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  • 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.
    Typ dokumentu:
    Reference
    Katalogové číslo produktu:
    MAB1501R
    Název katalogu produktů:
    Anti-Actin Antibody,clone C4

  • 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.
    Typ dokumentu:
    Reference
    Katalogové číslo produktu:
    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.
    Typ dokumentu:
    Reference
    Katalogové číslo produktu:
    07-1402

  • Characterization of NMDA receptor subunit-specific antibodies: distribution of NR2A and NR2B receptor subunits in rat brain and ontogenic profile in the cerebellum. 7790859

    Selective antisera for NMDA receptor subunits NR2A and NR2B have been developed. Each antiserum identifies a single band on an immunoblot at approximately 175 kDa that appears to be the appropriate subunit of the NMDA receptor. Using these antisera the relative densities of the subunits in eight areas of adult rat brain have been determined. The NR2A subunit was found to be at its highest level in hippocampus and cerebral cortex, to be at intermediate levels in striatum, olfactory tubercle, mid-brain, olfactory bulb, and cerebellum, and to be at lowest levels in the pons-medulla. The NR2B subunit was found to be expressed at its highest levels in the olfactory tubercle, hippocampus, olfactory bulb, and cerebral cortex. Intermediate levels were expressed in striatum and mid-brain, and low levels were detected in the pons-medulla. No signal for NR2B was found in the cerebellum. These regional distributions were compared with that for [3H]MK-801 binding sites. It was found that although the distribution of the NR2A subunit corresponds well with radioligand binding, the distribution of the NR2B subunit does not. The ontogenic profiles of NR2A and NR2B subunits in the rat cerebellum were also determined. Just following birth [postnatal day (P) 2] NR2A subunits are undetectable, whereas NR2B subunits are expressed at amounts easily measurable. Beginning at about P12 the levels of NR2A rise rapidly to reach adult levels by P22. At the same time (P12), levels of NR2B protein begin to decline rapidly to reach undetectable levels by 22 days after birth.(ABSTRACT TRUNCATED AT 250 WORDS)
    Typ dokumentu:
    Reference
    Katalogové číslo produktu:
    Multiple
    Název katalogu produktů:
    Multiple

  • Characterization of Progenitor Cells during Canine Retinal Development. 22567026

    We identify the presence of progenitor cells during retinal development in the dog, as this species represents a natural model for studying several breed-specific degenerative retinal disorders. Antibodies to detected progenitor cells (Pax6, C-kit, and nestin) and ganglion cells (BDNF, Brn3a, and Thy1) were used in combination with H3 for the purpose of identifying proliferating cells. Pax6, nestin, C-kit, and H3 were localized mainly in the neuroblastic layer of the retina during the embryonic stage. During the fetal stage, proteins were expressed in the inner neuroblastic layer (INL) as well as in the outer neuroblastic layer; BDNF, Thy1, and Brn3a were also expressed in the INL. During the neonatal stage only C-kit was not expressed. Proliferating cells were present in both undifferentiated and differentiated retina. These results suggest that, during canine retinogenesis, progenitor cells are distributed along the retina and some of these cells remain as progenitor cells of the ganglion cells during the first postnatal days.
    Typ dokumentu:
    Reference
    Katalogové číslo produktu:
    06-570
    Název katalogu produktů:
    Anti-phospho-Histone H3 (Ser10) Antibody, Mitosis Marker

  • 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.
    Typ dokumentu:
    Reference
    Katalogové číslo produktu:
    MAB1501
    Název katalogu produktů:
    Anti-Actin Antibody, clone C4

  • Characterization of the PRMT gene family in rice reveals conservation of arginine methylation. 21853042

    Post-translational methylation of arginine residues profoundly affects the structure and functions of protein and, hence, implicated in a myriad of essential cellular processes such as signal transduction, mRNA splicing and transcriptional regulation. Protein arginine methyltransferases (PRMTs), the enzymes catalyzing arginine methylation have been extensively studied in animals, yeast and, to some extent, in model plant Arabidopsis thaliana. Eight genes coding for the PRMTs were identified in Oryza sativa, previously. Here, we report that these genes show distinct expression patterns in various parts of the plant. In vivo targeting experiment demonstrated that GFP-tagged OsPRMT1, OsPRMT5 and OsPRMT10 were localized to both the cytoplasm and nucleus, whereas OsPRMT6a and OsPRMT6b were predominantly localized to the nucleus. OsPRMT1, OsPRMT4, OsPRMT5, OsPRMT6a, OsPRMT6b and OsPRMT10 exhibited in vitro arginine methyltransferase activity against myelin basic protein, glycine-arginine-rich domain of fibrillarin and calf thymus core histones. Furthermore, they depicted specificities for the arginine residues in histones H3 and H4 and were classified into type I and Type II PRMTs, based on the formation of type of dimethylarginine in the substrate proteins. The two homologs of OsPRMT6 showed direct interaction in vitro and further titrating different amounts of these proteins in the methyltransferase assay revealed that OsPRMT6a inhibits the methyltransferase activity of OsPRMT6b, probably, by the formation of heterodimer. The identification and characterization of PRMTs in rice suggests the conservation of arginine methylation in monocots and hold promise for gaining further insight into regulation of plant development.
    Typ dokumentu:
    Reference
    Katalogové číslo produktu:
    Multiple
    Název katalogu produktů:
    Multiple