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  • Expression of orexin A and its receptor 1 in the choroid plexuses from buffalo brain. 19250669

    The hypothalamic peptide orexin A, deriving from the proteolytic cleavage of the precursor molecule prepro-orexin, has a wide range of physiological effects including the regulation of feeding behaviour, neuroendocrine functions, sleep-wake cycle, and energy homeostasis. Lowered excretion of orexin A into the cerebrospinal fluid (CSF) plays a pathological role in animal and human narcolepsy. Altered levels of orexin A into the CSF have been also found in numerous disorders of the central nervous system, including Parkinson's and Huntington's disease, dementia, and depressive disorders. While the localization of orexin A and its receptor 1, OX(1), has been elicited in many regions of the mammalian brain and in peripheral organs, there are no information on the expression of the neuropeptide and its receptor 1 in the choroid plexuses (CPs) producing the CSF. In this study, we investigated the expression of orexin A and OX(1) in the CPs from the brain of an adult mammalian species, Bubalis bubalis, by immunogold-labelling in scanning electron microscopy. Both orexin A and OX(1) immuno-reactivity appeared to be widely distributed on the surface of choroid epithelium. Interestingly, a marked orexin A labelling was detected in the areas surrounding the CP blood capillaries. The expression of prepro-orexin and OX(1) mRNA transcripts of 200 and 300 bp, respectively, was assessed in the CPs by reverse-transcription polymerase chain reaction, while Western blotting analysis confirmed the presence of these two proteins in the tissue. Our findings provide the first evidence for orexin A and OX(1) expression in the CPs from mammalian brain, and suggest that the levels of orexin A into the CSF are probably regulated by CP activity.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB3096
    Nombre del producto:
    Anti-Orexin Antibody, Prepro

  • An evaluation of oligonucleotide-based therapeutic strategies for polyQ diseases. 22397573

    RNA interference (RNAi) and antisense strategies provide experimental therapeutic agents for numerous diseases, including polyglutamine (polyQ) disorders caused by CAG repeat expansion. We compared the potential of different oligonucleotide-based strategies for silencing the genes responsible for several polyQ diseases, including Huntington's disease and two spinocerebellar ataxias, type 1 and type 3. The strategies included nonallele-selective gene silencing, gene replacement, allele-selective SNP targeting and CAG repeat targeting.Using the patient-derived cell culture models of polyQ diseases, we tested various siRNAs, and antisense reagents and assessed their silencing efficiency and allele selectivity. We showed considerable allele discrimination by several SNP targeting siRNAs based on a weak G-G or G-U pairing with normal allele and strong G-C pairing with mutant allele at the site of RISC-induced cleavage. Among the CAG repeat targeting reagents the strongest allele discrimination is achieved by miRNA-like functioning reagents that bind to their targets and inhibit their translation without substantial target cleavage. Also, morpholino analog performs well in mutant and normal allele discrimination but its efficient delivery to cells at low effective concentration still remains a challenge.Using three cellular models of polyQ diseases and the same experimental setup we directly compared the performance of different oligonucleotide-based treatment strategies that are currently under development. Based on the results obtained by us and others we discussed the advantages and drawbacks of these strategies considering them from several different perspectives. The strategy aimed at nonallele-selective inhibiting of causative gene expression by targeting specific sequence of the implicated gene is the easiest to implement but relevant benefits are still uncertain. The gene replacement strategy that combines the nonallele-selective gene silencing with the expression of the exogenous normal allele is a logical extension of the former and it deserves to be explored further. Both allele-selective RNAi approaches challenge cellular RNA interference machinery to show its ability to discriminate between similar sequences differing in either single base substitutions or repeated sequence length. Although both approaches perform well in allele discrimination most of our efforts are focused on repeat targeting due to its potentially higher universality.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Comparative gene expression profiling between human cultured myotubes and skeletal muscle tissue. 20175888

