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The effect of the natural product diindolylmethane (DIM) on cytosolic Ca2+ concentrations ([Ca2+]i) and viability in MDCK renal tubular cells was explored. The Ca2+-sensitive fluorescent dye fura-2 was applied to measure [Ca2+]i. DIM at concentrations 1–50 μM induced a [Ca2+]i rise in a concentration-dependent manner. The response was reduced partly by removing Ca2+. DIM induced Mn2+ influx leading to quenching of fura-2 fluorescence. DIM-evoked Ca2+ entry was suppressed by nifedipine, econazole, SK&F96365 and protein kinase C modulators. In the absence of extracellular Ca2+, incubation with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) greatly inhibited DIM-induced [Ca2+]i rise. Incubation with DIM abolished TG or BHQ-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 reduced DIM-induced [Ca2+]i rise by 50%. At 1, 10, 40 and 50 μM, DIM slightly enhanced cell proliferation. The effect of 50 μM DIM was reversed by chelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. In sum, in MDCK cells, DIM induced a [Ca2+]i rise by evoking phospholipase C-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via protein kinase C-sensitive store-operated Ca2+ channels. DIM did not induce cell death. [ABSTRACT FROM PUBLISHER]

This paper proposes a non-orthogonal view iris recognition system comprising a new iris imaging module, an iris segmentation module, an iris feature extraction module and a classification module. A dual-charge-coupled device camera was developed to capture four-spectral (red, green, blue, and nearinfrared) iris images which contain useful information for simplifying the iris segmentation task. An intelligent random sample consensus iris segmentation method is proposed to robustly detect iris boundaries in a four-spectral iris image. In order to match iris images acquired at different off-axis angles, we propose a circle rectification method to reduce the off-axis iris distortion. The rectification parameters are estimated using the detected elliptical pupillary boundary. Furthermore, we propose a novel iris descriptor which characterizes an iris pattern with multiscale step/ridge edge-type maps. The edge-type maps are extracted with the derivative of Gaussian and the Laplacian of Gaussian filters. The iris pattern classification is accomplished by edge-type matching which can be understood intuitively with the concept of classifier ensembles. Experimental results show that the equal error rate of our approach is only 0.04% when recognizing iris images acquired at different off-axis angles within ±30°. [ABSTRACT FROM AUTHOR]

Riluzole is a drug used in the treatment of amyotrophic lateral sclerosis; however, its in vitro action is unclear. In this study, the effect of riluzole on intracellular Ca2+ concentration ([Ca2+]i) in Madin-Darby canine kidney (MDCK) cells was investigated using the Ca2+-sensitive fluorescent dye, fura-2. Riluzole (100–500 μM) caused a rapid and sustained increase of [Ca2+]i in a concentration-dependent manner (EC50 = 150 μM). Some 40 and 50% of this [Ca2+]i increase was prevented by the removal of extracellular Ca2+ and the addition of La3+, respectively, but was unchanged by dihydropyridines, verapamil and diltiazem. In Ca2+-free medium, thapsigargin – an inhibitor of the endoplasmic reticulum (ER) Ca2+-ATPase – caused a monophasic [Ca2+]i increase, after which the increasing effect of riluzole on [Ca2+]i was attenuated by 70%; in addition, pre-treatment with riluzole abolished thapsigargin-induced [Ca2+]i increases. U73122, an inhibitor of phospholipase C (PLC), abolished ATP (but not riluzole)-induced [Ca2+]i increases. At concentrations of 250 and 500 μM, riluzole killed 40 and 95% cells, respectively. The cytotoxic effect of riluzole (250 μM) was unaltered by pre-chelating cytosolic Ca2+ with BAPTA. Collectively, in MDCK cells, riluzole rapidly increased [Ca2+]i by stimulating extracellular Ca2+ influx via an La3+-sensitive pathway and intracellular Ca2+ release from the ER via, as yet, unidentified mechanisms. Furthermore, riluzole caused Ca2+-unrelated cytotoxicity in a concentration-dependent manner. [ABSTRACT FROM AUTHOR]

The effect of the carcinogen safrole on intracellular Ca 2+ mobilization and on viability of human PC3 prostate cancer cells was examined. Cytosolic free Ca 2+ levels ([Ca 2+ ] i ) were measured by using fura-2 as a probe. Safrole at concentrations above 10 μM increased [Ca 2+ ] i in a concentration-dependent manner with an EC 50 value of 350 μ M. The Ca 2+ signal was reduced by more than half after removing extracellular Ca 2+ but was unaffected by nifedipine, nicardipine, nimodipine, diltiazem, or verapamil. In Ca 2+ -free medium, after treatment with 650 μM safrole, 1 μM thapsigargin (an endoplasmic reticulum Ca 2+ pump inhibitor) failed to release Ca 2+ . Neither inhibition of phospholipase C with U73122 nor modulation of protein kinase C activity affected safrole-induced Ca 2+ release. Overnight incubation with 0.65–65 μM safrole did not affect cell viability, but incubation with 325–625 μM safrole decreased viability. Collectively, the data suggest that in PC3 cells, safrole induced a [Ca 2+ ] i increase by causing Ca 2+ release from the endoplasmic reticulum in a phospholipase C- and protein kinase C-independent fashion, and by inducing Ca 2+ influx. Safrole can decrease cell viability in a concentration-dependent manner. [ABSTRACT FROM AUTHOR]

