist, dihydrotestosterone (DHT), and the GR agonist, dexamethasone (Dex), was then tested. Whilst DHT (100 nM) had no effect on FS-induced cell death, Dex (1 microM) blocked FS-induced apoptosis. Cell cycle analysis revealed that 24 h exposure to FS prevented cells from leaving G(1) and 24-48 h later a marked sub-G(1) peak was observed. Dex was able to reduce the sub-G(1) peak, but it failed to reduce accumulation of cells in G(1). Further studies revealed that, in addition to blocking FS-induced apoptosis, Dex was able to block apoptosis of EA.hy 926 cells induced by serum deprivation, tumour necrosis factor-alpha, oxidants, DNA damage and mitochondrial disruption. This study strongly suggests that glucocorticoids have a role to play in preventing vascular injury and they may provide a reason why statins are apparently not toxic to vascular endothelial cells in vivo.

Primary Ewing sarcoma of the orbit.

PURPOSE: To report the clinicopathologic features of a 17-year-old patient with primary Ewing sarcoma of the orbit. METHODS: The patient was evaluated clinically before surgery with computed tomography scans of the orbit. After surgery, the patient was staged with computed tomography scans and bone scan and was treated with systemic chemotherapy and radiation therapy to the orbit. The orbital biopsy was evaluated with conventional light microscopy and immunohistochemistry. RESULTS: Clinical evaluation revealed proptosis and limited upgaze. Computed tomography scans disclosed a mass involving the superior orbit, anterior cranial fossa, and temporal fossa. Microscopic examination revealed small, poorly differentiated cells with medium-sized nuclei containing finely granular chromatin and small nucleoli. The cytoplasmic borders of the cells were indistinct. A PAS stain revealed modest glycogen in many of the tumor cells. The tumor stained positive for O-13 and vimentin and was negative for neural, skeletal, and lymphoid cell markers. Computed tomography scan, bone scan, and blood chemistries revealed no other site of involvement. After treatment, the clinical symptoms and signs resolved, and there has been no evidence of residual orbital tumor or metastasis. CONCLUSIONS: Primary Ewing sarcoma of the orbit should be considered in the differential diagnosis of children or young adults with proptosis, diplopia, or periorbital swelling. Immunohistochemistry is essential to distinguish Ewing sarcoma from other small round cell tumors.

Dexamethasone blocks antioestrogen- and oxidant-induced death of pituitary tumour cells.

The oestrogen receptor is fundamental to the growth and survival of the rat pituitary tumour cell line, GH(3). Our previous studies have shown that antioestrogens such as RU 58668 and ZM 182780 will reduce the rate of cell division and also induce cell death. Death of these cells in response to antioestrogen treatment appears to be due to a heightened sensitivity to reactive oxygen species (ROS). As part of a study to determine the cross-talk between steroid receptor systems in these cells, we have observed that the glucocorticoid, dexamethasone (Dex), inhibits antioestrogen-induced cell death. Cell death induced by H(2)O(2) is enhanced by ZM 182780 and this effect is also blocked by Dex. As apoptotic cell death in a number of systems involves an early loss of mitochondrial membrane potential (DeltaPsi(m)), we have performed detailed studies on the time-course of DeltaPsi(m) loss in relation to the loss in cell membrane function. These studies have indicated that a loss of DeltaPsi(m) parallels a loss of cell membrane function - this is more characteristic of necrosis than of apoptosis. From microscopic observations of these cells in response to H(2)O(2), it has been noted that early cell membrane blebbing, induced by H(2)O(2), is blocked in the presence of ZM 182780. Cell membrane blebbing can precede necrosis as well as apoptosis and it is thought to involve cytoskeletal changes, for which localised glycolytic reactions provide ATP. These observations, together with those showing that removal of glucose, but not inhibition of mitochondrial function, enhances ROS-induced cell death, prompted studies on the glycolytic pathway. As a strong candidate mechanism, it would appear that, via an effect on one of the rate-limiting glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase, Dex is able to overcome the antioestrogen-enhanced loss of glycolytic function following exposure of cells to ROS. This report contributes to the growing body of evidence showing that glucocorticoids provide a survival advantage to both normal and tumour cell types.

Response of estrogen receptor containing tumour cells to pure antiestrogens and the calmodulin inhibitor, calmidzolium chloride.

