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Supernatant Fluid (supernatant + fluid)
Selected AbstractsDifferential effects of arachidonoyl trifluoromethyl ketone on arachidonic acid release and lipid mediator biosynthesis by human neutrophilsFEBS JOURNAL, Issue 15 2002Evidence for different arachidonate pools The goal of this study was to determine the effects of a putative specific cytosolic phospholipase A2 inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3), on arachidonic acid (AA) release and lipid mediator biosynthesis by ionophore-stimulated human neutrophils. Initial studies indicated that AACOCF3 at concentrations 0,10 µm did not affect AA release from neutrophils. In contrast, AACOCF3 potently inhibited leukotriene B4 formation by ionophore-stimulated neutrophils (IC50 , 2.5 µm). Likewise, AACOCF3 significantly inhibited the biosynthesis of platelet activating factor. In cell-free assay systems, 10 µm AACOCF3 inhibited 5-lipoxygenase and CoA-independent transacylase activities. [3H]AA labeling studies indicated thatthe specific activities of cell-associated AA mimicked that of leukotriene B4 and PtdCho/PtdIns, while the specific activities of AA released into the supernatant fluid closely mimicked that of PtdEtn. Taken together, these data argue for the existence of segregated pools of arachidonate in human neutrophils. One pool of AA is linked to lipid mediator biosynthesis while another pool provides free AA that is released from cells. Additionally, the data suggest that AACOCF3 is also an inhibitor of CoA-independent transacylase and 5-lipoxygenase. Thus, caution should be exercised in using AACOCF3 as an inhibitor of cytosolic phospholipase A2 in whole cell assays because of the complexity of AA metabolism. [source] Annulus cells release ATP in response to vibratory loading in vitroJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2003Satoru Yamazaki Abstract Mechanical forces regulate the developmental path and phenotype of a variety of tissues and cultured cells. Vibratory loading as a mechanical stimulus occurs in connective tissues due to energy returned from ground reaction forces, as well as a mechanical input from use of motorized tools and vehicles. Structures in the spine may be particularly at risk when exposed to destructive vibratory stimuli. Cells from many tissues respond to mechanical stimuli, such as fluid flow, by increasing intracellular calcium concentration ([Ca2+]ic) and releasing adenosine 5,-triphosphate (ATP), extracellularly, as a mediator to activate signaling pathways. Therefore, we examined whether ATP is released from rabbit (rAN) and human (hAN) intervertebral disc annulus cells in response to vibratory loading. ATP release from annulus cells by vibratory stimulation as well as in control cells was quantitated using a firefly luciferin-luciferase assay. Cultured hAN and rAN cells had a basal level of extracellular ATP ([ATP]ec) in the range of 1,1.5 nM. Vibratory loading of hAN cells stimulated ATP release, reaching a net maximum [ATP] within 10 min of continuous vibration, and shortly thereafter, [ATP] declined and returned to below baseline level. [ATP] in the supernatant fluid of hAN cells was significantly reduced compared to the control level when the cells received vibration for longer than 15 min. In rAN cells, [ATP] was increased in response to vibratory loading, attaining a level significantly greater than that of the control after 30 min of continuous vibration. Results of the current study show that resting annulus cells secrete ATP and maintain a basal [ATP]ec. Annulus cells may use this nucleotide as a signaling messenger in an autocrine/paracrine fashion in response to vibratory loading. Rapid degradation of ATP to ADP may alternatively modulate cellular responses. It is hypothesized that exposure to repetitive, complex vibration regimens may activate signaling pathways that regulate matrix destruction in the disc. As in tendon cells, ATP may block subsequent responses to load and modulate the vibration response. Rabbit annulus cells were used as a readily obtainable source of cells in development of an animal model for testing effects of vibration on the disc. Human cells obtained from discarded surgical specimens were used to correlate responses of animal to human cells. © 2003 Wiley-Liss, Inc. [source] TT virus (TTV) loads associated with different peripheral blood cell types and evidence for TTV replication in activated mononuclear cellsJOURNAL OF MEDICAL VIROLOGY, Issue 2 2001Fabrizio Maggi Abstract TT virus (TTV) loads associated with the peripheral blood cells of seven patients known to carry the virus in plasma were investigated by real-time PCR. Whereas red cells/platelets were uniformly negative, six and four patients yielded positive peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes, respectively, but viral titres were generally low. Fractionation of PBMCs into monocyte- and B, T4, and T8 lymphocyte-enriched subpopulations showed no pattern in the viral loads that might suggest the preferential association of TTV to one or more specific cell types. TTV-negative PBMCs absorbed measurable amounts of virus when incubated with infected plasma at 4°C. Furthermore, cultures of TTV-negative phytohaemagglutinin-stimulated PBMCs exposed in vitro to virus-positive plasma and faecal extracts released considerable levels of infectious TTV into the supernatant fluid and the same was true for TTV-positive stimulated PBMCs. These results indicate that, whereas freshly harvested resting PBMCs seem to produce little, if any TTV, stimulated PBMCs actively replicate the virus. J. Med. Virol. 