Study design:In order to study the role of gender in recovery, we induced a thoracic compression spinal cord injury (SCI) separately in 2-month-old male and female C57Bl/6 mice.Objectives:We intended to assess effects of gender on recovery of hindlimb motor function and to correlate these with histomorphologic profiles of injured spinal cord tissue.Methods:Locomotor function was evaluated by three means: a modified locomotor scoring system for rodents, beam walking and computerized activity meter. Histology was analyzed by comparison of hematoxylin and eosin-stained perfused specimens.Results:Locomotor scores were 2.2±0.9 on day 1 in male mice, while, in contrast, they were significantly higher, 7.3±1.7, in females (P<0.02). On day 14 Basso, Beattie and Bresnahan scores were 9.5±2.2 in male mice and 16.0±2.2 in females (P<0.03). Terminal histology showed that the spinal cord architecture was relatively better preserved in female mice and that the extent of necrosis and infiltration of inflammatory cells was less compared to males.Setting:Neurobiology Research Laboratory of University of Kansas Medical School in US Department of Veterans Affairs Medical Center, Kansas City, Missouri.Conclusion:We found that the severity of the initial injury as well as the ultimate recovery of motor function after SCI is significantly influenced by gender, being remarkably better in females. The mechanism(s) of neuroprotection in females, although not yet elucidated, may be associated with the effects of estrogen on pathophysiological processes (blood flow, leukocyte migration inhibition, antioxidant properties, and inhibition of apoptosis).Sponsorship:Medical Research, US Department of Veterans Affairs, the Christopher Reeve Paralysis Foundation and NIH.Spinal Cord (2006) 44, 182–187. doi:10.1038/sj.sc.3101816; published online 30 August 2005 [ABSTRACT FROM AUTHOR]

Econazole is an antifungal drug with different in vitro effects. However, econazole's effect on osteoblast-like cells is unknown. In human MG63 osteosarcoma cells, the effect of econazole on intracellular Ca2+ concentrations ([Ca2+]i) was explored by using fura-2. At a concentration of 0.1 μM, econazole started to cause a rise in [Ca2+]i in a concentration-dependent manner. Econazole-induced [Ca2+]i rise was reduced by 74% by removal of extracellular Ca2+. The econazole-induced Ca2+ influx was mediated via a nimodipine-sensitive pathway. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a [Ca2+]i rise, after which the increasing effect of econazole on [Ca2+]i was abolished. Pretreatment of cells with econazole to deplete Ca2+ stores totally prevented thapsigargin from releasing Ca2+. U73122, an inhibitor of phospholipase C, abolished histamine (an inositol 1,4,5-trisphosphate-dependent Ca2+ mobilizer)-induced, but not econazole-induced, [Ca2+]i rise. Econazole inhibited 76% of thapsigargin-induced store-operated Ca2+ entry. These findings suggest that in MG63 osteosarcoma cells, econazole increases [Ca2+]i by stimulating Ca2+ influx and Ca2+ release from the endoplasmic reticulum via a phospholipase C-independent manner. In contrast, econazole acts as a potent blocker of store-operated Ca2+ entry. [ABSTRACT FROM AUTHOR]

The effect of celecoxib on renal tubular cells is largely unexplored. In Madin Darby canine kidney (MDCK) cells, the effect of celecoxib on intracellular Ca 2 + concentration ([Ca 2 + ] i ) and proliferation was examined by using the Ca 2 + -sensitive fluorescent dye fura-2 and the viability detecting fluorescent dye tetrazolium, respectively. Celecoxib (≥1 μ M) caused an increase of [Ca 2 + ] i in a concentration-dependent manner. Celecoxib-induced [Ca 2 + ] i increase was partly reduced by removal of extracellular Ca 2 + . Celecoxib-induced Ca 2 + influx was independently suggested by Mn 2 + influx-induced fura-2 fluorescence quench. In Ca 2 + -free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca 2 + -ATPase, caused a monophasic [Ca 2 + ] i increase, after which celecoxib only induced a tiny [Ca 2 + ] i increase; conversely, pretreatment with celecoxib completely inhibited thapsigargin-induced [Ca 2 + ] i increases. U73122, an inhibitor of phospholipase C, abolished ATP (but not celecoxib)-induced [Ca 2 + ] i increases. Overnight incubation with 1 or 10 μ M celecoxib decreased cell viability by 80% and 100%, respectively. These data indicate that celecoxib evokes a [Ca 2 + ] i increase in renal tubular cells by stimulating both extracellular Ca 2 + influx and intracellular Ca 2 + release and is highly toxic to renal tubular cells in vitro. [ABSTRACT FROM AUTHOR]