Cell survival is dependent on both external and internally generated signalling processes and current strategies for medical intervention in neoplastic disease are directed towards signal transduction blockade. Redundancy in signalling pathways may mean, however, that a combination of agents is required for the maximal therapeutic benefit. We have explored this idea with regard to the antiestrogen sensitivity of estrogen dependent tumours. Using estrogen receptor (ER) containing tumour cell lines, we have determined whether antiestrogens increase the cytotoxicity of the potent calmodulin inhibitior, calmidzolium chloride (CCl). For the pituitary tumour cell line GH(3), CCl induces a form of apoptotic cell death and co-treatment with the pure antiestrogen, ZM 182780, enhances sensitivity to the calmodulin inhibitor, by at least two fold. In contrast to the pure steroidal antiestrogens, the triphenylethylenes, tamoxifen and 4-hydroxytamoxifen give no enhancing effect on CCl induced cell death. Although CCl induces apoptosis of several ER containing breast cancer cell lines, unlike the pituitary tumour cells, ZM 182780 is unable to increase their sensitivity to calmodulin inhibition. Further studies strongly suggest that cell death in response to calmodulin inhibition is the result of metabolic disruption and that for GH(3) cells, this is enhanced by antiestrogen treatment.

Direct activation of oestrogen receptor-alpha by interleukin-6 in primary cultures of breast cancer epithelial cells.

Interleukin 6 (IL-6) is secreted by breast tumours and shows synergistic activity with 17beta-oestradiol (E2), leading to increases in reductive 17beta-hydroxysteroid dehydrogenase activity in breast cancer epithelial cells. However, the mechanisms involved are poorly understood. Using short-term epithelial cultures established from primary breast tumours, we have examined whether IL-6 could directly affect transcriptional activity of oestrogen reception alpha (ERalpha). Tumour epithelial cultures were established from 15 breast tumours, grown to 70% confluence and transiently transfected with a plasmid reporter containing the vitellogenin oestrogen response element and the luciferase coding sequence (ERE-TK-LUC). Following transfection, cells were incubated with E2, IL-6, the pure anti-oestrogen ZM 182780 or combinations of these substances for 48 h. Luciferase activity was then measured in cell lysates. E2 caused a dose-dependent increase in luciferase expression, causing a maximum threefold stimulation at 100 pM. In the presence of IL-6, transcriptional activity was increased by up to 2.5-fold in ERalpha+ cultures (11/15). In combination with E2, synergistic effects were observed with increases in luciferase activity of up to sixfold over controls. This effect could be blocked by treatment with ZM 182780. Pre-incubation of cells with an antibody directed against the signalling component of IL-6, gp130, was ineffective in blocking the E2 response. This antibody reduced, but did not completely block the effect of IL-6 either alone or in combination with E2, suggesting cross-talk between the two signalling pathways. In conclusion, these results provide evidence for direct transcriptional activation of ERalpha by IL-6.

Evidence for transcriptional activation of ERalpha by IL-1beta in breast cancer cells.

Estrogen is mitogenic in breast cancer where IL-1beta also fulfils a role. The aim of this study was to determine any relationship between IL-1beta and ERalpha in breast cancer. By RT-PCR, 26/77 tumours expressed IL-1beta, and 57/77 expressed ERalpha. Samples which were IL-1beta positive were categorised against those which expressed ERalpha. Of the 26 tumours which expressed IL-1beta, all were ERalpha positive. We next examined whether IL-1beta could directly activate ERalpha. MCF-7 cells stably transfected with a plasmid reporter (ERE-TK-LUC) were incubated with either 17beta-estradiol (E2, 10-9-10-13 M), IL-1beta (10 ng/ml), the pure antiestrogen ZM 182780 (10 nM) or combinations of these substances. Transcriptional activity was measured in cell lysates 48 h later. E2 caused a dose-dependent increase in luciferase activity. With IL-1beta, transcriptional activity was typically half of the E2 response. To determine the role of the IL-1 receptor, parallel cultures were incubated with IL-1 receptor antagonist. This reduced, but did not completely block the effect of IL-1beta, suggesting that IL-1beta was affecting transcriptional activity via another pathway. Confirmation that the effect was via ERalpha was verified using the pure antiestrogen, ZM 182370, which completely abrogated the effects of E2, when added alone or in combination with IL-1beta. These results provide compelling evidence for direct transcriptional activation of ERalpha by IL-1beta. Interactions of these factors may thus modulate hormonal activity in human breast tumours.

Functional inactivation of the oestrogen receptor by the antioestrogen, ZM 182780, sensitises tumour cells to reactive oxygen species.

