Wild-type human p53 and a temperature-sensitive mutant induce Fas/APO-1 expression.

Fas/APO-1 is a cell surface protein known to trigger apoptosis upon specific antibody engagement. Because wild-type p53 can activate transcription as well as induce apoptosis, we queried whether p53 might upregulate Fas/APO-1. To explore this possibility, we examined human p53-null (H358 non-small-cell lung adenocarcinoma and K562 erythroleukemia) and wild-type p53-containing (H460 non-small-cell lung adenocarcinoma) cell lines. When H358 or H460 cells were transduced with a replication-deficient adenovirus expression construct containing the human wild-type p53 gene but not with vector alone, a marked upregulation (approximately a three-to fourfold increase) of cell surface Fas/APO-1 was observed by flow cytometry. Similarly, K562, cells stably transfected with a plasmid vector containing the temperature-sensitive human p53 mutant Ala-143 demonstrated a four- to sixfold upregulation of Fas/APO-1 by flow-cytometric analysis at the permissive temperature of 32.5 degrees C. Temperature-sensitive upregulation of Fas/APO-1 in K562 Ala-143 cells was verified by immunoprecipitation and demonstrated to result from enhanced mRNA production by nuclear run-on and Northern (RNA) analyses. Stably transfected K562 cells expressing temperature-insensitive, transcriptionally inactive p53 mutants (His-175, Trp-248, His-273, or Gly-281) failed to upregulate Fas/APO-1 at either 32.5 degrees or 37.5 degrees C. The temperature-sensitive transcription of Fas/APO-1 occurred in the presence of cycloheximide, indicating that de novo protein synthesis was not required and suggested a direct involvement of p53. Collectively, these observations argue that Fas/APO-1 is a target gene for transcriptional activation by p53.

Lifestyle use of drugs by healthy people for enhancing cognition, creativity, motivation and pleasure.

Today, there is continued, and in some cases growing, availability of not only psychoactive substances, including treatments for mental health disorders such as cognitive enhancers, which can enhance or restore brain function, but also 'recreational' drugs such as novel psychoactive substances (NPS). The use of psychoactive drugs has both benefits and risks: whilst new drugs to treat cognitive symptoms in neuropsychiatric or neurodegenerative disorders could have great benefits for many patient groups, the increasing ease of accessibility to recreational NPS and the increasing lifestyle use of cognitive enhancers by healthy people means that the effective management of psychoactive substances will be an issue of increasing importance. Clearly, the potential benefits of cognitive enhancers are large and increasingly relevant, particularly as the population ages, and for this reason, we should continue to devote resources to the development of cognitive enhancers as treatments for neurodegenerative diseases and psychiatric disorders, including Alzheimer's disease, attention deficit hyperactivity disorder and schizophrenia. However, the increasing use of cognitive enhancers by healthy individuals raises safety, ethical and regulatory concerns, which should not be ignored. Similarly, understanding the short- and long-term consequences of the use of NPS, as well as better understanding the motivations and profiles of users could promote more effective prevention and harm reduction measures. Linked Articles This article is part of a themed section on Pharmacology of Cognition: a Panacea for Neuropsychiatric Disease? To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.19/issuetoc.

Divergent effect of fluoxetine on the response to physical or chemical stressors in zebrafish.

Fluoxetine is a selective serotonin reuptake inhibitor that increases serotonin concentration in the central nervous system and modulates various systems, including the control of sympathetic outflow and the hypothalamus-pituitary-adrenal. However, it is not yet established whether fluoxetine can modulate the responses to stressors stimulants (physical or chemical) that trigger cortisol response in zebrafish. We demonstrate that fluoxetine blunts the response to physical stress, but not to chemical stress.

Soluble Fas/APO-1 in tumor cells: a potential regulator of apoptosis?

