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  • Distribution of chromogranins A and B and secretogranin II (secretoneurin) in rat pelvic neurons and vas deferens. 9522381

    The family of chromogranins/secretogranin peptides comprises three major subtypes: chromogranin A, chromogranin B and secretogranin II. We have characterized these proteins in rat vas deferens and pelvic ganglia by using two approaches. Firstly, extracts of rat vas deferens were subjected to molecular sieve chromatography followed by radioimmunoassay. The results indicate that, in the peripheral nerves of this organ, chromogranin B and secretogranin II are processed to small peptides, i.e. PE-11 and secretoneuron, respectively. Secondly, we investigated the localization of each of these peptides in the rat pelvic ganglia and vas deferens. Comparisons with the distribution of tyrosine hydroxylase, choline acetyltransferase, vesicular acetylcholine transporter and SV2 were carried out in double labelling studies. All tyrosine hydroxylase-positive neurons contained neuropeptide Y, but many neuropeptide Y-containing neurons were negative for tyrosine hydroxylase. In the pelvic ganglia, chromogranin A was widely localized in the neuropeptide-positive neurons and 65% of chromogranin A-containing neurons were positive for tyrosine hydroxylase, suggesting their adrenergic nature. However, in nerve terminals of the vas deferens, chromogranin A was present at very low, or undetectable, levels. The chromogranin B-derived peptide PE-11, on the other hand, was absent from the large-sized, tyrosine hydroxylase-positive neurons, but present in some small-sized neurons that were choline acetyltransferase/vesicular acetylcholine transporter-positive and tyrosine hydroxylase-negative. In the vas deferens, PE-11 was present with intense immunoreactivity in nerve terminals of the lamina propria beneath the epithelium, but it was very sparse in the muscular layer and co-localized with vesicular acetylcholine transporter-like immunoreactivity, suggesting a cholinergic nature. The secretogranin II-derived peptide secretoneurin was distributed with strong immunoreactivity in the somata of pelvic ganglion neurons, 72% of which also contained tyrosine hydroxylase, as well as in nerve terminals in the muscular layer and the lamina propria of the vas deferens. Most, if not all, secretoneurin-positive terminals in the pelvic ganglia and the vas deferens were positive for choline acetyltransferase/vesicular acetylcholine transporter-like immunoreactivity. Retrograde tracing with FluoroGold demonstrated that the majority of FluoroGold-labelled neurons in the pelvic ganglia were positive for either chromogranin A or secretoneurin. The present study indicates that chromogranins A and B and secretogranin II are proteolytically processed to a high degree in the nerves of the rat vas deferens. Furthermore, they are heterogeneously localized in subsets of neurons of the pelvic ganglia and in different sets of nerve terminals in the vas deferens, suggesting that each of these peptides may play distinct roles in neurons of the autonomic nervous system to the vas deferens.
    Document Type:
    Reference
    Product Catalog Number:
    MAB305
    Product Catalog Name:
    Anti-Choline Acetyltransferase Antibody, clone 1E6

  • Preparative free-solution isotachophoresis for separation of human plasma lipoproteins: apolipoprotein and lipid composition of HDL subfractions. 10828082

    We have previously shown that plasma lipoproteins can be separated by analytical capillary isotachophoresis (ITP) according to their electrophoretic mobility in a defined buffer system. As in lipoprotein electrophoresis, HDL show the highest mobility followed by VLDL, IDL, and LDL. Chylomicrons migrate according to their net-charge between HDL and VLDL, because ITP has negligible molecular sieve effects. Three HDL subfractions were obtained which were designated fast-, intermediate-, and slow-migrating HDL. To further characterize these HDL subfractions, a newly developed free-solution ITP (FS-ITP)-system was used, that allows micro-preparative separation of human lipoproteins directly from whole plasma (Böttcher, A. et al. 1998. Electrophoresis. 19: 1110-1116). The fractions obtained by FS-ITP were analyzed for their lipid and apolipoprotein composition and by two-dimensional nondenaturing polyacrylamide gradient gel electrophoresis (2D-GGE) with subsequent immunoblotting. fHDL are characterized by the highest proportion of esterified cholesterol of all three subfractions and are relatively enriched in LpA-I. Together with iHDL they contain the majority of plasma apoA-I, while sHDL contain the majority of plasma apoA-IV, apoD, apoE, and apoJ. Pre-beta-HDL were found in separate fractions together with triglyceride-rich fractions between sHDL and LDL. In summary, ITP can separate the bulk of HDL into lipoprotein subfractions, which differ in apolipoprotein composition and electrophoretic mobility. While analytical ITP permits rapid separation and quantitation for diagnostic purposes, FS-ITP can be used to obtain these lipoprotein subfractions on a preparative scale for functional analysis. As FS-ITP is much better suited for preparative purposes than gel electrophoresis, it represents an important novel tool for the functional analysis of lipoprotein subclasses.
    Document Type:
    Reference
    Product Catalog Number:
    ABS1030
    Product Catalog Name:
    Anti-Apolipoprotein D Antibody

