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  • Calcium channels are involved in calcium oxalate crystal formation in specialized cells of Pistia stratiotes L. 15087302

    Pistia stratiotes produces large amounts of calcium (Ca) oxalate crystals in specialized cells called crystal idioblasts. The potential involvement of Ca(2+) channels in Ca oxalate crystal formation by crystal idioblasts was investigated.Anatomical, ultrastructural and physiological analyses were used on plants, fresh or fixed tissues, or protoplasts. Ca(2+) uptake by protoplasts was measured with (45)Ca(2+), and the effect of Ca(2+) channel blockers studied in intact plants. Labelled Ca(2+) channel blockers and a channel protein antibody were used to determine if Ca(2+) channels were associated with crystal idioblasts.(45)Ca(2+) uptake was more than two orders of magnitude greater for crystal idioblast protoplasts than mesophyll protoplasts, and idioblast number increased when medium Ca was increased. Plants grown on media containing 1-50 microM of the Ca(2+) channel blockers, isradipine, nifedipine or fluspirilene, showed almost complete inhibition of crystal formation. When fresh tissue sections were treated with the fluorescent dihydropyridine-type Ca(2+) channel blocker, DM-Bodipy-DHP, crystal idioblasts were intensely labelled compared with surrounding mesophyll, and the label appeared to be associated with the plasma membrane and the endoplasmic reticulum, which is shown to be abundant in idioblasts. An antibody to a mammalian Ca(2+) channel alpha1 subunit recognized a single band in a microsomal protein fraction but not soluble protein fraction on western blots, and it selectively and heavily labelled developing crystal idioblasts in tissue sections.The results demonstrate that Ca oxalate crystal idioblasts are enriched, relative to mesophyll cells, in dihydropyridine-type Ca(2+) channels and that the activity of these channels is important to transport and accumulation of Ca(2+) required for crystal formation.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB427
    Nombre del producto:
    Anti-Dihydropyridine-sensitive Calcium Channel α 1 Subunit Antibody, clone 1a

  • Assay of plasma oxalate with soluble oxalate oxidase. 7955374

    We use oxalate oxidase from barley seedlings for the colorimetric determination of oxalate in plasma. The oxalate is converted to hydrogen peroxide, which, in the presence of peroxidase, is detected by a Trinder-like chromogenic system. Optimization of the assay, including deproteinization and elimination of interferences from reducing substrates, is described. Ascorbate additions (200 mumol/L) did not affect oxalate concentration in plasma, even after long frozen storage. Mean analytical recovery of oxalate averaged 102% +/- 6.9%, imprecision (CV) at 2.0 mumol/L was 7.2%, and the lower limit of quantification (CV = 20%) was 0.6 mumol/L. Results correlated well with those by chromatography (r = 0.999, Sy/x = 0.29 mumol/L, n = 32, range for x, y = 0-140 mumol/L). Plasma oxalate concentrations measured in 32 healthy subjects ranged from 0.6 to 2.9 mumol/L (mean 1.28, SD 0.71 mumol/L), which agrees with those measurable by using indirect radioisotopic dilution methods. Patients with primary hyperoxaluria and chronic renal failure exhibited markedly greater plasma concentrations of oxalate.
    Tipo de documento:
    Referencia
    Referencia del producto:
    20-176
    Nombre del producto:
    100X GTPγS, 10mM

  • AC927, a σ receptor ligand, blocks methamphetamine-induced release of dopamine and generation of reactive oxygen species in NG108-15 cells. 22101517

    Methamphetamine is a highly addictive psychostimulant drug of abuse that causes neurotoxicity with high or repeated dosing. Earlier studies demonstrated the ability of the selective σ receptor ligand N-phenethylpiperidine oxalate (AC927) to attenuate the neurotoxic effects of methamphetamine in vivo. However, the precise mechanisms through which AC927 conveys its protective effects remain to be determined. With the use of differentiated NG108-15 cells as a model system, the effects of methamphetamine on neurotoxic endpoints and mediators such as apoptosis, necrosis, generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), and dopamine release were examined in the absence and presence of AC927. Methamphetamine at physiologically relevant micromolar concentrations caused apoptosis in NG108-15 cells. At higher concentrations of methamphetamine, necrotic cell death was observed. At earlier time points, methamphetamine caused ROS/RNS generation, which was detected with the fluorigenic substrate 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescin diacetate, acetyl ester, in a concentration- and time-dependent manner. N-Acetylcysteine, catalase, and l-N(G)-monomethyl arginine citrate inhibited the ROS/RNS fluorescence signal induced by methamphetamine, which suggests the formation of hydrogen peroxide and RNS. Exposure to methamphetamine also stimulated the release of dopamine from NG108-15 cells into the culture medium. AC927 attenuated methamphetamine-induced apoptosis, necrosis, ROS/RNS generation, and dopamine release in NG108-15 cells. Together, the data suggest that modulation of σ receptors can mitigate methamphetamine-induced cytotoxicity, ROS/RNS generation, and dopamine release in cultured cells.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Intracrystalline proteins and urolithiasis: a comparison of the protein content and ultrastructure of urinary calcium oxalate monohydrate and dihydrate crystals. 16104927