Reactive oxygen species (ROS) play a fundamental role in both apoptotic and necrotic cell death. Their importance is highlighted by studies showing that they mediate cell death in response to radiotherapy and to some forms of chemotherapy. Here we provide the first evidence for a role of ROS in response to an antiendocrine agent currently undergoing clinical trials. Using the oestrogen receptor (ER) containing rat pituitary GH3 cell line, we show that cell death is induced by the pure steroidal antioestrogen, ZM 182780, and that this is blocked by the antioxidant, N-acetyl cysteine (NAC). By flow cytometry, we show that, prior to the onset of DNA breakdown measured by ELISA, ZM 182780 exposure has no significant effect on intracellular oxidant concentrations. In contrast, ZM 182780 exposure greatly increases sensitivity to oxidants generated by blocking cellular antioxidant pathways and from exogenous administration of hydrogen peroxide (H2O2). As both necrosis and apoptosis are controlled by mitochondrial function, further experiments conducted to determine mitochondrial membrane potential (Delta|gWm) have indicated that the ZM 182780-induced loss of ER function increases the ease with which oxidants collapse mitochondrial activity and, as a consequence, cell death.

Development of differential preganglionic projections to pre- and paravertebral sympathetic ganglia.

Sympathetic preganglionic axons project to spatially distinct targets in the periphery. A precise topographic pattern exists within the thoracic preganglionic cell column relative to the direction of axonal projections within the sympathetic chain. In this study, the time course and pattern of axonal outgrowth from different populations of preganglionic neurons in the chicken embryo is examined in detail to clarify the origin of the topography in this system. Projections to prevertebral targets are established by development of the splanchnic nerves by stage 25, well after the earliest somatic motor projections at stage 19 but at least two stages before the reported onset of paravertebral projections. Further, preganglionic axons that project rostrally into the sympathetic chain may do so earlier than those that project caudally in the chain. The separation of preganglionic axons into prevertebral, rostral paravertebral or caudal paravertebral directions occurs at a common site in the ventral mesenchyme, established by the initial ventromedial projection of the splanchnic nerves. Analysis of the axonal trajectories of rostrally and caudally projecting cells reveals that preganglionic axons are not selectively fasciculated before their point of separation at the sympathetic chain. The patterning of the preganglionic cell column is specified before the establishment of functional connections within the chain, indicating that target contact is not a determinant of the segmental pattern. We suggest that the differential outgrowth of preganglionic axons to peripheral targets is determined by the unique identities of underlying subpopulations of preganglionic axons.

Neuroprotection against oxidative stress by estrogens: structure-activity relationship.

Oxidative stress-induced neuronal cell death has been implicated in different neurological disorders and neurodegenerative diseases; one such ailment is Alzheimer's disease. Using the Alzheimer's disease-associated amyloid beta protein, glutamate, hydrogen peroxide, and buthionine sulfoximine, we investigated the neuroprotective potential of estrogen against oxidative stress-induced cell death. We show that 17-beta-estradiol, its nonestrogenic stereoisomer, 17-alpha-estradiol, and some estradiol derivatives can prevent intracellular peroxide accumulation and, ultimately, the degeneration of primary neurons, clonal hippocampal cells, and cells in organotypic hippocampal slices. The neuroprotective antioxidant activity of estrogens is dependent on the presence of the hydroxyl group in the C3 position on the A ring of the steroid molecule but is independent of an activation of estrogen receptors.

Estrogen receptor blockade by the pure antiestrogen, ZM 182780, induces death of pituitary tumour cells.

Our previous studies have shown that even in the absence of estrogen, the estrogen receptor (ER) is still involved in growth by way of its conversion to a transcriptionally active state by growth inducing cytokines. The following paper now provides evidence that under more physiological conditions, the ER within the GH3 cell line used for the previous investigations, not only controls growth, but that transcriptional activity of the receptor is required for cell survival. Therefore when GH3 cells, maintained under serum and steroid replete conditions, are exposed to the pure antiestrogen ZM 182780 (10 nM), marked cell death is observed 72-120 h after first exposure. Studies on the nature of this cell death suggested that it had some of the reported characteristics of apoptosis or programmed cell death. Removal of steroids from the culture medium also resulted in cell death and this was enhanced by the addition of the pure antiestrogen. Both steroid withdrawal and ZM 182780 induced cell death was completely reversed by the inclusion of estrogens in the steroid free culture medium. In contrast, the non-steroidal antiestrogen, 4-hydroxytamoxifen (4-OHT) was not able to enhance steroid withdrawal death and at 1 microM, this compound was shown to have marked ER agonist activity. Further studies on the addition of conditioned medium from high density GH3 cell cultures, to low density steroid free cells, strongly suggested that the ER within these cells was responsible for the production of autocrine/paracrine survival factors.

Expression and localization of endothelin-1 and endothelin receptors in human meningiomas. Evidence for a role in tumoral growth.