64:190,194, 2001. © 2001 Wiley-Liss, Inc. [source] Coagulations in a water based magnetic fluid under magnetic fieldPHYSICA STATUS SOLIDI (C) - CURRENT TOPICS IN SOLID STATE PHYSICS, Issue 12 2004Y. Matsuno Abstract The paper deals with the coagulations of magnetic solid particles in a water based magnetic fluid under magnetic field. The coagulations can be detected by measuring the magnetization of supernatant fluid of magnetic fluid in test tubes under magnetic field. As the coagulations increase , the magnetization of supernatant fluid decreases due to the sedimentation of coagulations. So the more the coagulations, the lower the magnetization of supernatant fluid. Moreover, when test tubes are centrifuged before magnetization measurement, the sedimentation of coagulations is accelerated and the magnetization becomes lower and the detection of coagulations becomes easier. The test results show that latent coagulations can be detected by means of centrifuging, which can not be found out without it. (© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Anti-influenza virus activity of crude extract of Ribes nigrum L.PHYTOTHERAPY RESEARCH, Issue 2 2003Yoko M. Knox Abstract This experiment was designed to detect the antiviral activities of crude fruit extracts of wild Ribes nigrum L. (Kurokarin extract) against influenza virus types A and B. Kurokarin extract was prepared as follows: fruits of Ribes nigrum L. were heated at 50,°C in a heating tank, and then ground under anaerobic conditions. The extracts were centrifuged, and the supernatant fluid was filtered and sterilized by infrared rays. The crude extract was diluted with Eagle's minimum essential medium (MEM) and the solution was adjusted to a pH 7.2 with 0.1 N or 1 N NaOH. Proven anti-influenza virus effects of the extracts were shown. The concentration of extract required to inhibit the plaque formation of both IVA and IVB by 50% (IC50) was 3.2,,g/mL. Both IVA and IVB were directly inactivated up to 99% by 10,,g/mL of the extract at pH 2.8, and 95% to 98% by this dose at pH 7.2. The growth of IVA in cells treated with 10 and 100,,g/mL of the extract for 6,h after infection was completely suppressed. Virus titres in culture fluids of the cells treated with 100,,g/mL of Kurokarin extract for 1,h at 8 to 9,h after infection, were completely suppressed, indicating that the extract inhibited the virus release from the infected cells. Copyright © 2003 John Wiley & Sons, Ltd. [source] Mechanical loading stimulates ecto-ATPase activity in human tendon cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2005M. Tsuzaki Abstract Response to external stimuli such as mechanical signals is critical for normal function of cells, especially when subjected to repetitive motion. Tenocytes receive mechanical stimuli from the load-bearing matrix as tension, compression, and shear stress during tendon gliding. Overloading a tendon by high strain, shear, or repetitive motion can cause matrix damage. Injury may induce cytokine expression, matrix metalloproteinase (MMP) expression and activation resulting in loss of biomechanical properties. These changes may result in tendinosis or tendinopathy. Alternatively, an immediate effector molecule may exist that acts in a signal-dampening pathway. Adenosine 5,-triphosphate (ATP) is a candidate signal blocker of mechanical stimuli. ATP suppresses load-inducible inflammatory genes in human tendon cells in vitro. ATP and other extracellular nucleotide signaling are regulated efficiently by two distinct mechanisms: purinoceptors via specific receptor,ligand binding and ecto-nucleotidases via the hydrolysis of specific nucleotide substrates. ATP is released from tendon cells by mechanical loading or by uridine 5,-triphosphate (UTP) stimulation. We hypothesized that mechanical loading might stimulate ecto-ATPase activity. Human tendon cells of surface epitenon (TSC) and internal compartment (TIF) were cyclically stretched (1 Hz, 0.035 strain, 2 h) with or without ATP. Aliquots of the supernatant fluids were collected at various time points, and ATP concentration (ATP) was determined by a luciferin-luciferase bioluminescence assay. Total RNA was isolated from TSC and TIF (three patients) and mRNA expression for ecto-nucleotidase was analyzed by RT-PCR. Human tendon cells secreted ATP in vitro (0.5,1 nM). Exogenous ATP was hydrolyzed within minutes. Mechanical load stimulated ATPase activity. ATP was hydrolyzed in mechanically loaded cultures at a significantly greater rate compared to no load controls. Tenocytes (TSC and TIF) expressed ecto-nucleotidase mRNA (ENTPD3 and ENPP1, ENPP2). These data suggest that motion may release ATP from tendon cells in vivo, where ecto-ATPase may also be activated to hydrolyze ATP quickly. Ecto-ATPase may act as a co-modulator in ATP load-signal modulation by regulating the half-life of extracellular purine nucleotides. The extracellular ATP/ATPase system may be important for tendon homeostasis by protecting tendon cells from responding to excessive load signals and activating injurious pathways. © 2005 Wiley-Liss, Inc. [source] Evaluation of a reversed passive latex agglutination test for the detection of Verocytotoxin (VT) expressed by strains of VT-producing Escherichia coliLETTERS IN APPLIED MICROBIOLOGY, Issue 6 2001H. Chart Aims: To compare an experimental Reversed-Passive Latex Agglutination (RPLA) with Vero cells for the detection of Verocytotoxin expressed by VT-producing strains of Escherichia coli (VTEC). Methods and Results: The RPLA was used alongside a Vero cell tissue culture assay for the detection of VT in bacterial culture supernatant fluids and patients' faecal extracts. Conclusions: The RPLA was comparable with the Vero cell assay, although slightly less sensitive. Significance and Impact of the Study: The RPLA test proved to be a simple, rapid and convenient method of detecting VT in bacterial culture supernatant fluids and in the faeces of patients infected with VTEC. [source] | ||||||||||