Candida albicans has been implicated in human allergic disorders. However, many of its immunoglobulin E (IgE)-reacting components have not yet been identified. The purpose of the present study is to characterize a novel 29 kDa IgE-binding protein from C. albicans. The 29 kDa protein was partially purified and its tryptic digests subjected to mass spectrometric analysis. The cDNA encoding this protein was isolated and heterologously expressed in Escherichia coli. Monoclonal antibodies (MoAbs) were raised against the 29 kDa protein purified from C. abicans extracts. We isolated a 29 kDa IgE-reacting component from C. albicans. The protein was digested on-gel with trypsin and the masses of the resulting fragments were determined in a MALDI-TOF mass spectrometer. The data were searched against protein sequences deduced from the C. albicans genome. An open reading frame that possibly encodes the 29 kDa IgE-reacting component was identified. The cDNA corresponding to the open reading frame was isolated. It encodes a 236 residues protein that has 62% sequence identity to that of a hypothetical protein (YDR533c) from Saccharomyces cerevisiae. Conserved domain search suggests that the encoded protein belongs to the ThiJ/PfpI family. The cDNA isolated was inserted into a pQE-30 vector for protein expression in Escherichia coli. The recombinant protein can react with IgE antibodies in sera from asthmatic patients and two MoAbs that were generated against the purified native 29 kDa protein from C. albicans. We identified and cloned a novel 29 kDa IgE-reacting component (Cand a 3) from C. albicans. The recombinant proteins produced from this clone and the MoAbs prepared may be useful in the standardization of diagnostic extracts. They are also instrumental in elucidating the role of C. albicans in clinical allergy. [ABSTRACT FROM AUTHOR]

Objective: To test the effect of anticardiolipin antibodies (aCL) on cultured glomerular mesangial cells with regard to their expression of apoptosis-related genes.Methods: aCL purified from active lupus sera by cardiolipin micelles were incubated with cultured rodent mesangial cells (RMC). Morphological changes of the RMC were observed. The genomic DNA was extracted for the detection of apoptosis. The total cell RNA was extracted for detection of Fas, c-myc, p53, and bcl-2 transcripts by reverse transcription-polymerase chain reaction.Results: aCL (100 GPL-U/0.1 mg protein/ml) bound to RMC more prominent than human IgG (100 microg/ml). The antibodies suppressed RMC proliferation in a dose-dependent manner. The RMC were undergoing apoptosis as evidenced by morphologic changes, fluoresceinannexin V staining and appearance of nucleosome-sized DNA fragments. RMC spontaneously express p53 and c-myc but not Fas or bcl-2. aCL (100 GPL-U/ml) enhanced the expression of Fas but not other apoptosis-related genes and suppressed the intracellular tyrosine phosphorylation.Conclusions: Binding of aCL can induce apoptosis of the RMC. The aCL may be implicated in the pathogenesis of lupus nephritis. [ABSTRACT FROM AUTHOR]

Rabbit lymphoid cells transferred to newborn recipients synthesized donor-type immunoglobulin. Elimination of donor T cells did not affect this synthesis of donor immunoglobulin, while elimination of 8 cells abolished or significantly decreased the synthesis. The synthetic capacity of B cells from thymus was thirty-four times greater than the synthetic capacities of B cells from spleen, fifty-five times greater than that of mesenteric lymph node and 180 times greater than that of appendix cells. Synthetic activity of spleen cells ceased before the tenth day after transfer, while thymus cells might continue to synthesize immunoglobulin for a longer time. This was shown by comparing half-lives of donor immunoglobulin in the recipients" sofa, Increasing the number of injected spleen cells (from 2 × 106 to 120 × 106), resulted in a corresponding increase in donor IS synthesis. With thymus cells, donor immunoglobulin increased with cell numbers up to 2 × 107 cells, above this dose there was no further donor immunoglobulin increase in the recipients' serum. [ABSTRACT FROM AUTHOR]

