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  • Disruption of intermolecular disulfide bonds in PDGF-BB dimers by N-acetyl-L-cysteine does not prevent PDGF signaling in cultured hepatic stellate cells. 16289037

    Oxidative stress is important in the pathogenesis of liver fibrosis through its induction of hepatic stellate cell (HSC) proliferation and enhancement of collagen synthesis. Reactive oxygen species have been found to be essential second messengers in the signaling of both major fibrotic growth factors, platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta), in cultured HSC and liver fibrosis. The non-toxic aminothiol N-acetyl-L-cysteine (NAC) inhibits cellular activation and attenuates experimental fibrosis in liver. Prior reports show that NAC is capable of reducing the effects of TGF-beta in biological systems, in cultured endothelial cells, and HSC through its direct reducing activity upon TGF-beta molecules. We here analyzed the effects of NAC on PDGF integrity, receptor binding, and downstream signaling in culture-activated HSC. We found that NAC dose-dependently induces disintegration of PDGF in vitro. However, even high doses (>20mM) were not sufficient to prevent the phosphorylation of the PDGF receptor type beta, extracellular signal-regulated kinase, or protein kinase B (PKB/Akt). Therefore, we conclude that the PDGF monomer is still active. The described antifibrotic effects are therefore mainly attributable to the structural impairment of TGF-beta signaling components reported previously.
    Document Type:
    Reference
    Product Catalog Number:
    05-321
    Product Catalog Name:
    Anti-Phosphotyrosine Antibody, clone 4G10®

  • Nerve growth factor promoter activity revealed in mice expressing enhanced green fluorescent protein. 21456011

    Nerve growth factor (NGF) and its precursor proNGF are perhaps the best described growth factors of the mammalian nervous system. There remains, however, a paucity of information regarding the precise cellular sites of proNGF/NGF synthesis. Here we report the generation of transgenic mice in which the NGF promoter controls the ectopic synthesis of enhanced green fluorescent protein (EGFP). These transgenic mice provide an unprecedented resolution of both neural cells (e.g., neocortical and hippocampal neurons) and non-neural cells (e.g., renal interstitial cells and thymic reticular cells) that display NGF promoter activity from postnatal development to adulthood. Moreover, the transgene is inducible by injury. At 2 days after sciatic nerve ligation, a robust population of EGFP-positive cells is seen in the proximal nerve stump. These transgenic mice offer novel insights into the cellular sites of NGF promoter activity and can be used as models for investigating the regulation of proNGF/NGF expression after injury.
    Document Type:
    Reference
    Product Catalog Number:
    AB5220
    Product Catalog Name:
    Anti-Growth Associated Protein-43 (GAP-43) Antibody

  • Insulin down-regulates specific activity of aTP-binding cassette transporter a1 for high density lipoprotein biogenesis through its specific phosphorylation. 21402379

    Insulin resistance/hyperinsulinism is one of the major risks for atherosclerotic vascular diseases and low HDL may be involved in pathogenesis. We examined direct effects of insulin on HDL biosynthesis focusing on the activity of ATP-binding cassette transporter A1 (ABCA1) in culture cells and in experimental animals. Insulin impairs HDL biosynthesis through modulation of ABCA1 activity by two different mechanisms. Insulin enhances degradation of ABCA1. However, even after this effect was cancelled by blocking its specific signal, insulin still reduces HDL biogenesis. This effect was found due to phosphorylation of ABCA1 that leads to decrease of its specific activity. We identified a novel insulin-specific phosphorylation site Tyr1206 of ABCA1 to regulate its specific activity. The observation in a rat model of insulin resistance was consistent with these results. The findings demonstrate a new mechanism for regulation of ABCA1 activity and provide new insights into the link between development of atherosclerosis, and insulin resistance/hyperinsulinism.Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.
    Document Type:
    Reference
    Product Catalog Number:
    05-321
    Product Catalog Name:
    Anti-Phosphotyrosine Antibody, clone 4G10®

  • Platelet-derived growth factor isoform expression in carbon tetrachloride-induced chronic liver injury. 18663351

    Platelet-derived growth factor (PDGF) has an essential role in liver fibrogenesis, as PDGF-B and -D both act as potent mitogens on culture-activated hepatic stellate cells (HSCs). Induction of PDGF receptor type-beta (PDGFR beta) in HSC is well documented in single-dose carbon tetrachloride (CCl(4))-induced acute liver injury. Of the newly discovered isoforms PDGF-C and -D, only PDGF-D shows significant upregulation in bile duct ligation (BDL) models. We have now investigated the expression of PDGF isoforms and receptors in chronic liver injury in vivo after long-term CCl(4) treatment and demonstrated that isolated hepatocytes have the requisite PDGF signaling pathways, both in the naive state and when isolated from CCl(4)-treated rats. In vivo, PDGF gene expression showed upregulation of all PDGF isoforms and receptors, with values peaking at 4 weeks and decreasing to near basal levels by 8 and 12 weeks. Interestingly, PDGF-C increased significantly when compared to BDL-models. PDGF-A, PDGF-C and PDGF receptor type-alpha (PDGFR alpha) correlated closely with inflammation and steatosis. Immunohistochemistry revealed expression of PDGF-B, -C and -D in areas corresponding to centrilobular necrosis, inflammation and fibrosis, whereas PDGF-A localized in regenerative hepatocytes. PDGFR beta was identified along the fibrotic septa, whereas PDGFR alpha showed positive staining in fibrotic septa and regenerative hepatocytes. Despite a significant decline of PDGF isoforms, hepatocyte regeneration peaked at 8 weeks. A marked difference in the degree of fibrosis was observed amongst the individual animals. In summary, PDGF expression in liver damage primarily parallels mesenchymal cell proliferation and extracellular matrix production, rather than hepatocyte regeneration. We conclude that PDGF levels in chronic liver injury peak at 4 weeks after onset of injury, and that the outcome of chronic toxic liver injury strongly depends on the individual capacity for tissue regeneration in the weeks following the peak of PDGF expression.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Pro-fibrogenic potential of PDGF-D in liver fibrosis. 17397961