Fas/APO-1, a member of the NGF/TNF receptor superfamily expressed on the cell-surface of normal and malignant cells, is known to induce cell death by apoptosis. In the present study, we have investigated Fas/APO-1 gene defects in a human osteosarcoma cell line resistant to the apoptosis-inducing effects of anti-Fas. cDNA cloning and sequencing revealed that these cells contained both 'authentic' and mutant Fas/APO-1 containing a 63 base pair in-frame deletion spanning the transmembrane domain, designated DFas/APO-1. Direct evidence for the existence of a soluble Fas/APO-1 protein was obtained by immunoprecipitation and Western blotting. Taken together with prior studies demonstrating a role for Fas/APO-1 and Fas ligand, respectively, in tumor target cell killing by cytotoxic T-lymphocytes, production of soluble Fas/APO-1 might have significant implications in malignant disease pathogenesis.

Shear stress modulates the proliferation rate, protein synthesis, and mitogenic activity of arterial smooth muscle cells.

BACKGROUND: The aim of this study was to determine the correlation between hemodynamic forces and proliferation of smooth muscle cells (SMC). METHODS: Bovine arterial SMC were seeded in a fibronectin-coated polystyrene cylinder at 5 x 10(5) cells/tube and allowed to reach confluence and to adhere for 48 hours. The experimental groups were subjected to a laminar flow of 150 ml/min (9 dyne/cm2), 100 ml/min (6 dyne/cm2), and 50 ml/min (3 dyne/cm2) for 24 hours. The control group was subjected to similar incubation conditions without flow. The cells in the experiments remained attached and viable. All experiments were performed in triplicate or more. RESULTS: Shear stress significantly reduced (p < 0.001) the 24-hour incorporation of tritiated thymidine and cell proliferation. This effect was proportional to the level of shear stress and was still evident 24 hours after flow cessation. Results of flow cytometry confirmed a lower percentage of SMC in S phase with increasing shear stress. Synthesis of cell-associated proteins was increased twofold (p < 0.01) in SMC subjected to laminar flow. SMC subjected to shear stress released a higher quantity of mitogens, including a platelet-derived growth factor (PDGF)-like substance as detected by immunologic testing. Fifty percent volume per volume conditioned serum-free medium from SMC subjected to shear stress increased threefold the tritiated thymidine uptake in PDGF receptor-bearing Swiss 3T3 cells as compared with conditioned serum-free medium from control SMC not subjected to shear stress and twelvefold as compared with standard control. The release of mitogens was proportional to the level of shear stress and was still evident 24 hours after flow cessation. The mitogenic activity was partially reduced (30%, p < 0.01) by an excess of monospecific anti-PDGF antibody. CONCLUSIONS: We conclude that (1) increasing shear stress inhibits SMC proliferation and stimulates the synthesis of cell-associated proteins and the release of mitogens and (2) decreasing shear stress facilitates proliferation of SMC. Thus, in situations of arterial flow separation, the increased release of mitogens from SMC subjected to high shear stress and the increased proliferation rate and susceptibility to mitogens of SMC subjected to very low shear stress may generate a critical condition that predisposes to the development of atherosclerosis with early plaque formation in regions of low-flow shear stress.

Shear stress induces transforming growth factor-beta 1 release by arterial endothelial cells.

BACKGROUND: Myointimal hyperplasia is a common complication after vascular reconstruction. Increasing shear stress has been shown to reduce formation of myointimal hyperplasia. The aims of our study were (1) to analyze the correlation between shear stress and release of transforming growth factor (TGF)-beta 1 by endothelial cells and (2) to determine the effect of TGF-beta 1 on smooth muscle cell proliferation. METHODS: Bovine arterial endothelial cells were subjected to increasing shear stress in an in vitro serum-free system. The release of TGF-beta 1 by endothelial cells was assessed by enzyme-linked immunosorbent assay and Western blot analysis. The effect of TGF-beta 1 on the proliferation of the subconfluent monolayer of bovine smooth muscle cells was determined by tritiated thymidine uptake. RESULTS: Shear stress induced a significant increase of the release of TGF-beta 1 by endothelial cells (p < 0.001). This phenomenon was proportional to the level of shear stress. The amount of TGF-beta 1 released by endothelial cells subjected to shear stress had a significant inhibitory effect on growth rate and tritiated thymidine uptake of smooth muscle cells. CONCLUSIONS: On the basis of the results of our study, we conclude that increasing shear stress induces release of TGF-beta 1 by arterial endothelial cells in a concentration that has a clear inhibitory effect on smooth muscle cell proliferation. This phenomenon could explain the inhibitory effect of increasing shear stress on the formation of myointimal hyperplasia.

