Human fetal retinal pigment epithelium-induced cell cycle arrest, loss of mitochondrial membrane potential and apoptosis.
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PURPOSE: To investigate the mechanism of action of the soluble immune suppressive product secreted by human fetal retinal pigment epithelial (HFRPE) cells in a model system using the human T-cell line Jurkat (Jkt). METHODS: Pure HFRPE cells were isolated and cultured. The supernatants of both nonactivated and IFN-gamma-activated HFRPE cells were isolated. Cells from the human T-cell line Jkt were incubated either in standard culture medium or in the supernatant isolated from HFRPE cells. In the first assay Jkt cell proliferation was measured by [(3)H]thymidine incorporation. In the second assay Jkt cell apoptosis was examined for annexin V staining by flow cytometry. In the third assay Jkt cell division was evaluated with carboxyfluorescein succinimidyl ester (CFSE) fluorescent dye. In the last assay the mitochondrial transmembrane potential of Jkt cells was measured with the cationic lipophilic fluorochrome 3,3'-dihexyloxacarbocyanine iodide [DiOC(6)]. In all the assays the effect of supernatants isolated from both nonactivated and IFN-gamma-activated HFRPE cells were compared with standard culture medium. The involvement of antiapoptotic human gene bcl-x(L:)was determined by using a Jkt cell line that was stably transfected with bcl-x(L:). RESULTS: The supernatant isolated from HFRPE cells significantly suppressed the cell division in Jkt cells and induced apoptosis. These effects were stronger when the supernatant was isolated from IFN-gamma-activated HFRPE cells. The apoptosis pathway induced by the secreted product of HFRPE cells involved the early disruption of mitochondrial transmembrane potential. Although the overexpression of bcl-x(L) gene rescued the Jkt cells from supernatant-induced apoptosis, it could not restore the proliferation of Jkt cells. CONCLUSIONS: These data suggest that HFRPE cells secrete a product that initiates an early cell cycle arrest in the human T-cell line Jkt, which is followed by the activation of an apoptotic pathway that involves the loss of mitochondrial membrane potential. The latter could be prevented by bcl-x(L) overexpression. Also these data suggest that the HFRPE-induced T-cell apoptosis may play a significant role in maintaining the immune privilege in the subretinal space. (+info)
Production of betacyanins by a cell suspension culture of table beet (Beta vulgaris L.).
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A cell suspension culture of table beet (Beta vulgaris L.) was established for efficient betacyanin production from violet callus induced from the hypocotyls of aseptic seedlings. This suspension culture produced large amounts of betacyanins. The betacyanin content increased with increasing cell growth during the log phase. Reducing the total nitrogen concentration (30 mM) and modifying the ratio of ammonium to nitrate (1:14) resulted in an increased betacyanin content. Supplementation of Fe2+ to the LS medium also promoted betacyanin production. The maximal betacyanin yield was achieved with a 2 mM Fe2+ concentration. Combining these conditions, we established a revised LS medium to improve betacyanin productivity (250 mg/l for a 14-day culture). (+info)
Quantitative imaging of lateral ErbB1 receptor signal propagation in the plasma membrane.
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Evidence for a new signaling mechanism consisting of ligand-independent lateral propagation of receptor activation in the plasma membrane is presented. We visualized the phosphorylation of green fluorescent protein (GFP)-tagged ErbB1 (ErbB1-GFP) receptors in cells focally stimulated with epidermal growth factor (EGF) covalently attached to beads. This was achieved by quantitative imaging of protein reaction states in cells by fluorescence resonance energy transfer (FRET) with global analysis of fluorescence lifetime imaging microscopy (FLIM) data. The rapid and extensive propagation of receptor phosphorylation over the entire cell after focal stimulation demonstrates a signaling wave at the plasma membrane resulting in full activation of all receptors. (+info)
Simple two-color array-based approach for mutation detection.
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The ability to analyze multiple polymorphic/mutation sites rapidly and accurately is pivotal in all areas of genetic analysis. We have applied single nucleotide primer extension (SNE) for detection of multiple point mutations in a micro-array format using two-color, fluorescent dye-tagged dideoxynucleoside triphosphate terminators (ddNTPs). The oligonucleotide primer ending one nucleotide short of the mutation site being probed is bound to the slide and single-base extended in place with two different Cy5/Cy3 dye-tagged terminators using solution-phase, locus-specific, single-stranded complementary templates generated by PCR from genomic DNA. The composite fluorescence produced contains peaks of distinct wave lengths corresponding to each Cy dye-tagged terminator incorporated, resulting in a fluorescent 'fingerprint' for each DNA target. DNA polymerase-catalyzed incorporation of Cy dye-tagged dideoxynucleoside triphosphates was dependent on the particular dyes, the specific ddNTP, the DNA target concentration, sequence of the template, on-slide temperature cycling and washing conditions. Results from analysis of mutations in the human hemochromatosis and connexin 26 genes show that this approach has several advantages over existing methods and is simple, rapid, robust, cost effective and accurate with potential applications in many areas of genetic analysis. (+info)
Nucleotide effects on liver and muscle mitochondrial non-phosphorylating respiration and membrane potential.