In addition to its well-known homoeostatic actions in the cardiovascular system, ET-1 has been shown to constitute a potent growth regulatory peptide in various tissues. We have studied the expression of ET-1 and its receptors (ET-Ar and ET-Br) in human meningiomas (n = 35) as well as their involvement in cellular growth. By PCR of reverse-transcribed RNA we detected ET-1 mRNA in 91% (32 of 35), ET-Ar mRNA in 82% (29 of 35), and ET-Br mRNA in 42% (15 of 35) of human meningiomas examined. The localization of ET-1 mRNA, ET-Ar mRNA, and ET-1 peptide in tumoral cells was observed by in situ hybridization and immunohistochemistry, whereas ET-Br mRNA was expressed at low level only in cells belonging to blood vessels. In addition, we found that ET-1 stimulated [3H] thymidine incorporation in primary cell cultures of 20 meningiomas and that this effect could be blocked by BQ-123, a specific antagonist for ET-Ar. In contrast, RES-701-3, an antagonist of ET-Br, did not block the proliferative effect of ET-1. In conclusion, our data provide evidence that ET-1 constitutes an important growth factor for meningiomas acting via ET-Ar. We can hypothesize that ET-1, acting in concert with other growth factors and cytokines, is involved in the meningioma tumorigenesis.

Involvement of interleukin-1 and interleukin-1 receptor antagonist in rat pituitary cell growth regulation.

Interleukin-1 (IL-1), one of the mediators of the interaction between the immune and the neuroendocrine system, is well known to modulate anterior pituitary hormone secretion. As IL-1 influences growth of various cell types, we investigated whether IL-1 is also a growth factor of pituitary cells. We demonstrate that IL-1 dose and time dependently inhibits the growth of normal rat pituitary cells under serum-free conditions. The inhibitory potencies of IL-1 alpha (ED50, 8 pg/ml) and IL-1 beta (ED50, 6 pg/ml) were nearly identical. In the presence of low amounts of serum, both IL-1 alpha (ED50, 130 pg/ml) and IL-1 beta (ED50, 90 pg/ml) were less effective in inhibiting growth. The IL-1-induced growth inhibition was IL-1 receptor mediated, because both IL-1 alpha- and IL-1 beta-mediated growth suppression could be completely reversed by the IL-1 receptor antagonist, the physiological counterpart of IL-1. In the mammosomatotroph GH3 rat pituitary tumor cell line, IL-1 failed to influence growth, indicating that either IL-1 receptors are missing or the IL-1 signaling pathway is uncoupled from the growth regulation. In contrast to growth, in our rat pituitary monolayer cell culture system, IL-1 did not affect ACTH, GH, or PRL secretion as described previously. This discrepancy suggests the involvement of different mechanisms in the IL-1-induced mediation of growth and hormone secretion.

Halofantrine pharmacokinetics in Kenyan children with non-severe and severe malaria.

1. Kenyan children with uncomplicated malaria given oral halofantrine (HF; non-micronised suspension; 8 mg base kg-1 body weight 6 hourly for three doses) showed wide variation in the disposition of HF and desbutylhalofantrine (HFm). 2. Eight Kenyan children with severe (prostrate) falciparum malaria who were receiving intravenous quinine, were given the same HF regimen by nasogastric tube. One patient had undetectable HF and two had undetectable HFm at all times after drug administration. 3. The mean AUC(0,24 h) of HF in prostrate children was half (7.54 compared with 13.10 micrograms ml-1 h) (P = 0.06), and that for HFm one-third (0.84 compared with 2.51 micrograms ml-1 h) (P < 0.05) of the value in children with uncomplicated malaria. 4. Oral HF may be appropriate for some cases of uncomplicated falciparum malaria in Africa, but in patients with severe malaria, the bioavailability of HF and HFm may be inadequate.

Involvement of the estrogen receptor in the growth response of pituitary tumor cells to interleukin-2.

Having recently demonstrated in separate studies that the T-cell cytokine, interleukin-2, induces the growth of the pituitary tumor cell line GH3 and that in the same cells, the estrogen receptor mediates the mitogenic effect of growth factors, we sought here to determine whether the estrogen receptor was involved in the response to interleukin-2. We demonstrate that under steroid and serum free growth conditions, the pure antiestrogen, ZM 182780, blocks the mitogenic response of GH3 cells to interleukin-2. Transfection studies with a reporter plasmid responsive to the transcriptionally active estrogen receptor show that even in the absence of ligand, the estrogen receptor in these cells is transcriptionally active and this can be increased by interleukin-2. Further studies on the two estrogen receptor regulated proteins, the progesterone receptor and prolactin, showing that the levels of these proteins were increased by exposure of cells to interleukin-2, support the idea of a cross-talk between the estrogen receptor and interleukin-2 signal transduction.