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  • Proteomic characterization of an isolated fraction of synthetic proteasome inhibitor (PSI)-induced inclusions in PC12 cells might offer clues to aggresomes as a cellular ... 20704702

    Cooperation of constituents of the ubiquitin proteasome system (UPS) with chaperone proteins in degrading proteins mediate a wide range of cellular processes, such as synaptic function and neurotransmission, gene transcription, protein trafficking, mitochondrial function and metabolism, antioxidant defence mechanisms, and apoptotic signal transduction. It is supposed that constituents of the UPS and chaperone proteins are recruited into aggresomes where aberrant and potentially cytotoxic proteins may be sequestered in an inactive form.To determinate the proteomic pattern of synthetic proteasome inhibitor (PSI)-induced inclusions in PC12 cells after proteasome inhibition by PSI, we analyzed a fraction of PSI-induced inclusions. A proteomic feature of the isolated fraction was characterized by identification of fifty six proteins including twenty previously reported protein components of Lewy bodies, twenty eight newly identified proteins and eight unknown proteins. These proteins, most of which were recognized as a profile of proteins within cellular processes mediated by the UPS, a profile of constituents of the UPS and a profile of chaperone proteins, are classed into at least nine accepted categories. In addition, prolyl-4-hydroxylase beta polypeptide, an endoplasmic reticulum member of the protein disulfide isomerase family, was validated in the developmental process of PSI-induced inclusions in the cells.It is speculated that proteomic characterization of an isolated fraction of PSI-induced inclusions in PC12 cells might offer clues to appearance of aggresomes serving as a cellular defensive response against proteasome inhibition.
    Document Type:
    Reference
    Product Catalog Number:
    MAB2073

  • Molecular characterization and partial cDNA cloning of facilitative glucose transporters expressed in human articular chondrocytes; stimulation of 2-deoxyglucose uptake b ... 12554125

    OBJECTIVE: Recent evidence suggests that human chondrocytes express several facilitative glucose transporter (GLUT) isoforms and also that 2-deoxyglucose transport is accelerated by cytokine stimulation. The aim of the present investigation was to determine if human articular chondrocytes express any of the recently identified members of the GLUT/SLC2A gene family and to examine the effects of endocrine factors, such as insulin and IGF-I on the capacity of human chondrocytes for transporting 2-deoxyglucose. DESIGN/METHODS: PCR, cloning and immunohistochemistry were employed to study the expression of GLUT/SLC2A transporters in normal human articular cartilage. The uptake of 2-deoxyglucose was examined in monolayer cultured immortalized human chondrocytes following stimulation with TNF-alpha, insulin and IGF-I. Levels of MMP-2 were assessed by gelatin zymography following glucose deprivation of alginate cultures. RESULTS: Using PCR we detected transcripts for eight glucose transporter isoforms (GLUTs 1, 3, 6, 8, 9, 10, 11 and 12) and for a fructose transporter (GLUT5) in human articular cartilage. Expression of GLUT1, GLUT3 and GLUT9 proteins in normal human articular cartilage was confirmed by immunohistochemistry. The uptake of 2-deoxyglucose was dependent on time and temperature, inhibited by cytochalasin B and phloretin, and significantly accelerated in chondrocyte cultures stimulated with IGF-I. However, 2-deoxyglucose uptake was unaffected by short and long-term insulin treatment, which ruled out a functional role for insulin-sensitive GLUT4-mediated glucose transport. Furthermore, secretion of MMP-2 was increased in alginate cultures deprived of glucose. CONCLUSIONS: The data supports a critical role for glucose transport and metabolism in the synthesis and degradation of cartilage.
    Document Type:
    Reference
    Product Catalog Number:
    07-1402

  • An in vivo characterization of trophic factor production following neural precursor cell or bone marrow stromal cell transplantation for spinal cord injury. 22085254