    A high-sensitivity DNA microarray platform requiring nanograms of RNA input facilitates the application of transcriptome analysis to individual skeletal muscle (SM) tissue samples. Culturing myotubes from SM-biopsies enables investigating transcriptional defects and assaying therapeutic strategies. This study compares the transcriptome of aneurally cultured human SM cells versus that of tissue biopsies.We used the Illumina expression BeadChips to determine the transcriptomic differences between tissue and cultured SM samples from five individuals. Changes in the expression of several genes were confirmed by QuantiGene Plex assay or reverse transcription real-time PCR. In cultured myotubes compared to the tissue, 1216 genes were regulated: 583 down and 633 up. Gene ontology analysis showed that downregulated genes were mainly associated with cytoplasm, particularly mitochondria, and involved in metabolism and the muscle-system/contraction process. Upregulated genes were predominantly related to cytoplasm, endoplasmic reticulum, and extracellular matrix. The most significantly regulated pathway was mitochondrial dysfunction. Apoptosis genes were also modulated. Among the most downregulated genes detected in this study were genes encoding metabolic proteins AMPD1, PYGM, CPT1B and UCP3, muscle-system proteins TMOD4, MYBPC1, MYOZ1 and XIRP2, the proteolytic CAPN3 and the myogenic regulator MYF6. Coordinated reduced expression of five members of the GIMAP gene family, which form a cluster on chromosome 7, was shown, and the GIMAP4-reduction was validated. Within the most upregulated group were genes encoding senescence/apoptosis-related proteins CDKN1A and KIAA1199 and potential regulatory factors HIF1A, TOP2A and CCDC80.Cultured muscle cells display reductive metabolic and muscle-system transcriptome adaptations as observed in muscle atrophy and they activate tissue-remodeling and senescence/apoptosis processes.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB907
    Nombre del producto:
    Anti-Desmin Antibody

  • P2X4 is up-regulated in gingival fibroblasts after periodontal surgery. 17251520

    Several studies have shown that surgical detachment of marginal gingiva close to the cervical cementum of molar teeth in a rat mandible is a distinct stimulus for alveolar bone resorption. Recently, we found that P2X4, an ATP-receptor, is significantly up-regulated in marginal gingival cells soon after surgery. We hypothesized that local release of ATP signaling through P2X4 elicits activation of osteoclasts on the alveolar bone surface. In this study, we identified intense immunoreactivity of gingival fibroblasts to P2X4-specific antibodies and a 6.4-fold increase in expression by real-time RT-PCR. Moreover, a single local application, at the time of surgery, of Apyrase (which degrades ATP) or Coomassie Brilliant Blue (an antagonist of purinoreceptors) significantly reduced alveolar bone loss. We propose that ATP flowing from cells after surgery can directly activate P2X4 receptors in the sensor cells of marginal gingiva through Ca(2+) signaling, or by direct activation of osteoclasts on the bone surface.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AP182B
    Nombre del producto:
    Donkey Anti-Rabbit IgG Antibody, biotin-SP conjugate, Species Adsorbed

  • Morphologic and neurotoxic effects of ethanol vary with timing of exposure in vitro. 12551761

    Results of investigations with animal models of fetal alcohol syndrome (FAS) seem to indicate that neuronal vulnerability to ethanol-induced cell death may be correlated with specific developmental events. In the present study, we sought to test this observation in a cell culture model of neuronal development in which morphogenesis as well as survival could be assessed. Using embryonic rat hippocampal pyramidal neurons in primary cultures, we compared the sensitivity of neurons to ethanol added, at 400 mg/dl, to the medium at different times relative to the development of axons and dendrites. Quantitative morphometric analysis was performed by using phase contrast at 12 h (0.5 day) and 24 h (1 day), or fluorescence microscopy after microtubule-associated protein-2 (MAP2) immunostaining at 6 and 14 days. Survival was assessed by counting the number of neurons per unit area of the substrate at 14 days. Addition of ethanol 1 day after plating, when most neurons had developed an axon, had no effect on survival up to 14 days in vitro, but resulted in significantly shorter, less branched dendrites than observed when ethanol was added 2 h after plating. Despite the shorter duration of ethanol exposure, the addition of ethanol on day 6, after rapid growth of dendrites and synapses had begun, resulted in loss of all but about one third of the neurons by 14 days. This supports the suggestion that increased neuronal vulnerability to the morphoregulatory effects of ethanol is correlated with the establishment of polarity, but that the sensitivity of neurons to the cytotoxic effects of ethanol occurs later, when dendrites and synapses are rapidly forming.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB3418
    Nombre del producto:
    Anti-MAP2 Antibody, clone AP20