Shear stress increases the release of interleukin-1 and interleukin-6 by aortic endothelial cells.

BACKGROUND: The aim of this study was to determine the correlation between shear stress and the release of interleukin-1 (IL-1) and interleukin-6 (IL-6) by endothelial cells (EC). METHODS: Bovine aortic EC were seeded in fibronectin-coated cylinders at 1.0 x 10(6) cells/tube and allowed to reach confluence and to adhere for 48 hours. The experimental groups were subjected to a laminar flow of 100 ml/min (6 dyne/cm2). The control group was subjected to similar incubation conditions without flow. The release of IL-1 and IL-6 by EC was measured by enzyme-linked immunosorbent assay. RESULTS: Shear stress increased significantly (p < 0.01) the release of IL-1 and IL-6 by EC. The release of these two cytokines had different kinetics. CONCLUSIONS: Increasing shear stress facilitates release of IL-1 and IL-6 by EC. Previous reports have shown that IL-1 and IL-6 promote vascular smooth-muscle cell proliferation. Thus abnormal flow conditions with increasing shear stress may predispose to smooth-muscle cell proliferation that characterizes early atherosclerotic plaque development by an interleukin-mediated mechanism.

Formation of myointimal hyperplasia and cytokine production in experimental vein grafts.

BACKGROUND: The purpose of this study was to determine the correlation between progression and regression of myointimal hyperplasia (MH) and cytokine production in experimental vein grafts. Although the autologous vein is the best suitable bypass conduit for reconstruction of peripheral arteries, at the end of the first year thrombosis in the coronary and lower extremity circulation ranges from 20% to 50%. Many of these failures are caused by MH. METHODS: In 76 inbred Lewis rats, a 1 cm long segment of inferior vena cava was inserted at the level of the abdominal aorta. The segments of inferior vena cava were obtained from syngeneic Lewis rats. In 56 animals the arterial vein graft was explanted 3 days (n = 10), 7 days (n = 10), 4 weeks (n = 26), and 12 weeks (n = 10) after operation. In 20 animals the vein graft was explanted 4 weeks after being in the arterial system and reimplanted as iliac venovenous bypass in syngeneic Lewis rats. These grafts were explanted 2 weeks (n = 10) and 8 weeks (n = 10) later. Grafts were analyzed by light and electron microscopy, morphometric study, and histochemical analysis and were put in an organ culture to assess cytokine production. RESULTS: We observed MH formation in arterial vein grafts and MH regression in reimplanted vein grafts (p < 0.001). MH formation was correlated with production of platelet-derived growth factor, basic fibroblast growth factor, interleukin-1, and tumor necrosis factor-alpha. MH regression was correlated with transforming growth factor-beta 1 production. CONCLUSIONS: On the basis of the results of our study, we conclude that MH formation in experimental vein grafts depends on production of platelet-derived growth factor, basic fibroblast growth factor, interleukin-1, and tumor necrosis factor-alpha, and MH regression depends on transforming growth factor-beta 1 production. Cytokine therapy may represent a valuable new treatment to prevent vein bypass failures caused by MH.

Selection, establishment and characterization of cell lines derived from a chemically-induced rat mammary heterogeneous tumor, by flow cytometry, transmission electron microscopy, and immunohistochemistry.

In order to isolate, characterize, and establish culture cell lines with different diagnostic and prognostic significance, derived from multiclonal neoplasms, a ductal infiltrating mammary tumor was induced in rats by 7,12-dimethylbenz[a]anthracene. Clones with different DNA/protein content, being the DI of 1.16, 1.30, and 1.60, respectively, were observed in the primary tumor. Biparametric flow cytometry suggested that the clone at 1.30 is made up of two subpopulations with different protein and slightly different DNA contents. The culture, after a few passages, exhibited the presence of aneuploid cells and the absence of diploid components, demonstrating that only tumor cells survived. The limiting dilution method gave rise to four lines with DI of 1.16, 1.25, 1.30, and 1.50; a mean chromosome number of 45, 46, 47, and 88, respectively; and different morphological and ultrastructural features. These characteristics were stable during the experimental procedure, that is, for about 20 passages. Conversely, the detection of cytoskeletal proteins indicated that the tumor epithelial cells underwent early dedifferentiation into sarcoma-like cells showing markers of stromal cell type and thus exhibiting phenotypic instability in vitro, a feature reported in many advanced human breast cancers in vivo. In conclusion, this cellular model represents the in vivo situation and appears suitable for in vitro studies of tumor cell characteristics and might be used to predict clinical behavior.