    Cellular transplantation strategies for repairing the injured spinal cord have shown consistent benefit in preclinical models, and human clinical trials have begun. Interactions between transplanted cells and host tissue remain poorly understood. Trophic factor secretion is postulated a primary or supplementary mechanism of action for many transplanted cells, however, there is little direct evidence to support trophin production by transplanted cells in situ. In the present study, trophic factor expression was characterized in uninjured, injured-untreated, injured-treated with transplanted cells, and corresponding control tissue from the adult rat spinal cord. Candidate trophic factors were identified in a literature search, and primers were designed for these genes. We examined in vivo trophin expression in 3 paradigms involving transplantation of either brain or spinal cord-derived neural precursor cells (NPCs) or bone marrow stromal cells (BMSCs). Injury without further treatment led to a significant elevation of nerve growth factor (NGF), leukemia inhibitory factor (LIF), insulin-like growth factor-1 (IGF-1), and transforming growth factor-β1 (TGF-β1), and lower expression of vascular endothelial growth factor isoform A (VEGF-A) and platelet-derived growth factor-A (PDGF-A). Transplantation of NPCs led to modest changes in trophin expression, and the co-administration of intrathecal trophins resulted in significant elevation of the neurotrophins, glial-derived neurotrophic factor (GDNF), LIF, and basic fibroblast growth factor (bFGF). BMSCs transplantation upregulated NGF, LIF, and IGF-1. NPCs isolated after transplantation into the injured spinal cord expressed the neurotrophins, ciliary neurotrophic factor (CNTF), epidermal growth factor (EGF), and bFGF at higher levels than host cord. These data show that trophin expression in the spinal cord is influenced by injury and cell transplantation, particularly when combined with intrathecal trophin infusion. Trophins may contribute to the benefits associated with cell-based repair strategies for spinal cord injury.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1501R
    Product Catalog Name:
    Anti-Actin Antibody,clone C4

  • Characterization of a protein phosphatase 2A holoenzyme that dephosphorylates the clathrin adaptors AP-1 and AP-2. 18158287

    The AP-2 complex is a key factor in the formation of endocytic clathrin-coated vesicles (CCVs). AP-2 sorts and packages cargo membrane proteins into CCVs, binds the coat protein clathrin, and recruits numerous other factors to the site of vesicle formation. Structural information on the AP-2 complex and biochemical work have allowed understanding its function on the molecular level, and recent studies showed that cycles of phosphorylation are key steps in the regulation of AP-2 function. The complex is phosphorylated on both large subunits (alpha- and beta2-adaptins) as well as at a single threonine residue (Thr-156) of the medium subunit mu2. Phosphorylation of mu2 is necessary for efficient cargo recruitment, whereas the functional context of the large subunit phosphorylation is unknown. Here, we show that the subunit phosphorylation of AP-2 exhibits striking differences, with calculated half-lives of <1 min for mu2, approximately 25 min for beta2, and approximately 70 min for alpha. We were also able to purify a phosphatase that dephosphorylates the mu2 subunit. The enzyme is a member of the protein phosphatase 2A family and composed of a catalytic Cbeta subunit, a scaffolding Abeta subunit, and a regulatory Balpha subunit. RNA interference knock down of the latter subunit in HeLa cells resulted in increased levels of phosphorylated adaptors and altered endocytosis, showing that a specific PP2A holoenzyme is an important regulatory enzyme in CCV-mediated transport.
    Document Type:
    Reference
    Product Catalog Number:
    AP180
    Product Catalog Name:
    Donkey Anti-Goat IgG Antibody, Species Adsorbed

  • Characterization of cytokine-producing cells in mucosal effector sites: CD3+ T cells of Th1 and Th2 type in salivary gland-associated tissues. 7957557

    The major purpose of this study was to elucidate Th1 [interferon (IFN)-gamma and interleukin (IL)-2] and Th2 (IL-4, IL-5 and IL-6) cytokine-producing CD3+ T cells in salivary glands, which are the major mucosal effector tissues in the oral region. Thus, CD3+ T cells were isolated from salivary gland-associated tissues (SGAT) which consist of the submandibular gland (SMG: approximately 46%), the periglandular lymph node (PGLN: approximately 72%), and the cervical lymph node (CLN: approximately 90%). When SMG CD3+ T cells were examined by Th1 and Th2 cytokine-specific ELISPOT and reverse transcriptase-polymerase chain reaction assay, high levels of both cytokine-specific spot-forming cells (SFC) and mRNA for IFN-gamma, and for IL-5 and IL-6 were noted as representative Th1 or Th2 cytokines, respectively. Following stimulation with concanavalin A (Con A), SMG CD3+ T cells expressed mRNA and produced lymphokines for an array of Th1 (IFN-gamma and IL-2) and Th2 (IL-4, IL-5 and IL-6) cytokines. In comparison to the SMG CD3+ T cells, PGLN and CLN contain lower numbers of IFN-gamma-, IL-5 and IL-6-producing T cells. When these two tissues were compared, PGLN CD3+ T cells contained higher numbers of cytokine-secreting cells than CLN. Further, IL-2 and IL-4 SFC and mRNA were also noted in addition to IFN-gamma, IL-5 and IL-6 after Con A activation. These findings showed that CD3+ T cells in SGAT, especially the SMG, are programmed to produce IFN-gamma, and IL-5 and IL-6 as Th1 and Th2 cytokines, respectively in vivo, although these cells are capable of producing other Th1 and Th2 cytokines after receiving appropriate T cell activation signals.
    Document Type:
    Reference
    Product Catalog Number:
    17-191
    Product Catalog Name:
    MAP Kinase/Erk Assay Kit, non-radioactive