  • Basement membrane complexes with biological activity. 2937447

    We have studied the reconstitution of basement membrane molecules from extracts prepared from the basement membrane of the EHS tumor. Under physiological conditions and in the presence of added type IV collagen and heparan sulfate proteoglycan, gellike structures form whose ultrastructure appears as interconnected thin sheets resembling the lamina dense zone of basement membrane. The major components of the reconstituted structures include laminin, type IV collagen, heparan sulfate proteoglycan, entactin, and nidogen. These components polymerize in constant proportions on reconstitution, suggesting that they interact in defined proportions. Molecular sieve studies on the soluble extract demonstrate that laminin, entactin, and nidogen are associated in large but dissociable complexes which may be a necessary intermediate in the deposition of basement membrane. The reconstituted matrix was biologically active and stimulated the growth and differentiation of certain cells.
    Document Type:
    Reference
    Product Catalog Number:
    08-110
    Product Catalog Name:
    ECL Cell Attachment Matrix

  • Neurokinin-1 receptor (NK1-R) expression in the brains of SIV-infected rhesus macaques: implications for substance P in NK1-R immune cell trafficking into the CNS. 20671267

    Recent studies suggest a link between neuropsychiatric disorders and HIV/SIV infection. Most evidence indicates that monocytes/macrophages are the primary cell type infected within the CNS and that they contribute to CNS inflammation and neurological disease. Substance P (SP), a pleotropic neuropeptide implicated in inflammation, depression, and immune modulation via interaction with its cognate receptor, the neurokinin 1 receptor (NK1-R), is produced by monocyte/macrophages. While the presence of NK1-R on neurons is well known, its role on cells of the immune system such as monocyte/macrophages is just beginning to emerge. Therefore, we have examined the expression of SP and NK1-R and their relationship to SIV/HIV encephalitis (SIVE/HIVE) lesions and SIV-infected cells. These studies demonstrated intense expression of SP and NK1-R in SIVE lesions, with macrophages being the principal cell expressing NK1-R. Interestingly, all of the SIV-infected macrophages expressed NK1-R. Additionally, we examined the functional role of SP as a proinflammatory mediator of monocyte activation and chemotaxis. These studies demonstrated that treatment of monocytes with SP elicited changes in cell-surface expression for CCR5 and NK1-R in a dose-dependent manner. Moreover, pretreatment with SP enhanced both SP- and CCL5-mediated chemotaxis. All of these findings suggest that SP and NK1-R are important in SIV infection of macrophages and the development of SIVE lesions.
    Document Type:
    Reference
    Product Catalog Number:
    S7100
    Product Catalog Name:
    ApopTag® Peroxidase In Situ Apoptosis Detection Kit

  • In vitro differentiation of size-sieved stem cells into electrically active neural cells. 12456960

    Size-sieved stem (SS) cells isolated from human bone marrow and propagated in vitro are a population of cells with consistent marker typing, and can form bone, fat, and cartilage. In this experiment, we demonstrated that SS cells could be induced to differentiate into neural cells under experimental cell culture conditions. Five hours after exposure to antioxidant agents (beta-mercaptoethanol +/- retinoic acid) in serum-free conditions, SS cells expressed the protein for nestin, neuron-specific enolase (NSE), neuron-specific nuclear protein (NeuN), and neuron-specific tubulin-1 (TuJ-1), and the mRNA for NSE and Tau. Immunofluorescence showed that almost all the cells (>98%) expressed NeuN and TuJ-1. After 5 days of beta-mercaptoethanol treatment, the SS cells expressed neurofilament high protein but not mitogen-activated protein-2, glial filament acidic protein, and galactocerebroside. For such long-term-treated cells, voltage-sensitive ionic current could be detected by electrophysiological recording, and the intracellular calcium ion, Ca(2+), concentration can be elevated by high potassium (K(+)) buffer and glutamate. These findings suggest that SS cells may be an alternative source of undifferentiated cells for cell therapy and gene therapy in neural dysfunction.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • An integrated systems analysis implicates EGR1 downregulation in simian immunodeficiency virus encephalitis-induced neural dysfunction. 19812322

    Human immunodeficiency virus (HIV)-associated dementia (HAD) is a syndrome occurring in HIV-infected patients with advanced disease that likely develops as a result of macrophage and microglial activation as well as other immune events triggered by virus in the central nervous system. The most relevant experimental model of HAD, rhesus macaques exhibiting simian immunodeficiency virus (SIV) encephalitis (SIVE), closely reproduces the human disease and has been successfully used to advance our understanding of mechanisms underlying HAD. In this study we integrate gene expression data from uninfected and SIV-infected hippocampus with a human protein interaction network and discover modules of genes whose expression patterns distinguish these two states, to facilitate identification of neuronal genes that may contribute to SIVE/HIV cognitive deficits. Using this approach we identify several downregulated candidate genes and select one, EGR1, a key molecule in hippocampus-related learning and memory, for further study. We show that EGR1 is downregulated in SIV-infected hippocampus and that it can be downregulated in differentiated human neuroblastoma cells by treatment with CCL8, a product of activated microglia. Integration of expression data with protein interaction data to discover discriminatory modules of interacting proteins can be usefully used to prioritize differentially expressed genes for further study. Investigation of EGR1, selected in this manner, indicates that its downregulation in SIVE may occur as a consequence of the host response to infection, leading to deficits in cognition.
    Document Type:
    Reference
    Product Catalog Number:
    MAB374
    Product Catalog Name:
    Anti-Glyceraldehyde-3-Phosphate Dehydrogenase Antibody, clone 6C5
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