Response of arterial smooth muscle cells to laminar flow.

This study was designed to determine whether laminar flow influences the proliferation rate and morphology of smooth muscle cells (SMC) in culture. Bovine aortic SMC were subjected to a shear stress of 6 dyne/cm2. The control group was subjected to similar incubation conditions without flow. Flow cytometry demonstrated decreased proliferation rate in SMC subjected to laminar flow. This phenomenon was still evident 24 hours after flow cessation. SMC were examined by light and electron microscopy. SMC subjected to laminar flow aligned along the direction of flow assuming a spheric morphology. These changes were reversible after a 48-hour resting period. The degree of organization of actin, tubulin and other microfilaments was evaluated by indirect immunofluorescence. SMC subjected to shear stress showed a clear reorganization of the cytoskeleton with expression of stress fibres. These changes were reversible after a 48-hour resting period. These findings may contribute to understanding the mechanisms by which SMC in vivo respond to forces generated by blood flowing under pressure.

Memory retrieval improvement by Ptychopetalum olacoides in young and aging mice.

Amazonian peoples use traditional remedies prepared with Ptychopetalum olacoides (PO) roots for treating various age-related conditions. This study shows that a single intraperitoneally (i.p.) administration of Ptychopetalum olacoides ethanol extract (POEE, 50 and 100mg/kg) improved memory retrieval in step-down inhibitory avoidance (P

An anti-LFA-1 monoclonal antibody (LDA-8) induces cellular aggregation of human lymphoblastoid cell lines and peripheral blood lymphocytes.

The human leukocyte-function-associated antigen-1 (LFA-1) plays a key role in intercellular adhesion interactions of the immune response. A monoclonal antibody (mab), designated LDA-8, is described that recognizes LFA-1. In contrast to nearly all other anti-LFA-1 mabs, which inhibit cellular aggregation, LDA-8 induces cell-cell aggregation. The LDA-8 mab was generated by immunizing mice with membrane fragments from the Jurkat T-cell line. The LDA-8 mab stained peripheral blood mononuclear cells (PBMC), monocyte-depleted peripheral blood lymphocytes, purified monocytes, and a number of T and B tumor cell lines. The LDA-8 mab induced aggregation of PBMC from normal donors, as well as of cells from T-cell lines (MOLT4 and CEM). Control mabs directed against HLA class 1 or CD4 did not induce aggregation. Aggregation was concentration- and time-dependent. EDTA added to the cultures 1 hour prior to or together with the LDA-8 mab did not inhibit LDA-8-induced aggregate formation. Anti LFA-1 alpha-chain mab added to the cells 1 hour prior to LDA-8 mab, or together with the LDA-8 mab, also did not inhibit LDA-8-induced aggregation. In contrast, anti-LFA-1 beta-chain mab, added to the cells together with or 1 hour prior to the LDA-8 mab, significantly inhibited LDA-8-induced aggregate formation. The LDA-8 mab immunoprecipitated two polypeptide chains of 110 kDa and 160 kDa under non-reducing conditions and of 92 kDa and 162 kDa under reducing conditions, from cells of the MOLT-4 or CEM T-cell lines or phytohemagglutinin (PHA)-stimulated PBMC. The molecular mass of these polypeptides was identical to that of polypeptides immunoprecipitated by the anti-LFA-1 TS1.22 mab, suggesting that the LDA-8 mab and the anti-LFA-1 mab recognize the same molecule. This was confirmed by sequential immunoprecipitation. The LDA-8 mab recognizes a unique epitope on LFA-1 and induces cell aggregation that is blocked by mabs recognizing the beta-chain, but not the alpha-chain of the LDA-1 molecule.