  • Histological characterization of gonadotropin-releasing hormone (GnRH) in the hypothalamus of the South American plains vizcacha (Lagostomus maximus). 21660456

    In contrast to most mammalian species, females of the South American plains vizcacha, Lagostomus maximus, show an extensive suppression of apoptosis-dependent follicular atresia, continuous folliculogenesis, and massive polyovulation. These unusual reproductive features pinpoint to an eventual peculiar modulation of the hypothalamo-hypophyseal-gonadal axis through its main regulator, the gonadotropin-releasing hormone (GnRH). We explored the hypothalamic histological landscape and cellular and subcellular localization of GnRH in adult non-pregnant L. maximus females. Comparison to brain atlases from mouse, rat, guinea pig and chinchilla enabled us to histologically define and locate the preoptic area (POA), the ventromedial nucleus, the median eminence (ME), and the arcuate nucleus (Arc) of the hypothalamus in vizcacha's brain. Specific immunolocalization of GnRH was detected in soma of neurons at medial POA (MPA), ventrolateral preoptic nucleus, septohypothalamic nucleus (SHy) and Arc, and in beaded fibers of MPA, SHy, ventromedial hypothalamic nucleus, anterior hypothalamic area and ME. Electron microscopy examination revealed GnRH associated to cytoplasmic vesicles of the ME and POA neurons, organized both in core and non-core vesicles within varicosities, and in neurosecretory vesicles within the myelinated axons of the MPA. Besides the peculiar and unusual features of folliculogenesis and ovulation in the vizcacha, these results show that hypothalamus histology and GnRH immune-detection and localization are comparable to those found in other mammals. This fact leads to the possibility that specific regulatory mechanisms should be in action to maintain continuous folliculogenesis and massive polyovulation.
    Document Type:
    Reference
    Product Catalog Number:
    MAB5456
    Product Catalog Name:
    Anti-Gonadotropin-Releasing Hormone Antibody, NT, clone HU11B

  • Characterization of the C. elegans erlin homologue. 22269071

    Erlins are highly conserved proteins associated with lipid rafts within the endoplasmic reticulum (ER). Biochemical studies in mammalian cell lines have shown that erlins are required for ER associated protein degradation (ERAD) of activated inositol-1,4,5-trisphosphate receptors (IP3Rs), implying that erlin proteins might negatively regulate IP3R signalling. In humans, loss of erlin function appears to cause progressive intellectual disability, motor dysfunction and joint contractures. However, it is unknown if defects in IP3R ERAD are the underlying cause of this disease phenotype, whether ERAD of activated IP3Rs is the only function of erlin proteins, and what role ERAD plays in regulating IP3R-dependent processes in the context of an intact animal or embryo. In this study, we characterize the erlin homologue of the nematode Caenorhabditis elegans and examine erlin function in vivo. We specifically set out to test whether C. elegans erlin modulates IP3R-dependent processes, such as egg laying, embryonic development and defecation rates. We also explore the possibility that erlin might play a more general role in the ERAD pathway of C. elegans.We first show that the C. elegans erlin homologue, ERL-1, is highly similar to mammalian erlins with respect to amino acid sequence, domain structure, biochemical properties and subcellular location. ERL-1 is present throughout the C. elegans embryo; in adult worms, ERL-1 appears restricted to the germline. The expression pattern of ERL-1 thus only partially overlaps with that of ITR-1, eliminating the possibility of ERL-1 being a ubiquitous and necessary regulator of ITR-1. We show that loss of ERL-1 does not affect overall phenotype, or alter brood size, embryonic development or defecation cycle length in either wild type or sensitized itr-1 mutant animals. Moreover we show that ERL-1 deficient worms respond normally to ER stress conditions, suggesting that ERL-1 is not an essential component of the general ERAD pathway.Although loss of erlin function apparently causes a strong phenotype in humans, no such effect is seen in C. elegans. C. elegans erlin does not appear to be a ubiquitous major modulator of IP3 receptor activity nor does erlin appear to play a major role in ERAD.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1501
    Product Catalog Name:
    Anti-Actin Antibody, clone C4