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Uncoupling protein-1 homologs are hypothesized to mediate mitochondrial proton leak. To test this hypothesis, we determined the effects of ATP and other nucleotides on liver and skeletal muscle mitochondrial non-phosphorylating respiration (VO(2)), membrane potential, FCCP-stimulated respiratory control ratios, and swelling. Neither ATP nor CTP affected liver or muscle proton leak, but both inhibited the respiratory chain. Unexpectedly, CMP stimulated liver proton leak (EC(50) approximately 4.4+/-0.5 mM). Using CMP chromatography, we identified two proteins (M(r)=31.2 and 32.6 kDa) from liver mitochondria that are similar in size to members of the mitochondrial carrier protein family. We conclude (a) liver and muscle mitochondrial proton leak is insensitive to ATP and CTP, and (b) CMP activates a leak in liver mitochondria. The CMP-inducible leak may be mediated by a 30-32 kDa protein. Based on the high concentrations required, CMP is unlikely to be a physiologically important leak regulator. Nonetheless, our results show that tissues other than brown fat have inducible leaks that may be protein-mediated. (+info)
Diffusion barriers in ram and boar sperm plasma membranes: directionality of lipid diffusion across the posterior ring.
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The plasma membrane of mammalian spermatozoa, like that of other differentiated cells, is compartmentalized into discrete regions or domains that are biochemically and functionally distinct from one another. Physical structures within the membrane, such as the posterior ring at the juncture of the sperm head and tail, have long been thought to act as diffusion barriers to help segregate important molecules required for fertilization within specific domains and to regulate migration of molecules between domains. In this investigation, we used a quantitative photobleaching technique (video-FRAP) to assess the efficacy of the posterior ring as a barrier to exchange of lipids between the postacrosomal and midpiece plasma membranes. A lipid reporter probe (1,1'-diduodecyl-3,3,3', 3'-tetramethylindocarbocyanine; DiIC(12)) was incorporated into the plasma membrane of live ram and boar spermatozoa, and the directionality of its diffusion across the posterior ring was measured by line-profile analysis. Results showed that DiIC(12) was able to traverse the posterior ring from the direction of the postacrosomal plasma membrane and to diffuse onto the midpiece plasma membrane. These results suggest that the posterior ring is not an immutable barrier to lipid exchange in mature spermatozoa and that there are other mechanisms for maintaining in-plane lipid asymmetry, such as differential phase behavior and interaction with the submembranous cytoskeleton. (+info)
The sub-lip domain--a distinct pathway for myotome precursors that demonstrate rostral-caudal migration.
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We have previously reported that the myotome is formed by a first wave of pioneer cells generated from all along the dorsomedial portion of the epithelial somite and a second wave of cells issued from all four edges of the dermomyotome. Cells from the extreme rostral and caudal edges directly generate myofibers that elongate towards the opposite pole of each segment and along the pre-existing myotomal scaffold. In contrast, cells from the dorsomedial and ventrolateral lips first reach the extreme edges and then contribute to myofiber formation. The mechanism by which these epithelial cells translocate remained unknown and was the goal of the present study. We have found that epithelial cells along the dorsomedial and ventrolateral lips of the dermomyotome first delaminate into the immediate underlayer of the corresponding lips, the sub-lip domain, then migrate longitudinally along this pathway until reaching the extreme edges from which they differentiate into myofibers. Cells of the sub-lip domain are negative for Pax3 and desmin but express MyoD, Myf5 and FREK, suggesting that they are specific myogenic progenitors. (+info)
JC-1: a very sensitive fluorescent probe to test Pgp activity in adult acute myeloid leukemia.
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One of the best-characterized resistance mechanisms in acute myeloid leukemia (AML) is the drug extrusion mediated by P-glycoprotein (Pgp). Recently the results of workshops organized by several groups concluded that accurate measurement of low activity of Pgp is a difficult goal in clinical samples. Therefore, highly sensitive and specific assays were developed to assess the functionality of Pgp using JC-1, a fluorescent molecule with the different emission wavelength (green and red fluorescence) according to its concentration in 129 AML samples. It was shown that JC-1 (green and red bands) may define 3 groups of patients: resistant (R) (29% of patients), intermediate (I) (36%), and sensitive (S) (35%). In contrast, rhodamine 123 assay detected only the R group defined by JC-1. Nevertheless, the I group has an intermediate expression of Pgp (0.39, 0.29, and 0.19 for the R, I, and S groups, respectively, P =.002), an intermediate biologic profile (percentage of CD34, 95%, 67%, and 44%, respectively, P <.0001; in vitro resistance to daunorubicin, 94 microM, 20 microM, and 12 microM, respectively, P =. 02), and an intermediate prognosis (achievement of complete remission, 55%, 65%, and 87%, P =.006; 3-year disease-free survival, 11%, 16%, and 36%, respectively, P =.005; and 3-year overall survival, 0%, 20%, and 51%, respectively, P <.0001). Therefore, JC-1 appeared to be a more convenient and simple way to detect a functional Pgp in clinical AML samples than rhodamine 123. (Blood. 2001;97:502-508) (+info)