Removal of adherent cells or complement-mediated killing of rabbit thymus lymphocyte antigen (RTLA) bearing rabbit T lymphocytes did not abolish the responsiveness (increased thymidine incorporation) of lymphoid cells to antibody against immunoglobulin allotype, Nocardia water-soluble mitogen (NWSM), pneumococcal polysaccharide SIII (PPSIII), S. abortus lipopolysaccharide (LPS), lipid A conjugated to bovine serum albumin and a crude preparation containing C polysaccharide from the cell wall of Diplococcus pneumoniae. Isologous and heterologous antisera, directed against different portions of the Ig receptor, differed in their capacity to enhance thymidine incorporation. The difference in mitogenicity of these antisera was discussed in terms of the accessibility of cell-bound immunoglobulin receptor sites. Spleen cells, responsive to anti-allotype (Ab4) antiserum and B-cell mitogens, NWSM and PPSIII, were characterized by velocity sedimentation. The mean volume of NWSM- and PPSIII-responsive cells was larger than that of the cells responsive to anti-allotype antiserum. Fractionation of spleen cells on glass bead columns yielded a population of non-adherent cells which were two to six times as responsive to anti-Ab4 antiserum as the original spleen cell suspension. The responsiveness of peripheral blood lymphocytes to anti-Ab4 antiserum was significantly greater than that of spleen cells. On the other hand, spleen cells were more responsive to PPSIII than were cells from the peripheral blood, the popliteal and the mesenteric lymph nodes. [ABSTRACT FROM AUTHOR]

Thymus-derived cell populations were characterized by sedimentation velocity in the Earth's gravitational field, by adherence properties and in terms of the thymus-specific antigen RTLA. T cells, responding to mitogens, could be shown to be a subpopulation of RTLA-bearing cells and to have a relatively large volume. The size distribution of mitogen-responsive cells from different individuals showed some variation, but in general large spleen cells took up less thymidine per 106 cells than did large cells from other lymphoid organs. Upon stimulation with concanavalin A (con A)small popliteal lymph node cells took up more thymidine per 106 cells than did small thymus cells. The corresponding small cells of mesenteric lymph node and spleen took up intermediate quantities of thymidine. Thymusderived cells that were resistant to complementmediated cell kill with RTLA antiserum showed greater responsiveness to con A than did the original cell preparations. Non-adherent cells incorporated more thymidine when stimulated with phytohaemagglutinin (PHA) than when they were stimulated with con A. Lightly adherent cells were relatively more responsive to con A stimulation and firmly adherent cells were less responsive to both. The findings were discussed in terms of the density of RTLA on the membranes of T cells, in adherence and in sedimentation velocity fractionation, the relative yield of nucleated cells always exceeded the yield of PHAand con A-responsive cells. The differential separation of mitogen responsive and of co-operating cells were considered as a possible cause of this deficit. [ABSTRACT FROM AUTHOR]

Using resetting methods. We have purified rabbit B cells and studied their interactions with T cells purified by passage over an anti-immunoglobulin-coated Degalan beads column. B cells enhance the response of T cells to concanavalin A (Con A) and phytohaemagglutinn. In regulation of the response to Con A, an adhernt cell is a third participating cell. B-cell preparations contain a minority of cells that can respond to T mitogens with the help of non-proliferating T cells, but the proportion of these responding cells is small, and the involvement of a T-cell impurity cannot be excluded. [ABSTRACT FROM AUTHOR]

The proliferative response, induced in rabbit spleen cells by concanavalin A (Con A) and phytohaemagglutinin (PHA), is abolished when T cells are killed with antibody against rabbit thymus lymphocyte antigen (RTLA) in the presence of complement. The response was examined with purified spleen T cells, to which various helper cell factions were added: it could be shown that B cells help the responding T cells. The helper effect in the response to PH A is abolished and the response to Con A is reduced by any manoeuvre which destroys or removes B cells. Help by B cells is given when helper cells have lost proliferative capacity as a consequence of mitomycin-C treatment. Spleen cells adhering to the walls of cullure tuhes help suspended T spleen cells in their response to Con A. This help could be abolished by complement mediated cell kill with antibody to rabbit bursal equivalent lymphocyte antigen (RABELA). On the other hand, the helper effect in Con A response was increased when T cells were removed. Thus the response of T cells to Con A is regulated by helper B cells and suppressor T cells. [ABSTRACT FROM AUTHOR]

Reagents for the identification of rabbit cell markers have been developed at a relatively slow rate. In this paper, rabbit cells are being characterized by polyclonal antibodies against a Tall antigen (RTLA), a B-cell antigen (RABELA) and an analogue of murine Ia antigen. A number of monoclonal antibodies, specific for lymphocytes and/or bone marrow and/or polymorphonuclear leucocytes, have been used for the analysis of cells with identifiable membrane antigens. Populations that have cells with two of the above antigens in the membranes were identified. To these ends, complement-mediated cell kill by antisera alone and in mixtures was employed. [ABSTRACT FROM AUTHOR]