  • The involvement of integrin β1 signaling in the migration and myofibroblastic differentiation of skin fibroblasts on anisotropic collagen-containing nanofibers. 22136719

    Utilization of nanofibrous matrices for skin wound repair holds great promise due to their morphological and dimensional similarity to native extracellular matrix (ECM). It becomes highly desired to understand how various nanofibrous matrices regulate skin cell behaviors and intracellular signaling pathways, important to tuning the functionality of tissue-engineered skin grafts and affecting the wound healing process. In this study, the phenotypic expressions of normal human dermal fibroblasts (NHDFs) on collagen-containing nanofibrous matrices with either isotropic (i.e., fibers collected randomly with no alignment) or anisotropic (i.e., fibers collected with alignment) fiber organizations were studied by immunostaining, migration assay and molecular analyses. Results showed that both nanofibrous matrices supported the attachment and growth of NHDFs similarly, while showing different cell morphology with distinct variation in focal adhesion formation and distribution. Anisotropic nanofibers significantly triggered the integrin β1 signaling pathway in NHDFs as evidenced by an increase of active integrin β1 (130 kD mature form) and phosphorylation of focal adhesion kinase (FAK) at Tyr-397. Anisotropic matrices also promoted the migration of NHDFs along the fibers, while neutralization of the integrin β1 activity abolished this promotion. Moreover, the fibroblast-to-myofibroblast differentiation was greatly enhanced for the NHDFs cultured on anisotropic nanofibrous matrices over a period of 48 h. Inhibition of cellular integrin β1 activity by neutralizing antibody eliminated this enhancement. These findings suggest the important role of integrin β1 signaling pathway in regulating the nanofiber-induced fibroblast phenotypic alteration and providing insightful understanding of the possible application of collagen-containing nanofibrous matrices for skin regeneration.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Lead exposure during synaptogenesis alters NMDA receptor targeting via NMDA receptor inhibition. 21192972

    N-methyl-D-aspartate receptor (NMDAR) ontogeny and subunit expression are altered during developmental lead (Pb²+) exposure. However, it is unknown whether these changes occur at the synaptic or cellular level. Synaptic and extra-synaptic NMDARs have distinct cellular roles, thus, the effects of Pb²+ on NMDAR synaptic targeting may affect neuronal function. In this communication, we show that Pb²+ exposure during synaptogenesis in hippocampal neurons altered synaptic NMDAR composition, resulting in a decrease in NR2A-containing NMDARs at established synapses. Conversely, we observed increased targeting of the obligatory NR1 subunit of the NMDAR to the postsynaptic density (PSD) based on the increased colocalization with the postsynaptic protein PSD-95. This finding together with increased binding of the NR2B-subunit specific ligand [³H]-ifenprodil, suggests increased targeting of NR2B-NMDARs to dendritic spines as a result of Pb²+ exposure. During brain development, there is a shift of NR2B- to NR2A-containing NMDARs. Our findings suggest that Pb²+ exposure impairs or delays this developmental switch at the level of the synapse. Finally, we show that alter expression of NMDAR complexes in the dendritic spine is most likely due to NMDAR inhibition, as exposure to the NMDAR antagonist aminophosphonovaleric acid (APV) had similar effects as Pb²+ exposure. These data suggest that NMDAR inhibition by Pb²+ during synaptogensis alters NMDAR synapse development, which may have lasting consequences on downstream signaling.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB1557P
    Nombre del producto:
    Anti-NMDAR2B Antibody

  • The mouse cyclophosphamide model of bladder pain syndrome: tissue characterization, immune profiling, and relationship to metabotropic glutamate receptors. 24760514