    To compare the ultrastructure and protein content, particularly prothrombin fragment 1 and osteopontin, of calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) crystals precipitated from human urine, and their susceptibility to proteolysis, to try to clarify the role of intracrystalline proteins in urolithiasis, as differences between these types of crystal may determine whether calcium oxalate crystals nucleated in urine progress to stone formation.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AP136P
    Nombre del producto:
    Goat Anti-Rat IgG Antibody, HRP conjugate

  • Plasma and urinary oxalate and glycolate in healthy subjects. 8419038

    High-performance ion chromatography (HPIC) has been widely used for oxalate analysis and, more recently, for glycolate analysis. We describe a procedure for sample preparation in which the plasma ultrafiltrate is acidified during harvesting with a cation-exchange resin, and the chloride is removed before the ion chromatography, which is performed with a newly developed AS10 column. The same ultrafiltrate sample is analyzed for glycolate. For plasma oxalate, the mean recovery of sample in eluted fractions was 95-96%, and intraassay CV was 6.2-8.1%. The reference interval (mean +/- 2 SD) for men was 0.8-3.2 mumol/L and for women, 1.0-2.6 mumol/L. For urinary oxalate, the reference interval for men was 175-560 mumol/day and for women, 107-432 mumol/day. For plasma glycolate, the mean analytical recovery was 96-98%, and the intra-assay CV was 2.4-6.2%. The reference interval for men was 1.9-7.5 mumol/L and for women, 1.4-7.4 mumol/L. For urinary glycolate, the reference interval for men was 0-1400 mumol/day and for women, 91-1001 mumol/day.
    Tipo de documento:
    Referencia
    Referencia del producto:
    20-176
    Nombre del producto:
    100X GTPγS, 10mM

  • Cholinergic signaling inhibits oxalate transport by human intestinal T84 cells. 21956166

    Urolithiasis remains a very common disease in Western countries. Seventy to eighty percent of kidney stones are composed of calcium oxalate, and minor changes in urinary oxalate affect stone risk. Intestinal oxalate secretion mediated by anion exchanger SLC26A6 plays a major constitutive role in limiting net absorption of ingested oxalate, thereby preventing hyperoxaluria and calcium oxalate urolithiasis. Using the relatively selective PKC-δ inhibitor rottlerin, we had previously found that PKC-δ activation inhibits Slc26a6 activity in mouse duodenal tissue. To identify a model system to study physiologic agonists upstream of PKC-δ, we characterized the human intestinal cell line T84. Knockdown studies demonstrated that endogenous SLC26A6 mediates most of the oxalate transport by T84 cells. Cholinergic stimulation with carbachol modulates intestinal ion transport through signaling pathways including PKC activation. We therefore examined whether carbachol affects oxalate transport in T84 cells. We found that carbachol significantly inhibited oxalate transport by T84 cells, an effect blocked by rottlerin. Carbachol also led to significant translocation of PKC-δ from the cytosol to the membrane of T84 cells. Using pharmacological inhibitors, we observed that carbachol inhibits oxalate transport through the M(3) muscarinic receptor and phospholipase C. Utilizing the Src inhibitor PP2 and phosphorylation studies, we found that the observed regulation downstream of PKC-δ is partially mediated by c-Src. Biotinylation studies revealed that carbachol inhibits oxalate transport by reducing SLC26A6 surface expression. We conclude that carbachol negatively regulates oxalate transport by reducing SLC26A6 surface expression in T84 cells through signaling pathways including the M(3) muscarinic receptor, phospholipase C, PKC-δ, and c-Src.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo

  • Immuno-localization of CD44 and osteopontin in developing human kidney. 18787423

    CD44 is observed in ureteric bud structures and is implicated in branching morphogenesis during early mouse renal development. Healthy adult kidney demonstrates minimal CD44, but CD44 is up-regulated in renal diseases. CD44 may mediate binding of calcium oxalate crystals to tubular epithelia via the ligands osteopontin (OPN) and hyaluronan. Because 15% of premature infants develop nephrocalcinosis, developmental tubular CD44 expression might promote nephrocalcinosis. We studied CD44 and OPN immuno-localization in developing human kidney by immunohistochemical analysis. Human renal tissue between 18 and 40 wk of gestation showed CD44 immuno-localization in ureteric buds, with staining decreasing with increasing gestational age; CD44 was rarely observed in developing renal tubules. OPN was diffusely observed in proximal tubules, rarely observed in distal tubules, ureteric buds or metanephric structures. These data support the role of CD44 in early human nephron formation and branching morphogenesis. Rare CD44 staining in developing tubular epithelium suggests no role for CD44 in promoting calcium oxalate adherence to tubular epithelia in premature infants. Immuno-localization of OPN in tubules supports its role in tubular differentiation, but OPN does not seem to be necessary during early nephron formation.
    Tipo de documento:
    Referencia
    Referencia del producto:
    07-068
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