Growth factor release by smooth muscle cells is dependent on haemodynamic factors.

Abnormal proliferation of smooth muscle cells (SMCs) is a key event in the development of the atherosclerotic plaque. The early atherosclerotic plaque localises preferentially in areas of flow separation. The basic mechanisms which correlate haemodynamic forces and atherosclerotic plaque formation are not known. Bovine arterial SMCs were subjected to increasing levels of shear stress for 24 h in an in vitro system. The control group was subjected to similar incubation conditions without flow. SMCs subjected to shear stress released a higher quantity of mitogens, including a platelet derived growth factor (PDGF)-like substance. This mitogenic activity was partially reduced (30%, p < 0.01) by an excess of monospecific anti-PDGF antibody. The release of mitogens was proportional to the level of shear stress and was still evident 24 h after flow cessation. In conclusion, shear stress influences the release of mitogens; this might represent a mechanism which links haemodynamic forces and atherosclerotic plaque formation.

Progression and regression of myointimal hyperplasia in experimental vein grafts depends on platelet-derived growth factor and basic fibroblastic growth factor production.

PURPOSE: The factors that lead to myointimal hyperplasia (MH) in arterial vein grafts (AVGs) are unknown. Platelet-derived growth factor (PDGF) and basic fibroblastic growth factor (bFGF) are two powerful mitogens for smooth muscle cells that have been implicated in the genesis of MH. The aim of this study was to analyze the correlation between progression and regression of MH and production of PDGF and bFGF in experimental vein grafts. MATERIALS: In 64 inbred Lewis rats, a 1-cm segment of inferior vena cava was inserted at the level of the abdominal aorta. The segments of inferior vena cava were obtained from syngenic rats. In 48 rats, the AVG was explanted 3 days (n = 8), 7 days (n = 8), 4 weeks (n = 24), and 12 weeks (n = 8) after surgery. In 16 rats the vein graft was explanted after being in the arterial system for 4 weeks and was reimplanted as a venous-venous bypass in syngenic Lewis rats. Reimplanted vein grafts (RVGs) were explanted 2 weeks (n = 8) and 8 weeks (n = 8) later. Grafts were analyzed by light and electron microscopy, morphometry, and histochemistry, and were put in organ culture to assess PDGF and bFGF production and mitogenic activity. RESULTS: We observed MH formation in AVGs and MH regression in RVGs (p < 0.001).PDGF and bFGF production correlated with the degree of MH (p < 0.01). Histochemistry showed PDGF and bFGF in the area of MH in AVG, which disappeared in RVG. Conditioned media from AVG had greater mitogenic activity than RVG or control veins. CONCLUSION: MH formation and regression in experimental vein grafts correlate with PDGF and bFGF production.

The degree of porosity influences the release of growth factors by healing polytetrafluoroethylene (PTFE) grafts.

OBJECTIVES: The aim of this study was to determine the influence of the degree of porosity on the release of growth factors (PDGF AA, PDGF BB, bFGF) by healing PTFE grafts. DESIGN AND SETTING: Laboratory animal study. MATERIALS: 1 cm long segments of non-reinforced PTFE grafts (30 microns fibril length, 2 mm internal diameter, 0.39 mm thick) were placed as an abdominal aortic interposition in Lewis rats. Fifteen grafts served as control (Group A; porous grafts); in eight rats (Group B; non porous grafts) the PTFE graft was completely wrapped by a non-porous plastic envelope. Animals were killed 4 weeks after surgery. OUTCOME MEASURES: The release of PDGF AA, PDGF BB and bFGF was assessed by Enzyme Linked Immunosorbent Assay (ELISA). RESULTS: The release of PDGF AA, PDGF BB and bFGF was statistically higher in porous grafts. The only histological difference between the two groups was that porous PTFE grafts were invaded by many tufts of capillaries from the surrounding tissue, whereas this phenomenon was absent in non porous PTFE grafts. CONCLUSIONS: The degree of porosity influences the release of growth factors by healing PTFE grafts. This fact may have implication in the endothelisation of PTFE grafts and in myointimal hyperplasia formation as well.