    Abstract Painful bladder syndrome/Interstitial cystitis (PBS/IC) is a chronic disorder characterized clinically by recurring episodes of pelvic pain and increased urination frequency, significantly impairing patients' quality of life. Despite this, there is an unmet medical need in terms of effective diagnostics and treatment. Animal models are crucial in this endeavor. Systemic chronic administration of cyclophosphamide (CYP) in mice has been proposed as a relevant preclinical model of chronic bladder pain. However, molecular mechanisms underlying the pathogenesis of this model are lacking. Here, we show that mice, subjected to repetitive systemic injections of CYP, developed mild inflammatory response in bladder tissue characterized by submucosal edema, moderate increase in proinflammatory cytokine gene expression, and mastocytosis. No signs of massive inflammatory infiltrate, tissue hemorrhages, mucosal ulcerations and urothelium loss were observed. Instead, CYP treatment induced urothelium hyperplasia, accompanied by activation of proliferative signaling cascades, and a decrease in the expression of urothelium-specific markers. Metabotropic glutamate (mGlu) receptors have been implicated in chronic pain disorders. CYP administration induced differential changes in mGlu receptors mRNA levels in bladder tissue, without affecting gene expression at spinal cord level, pointing to the potential link between peripheral mGlu receptors and inflammation-induced bladder malfunction and hyperalgesia. Taken together, these data indicate that chronic CYP treatment in mice is a model of PBS mostly relevant to the major, nonulcerative subtype of the syndrome, characterized by a relatively unaltered mucosa and a sparse inflammatory response. This model can help to elucidate the pathogenetic mechanisms of the disease.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Heregulin beta1 drives gefitinib-resistant growth and invasion in tamoxifen-resistant MCF-7 breast cancer cells. 17686159

    Resistance to anti-epidermal growth factor receptor (anti-EGFR) therapies is an emerging clinical problem. The efficacy of anti-EGFR therapies can be influenced by the presence of heregulins (HRGs), which can bind erbB3/4 receptors and can activate alternative signalling pathways. In the present study we have examined whether HRG signalling can circumvent EGFR blockade in an EGFR-positive tamoxifen-resistant MCF-7 (Tam-R) breast cancer cell line.Tam-R cells, incubated with the selective EGFR tyrosine kinase inhibitor gefitinib ('Iressa', ZD1839), were exposed to HRGbeta1 and the effects on erbB receptor dimerization profiles and on activation of associated downstream signalling components were assessed by immunoprecipitation, western blotting and immunocytochemistry. The effects of HRGbeta1 on gefitinib-treated Tam-R cell growth and invasion were also examined, and HRGbeta1 expression levels were assessed in breast cancer tissue by immunohistochemistry to address the potential clinical relevance of such a resistance mechanism.In Tam-R cells, HRGbeta1 promoted erbB3/erbB2 and erbB3/EGFR heterodimerization, promoted ERK1/2 and AKT pathway activation and increased cell proliferation and invasion. Gefitinib prevented HRGbeta1-driven erbB3/EGFR heterodimerization, ERK1/2 activation and Tam-R cell proliferation, but HRGbeta1-driven erbB3/erbB2 heterodimerization, AKT activation and Tam-R cell invasion were maintained. A combination of gefitinib and the phosphatidylinositol 3-kinase inhibitor LY294002 effectively blocked HRGbeta1-mediated intracellular signalling activity, growth and invasion in Tam-R cells. Similarly, targeting erbB2 with trastuzumab in combination with gefitinib in Tam-R cells reduced HRGbeta1-induced erbB2 and ERK1/2 activity; however, HRGbeta1-driven AKT activity and cell growth were maintained while cell invasion was significantly enhanced with this combination. In clinical tissue all samples demonstrated cytoplasmic tumour epithelial HRGbeta1 protein staining, with expression correlating with EGFR positivity and activation of both AKT and ERK1/2.HRGbeta1 can overcome the inhibitory effects of gefitinib on cell growth and invasion in Tam-R cells through promotion of erbB3/erbB2 heterodimerization and activation of the phosphatidylinositol 3-kinase/AKT signalling pathway. This may have implications for the effectiveness of anti-EGFR therapies in breast cancer as HRGbeta1 is enriched in many EGFR-positive breast tumours.
    Tipo de documento:
    Referencia
    Referencia del producto:
    06-229
    Nombre del producto:
    Anti-phospho-erbB-2/HER-2 (Tyr1248) Antibody