    We analyzed the expression of platelet-derived growth factor D (PDGF-D) in an experimental bile duct-ligated (BDL) rat model and assessed its biological function in cultured hepatic stellate cells (HSC) and myofibroblasts (MFB).
    Document Type:
    Reference
    Product Catalog Number:
    AB1954
    Product Catalog Name:
    Anti-Fibronectin Antibody

  • Delayed treatment with a novel neurotrophic compound reduces behavioral deficits in rabbit ischemic stroke. 21054387

    Acute ischemic stroke is a major risk for morbidity and mortality in our aging population. Currently only one drug, the thrombolytic tissue plasminogen activator, is approved by the US Food and Drug Administration to treat stroke. Therefore, there is a need to develop new drugs that promote neuronal survival following stroke. We have synthesized a novel neuroprotective molecule called CNB-001 (a pyrazole derivative of curcumin) that has neurotrophic activity, enhances memory, and blocks cell death in multiple toxicity assays related to ischemic stroke. In this study, we tested the efficacy of CNB-001 in a rigorous rabbit ischemic stroke model and determined the molecular basis of its in vivo activity. CNB-001 has substantial beneficial properties in an in vitro ischemia assay and improves the behavioral outcome of rabbit ischemic stroke even when administered 1 h after the insult, a therapeutic window in this model comparable to tissue plasminogen activator. In addition, we elucidated the protein kinase pathways involved in neuroprotection. CNB-001 maintains the calcium-calmodulin-dependent kinase signaling pathways associated with neurotrophic growth factors that are critical for the maintenance of neuronal function. On the basis of its in vivo efficacy and novel mode of action, we conclude that CNB-001 has a great potential for the treatment of ischemic stroke as well as other CNS pathologies.
    Document Type:
    Reference
    Product Catalog Number:
    05-591
    Product Catalog Name:
    Anti-Akt/PKB Antibody, PH Domain, clone SKB1

  • Involvement of the PRKCB1 gene in autistic disorder: significant genetic association and reduced neocortical gene expression. 18317465

    Protein kinase C enzymes play an important role in signal transduction, regulation of gene expression and control of cell division and differentiation. The fsI and betaII isoenzymes result from the alternative splicing of the PKCbeta gene (PRKCB1), previously found to be associated with autism. We performed a family-based association study in 229 simplex and 5 multiplex families, and a postmortem study of PRKCB1 gene expression in temporocortical gray matter (BA41/42) of 11 autistic patients and controls. PRKCB1 gene haplotypes are significantly associated with autism (P0.05) and have the autistic endophenotype of enhanced oligopeptiduria (P0.05). Temporocortical PRKCB1 gene expression was reduced on average by 35 and 31% for the PRKCB1-1 and PRKCB1-2 isoforms (P0.01 and 0.05, respectively) according to qPCR. Protein amounts measured for the PKCbetaII isoform were similarly decreased by 35% (P=0.05). Decreased gene expression characterized patients carrying the 'normal' PRKCB1 alleles, whereas patients homozygous for the autism-associated alleles displayed mRNA levels comparable to those of controls. Whole genome expression analysis unveiled a partial disruption in the coordinated expression of PKCbeta-driven genes, including several cytokines. These results confirm the association between autism and PRKCB1 gene variants, point toward PKCbeta roles in altered epithelial permeability, demonstrate a significant downregulation of brain PRKCB1 gene expression in autism and suggest that it could represent a compensatory adjustment aimed at limiting an ongoing dysreactive immune process. Altogether, these data underscore potential PKCbeta roles in autism pathogenesis and spur interest in the identification and functional characterization of PRKCB1 gene variants conferring autism vulnerability.
    Document Type:
    Reference
    Product Catalog Number:
    AP124P
    Product Catalog Name:
    Goat Anti-Mouse IgG Antibody, Peroxidase Conjugated, H+L

  • Enhanced bone regeneration associated with decreased apoptosis in mice with partial HIF-1alpha deficiency. 17181398

    HIF-1alpha activates genes under hypoxia and was hypothesized to regulate bone regeneration. Surprisingly, HIF-1alpha+/- fracture calluses are larger, stronger, and stiffer than HIF-1alpha+/+ calluses because of decreased apoptosis. These data identify apoptosis inhibition as a means to enhance bone regeneration. INTRODUCTION: Bone regeneration subsequent to fracture involves the synergistic activation of multiple signaling pathways. Localized hypoxia after fracture activates hypoxia-inducible factor 1alpha (HIF-1alpha), leading to increased expression of HIF-1 target genes. We therefore hypothesized that HIF-1alpha is a key regulator of bone regeneration. MATERIALS AND METHODS: Fixed femoral fractures were generated in mice with partial HIF-1alpha deficiency (HIF-1alpha+/-) and wildtype littermates (HIF-1alpha+/+). Fracture calluses and intact contralateral femurs from postfracture days (PFDs) 21 and 28 (N=5-10) were subjected to microCT evaluation and four-point bending to assess morphometric and mechanical properties. Molecular analyses were carried out on PFD 7, 10, and 14 samples (N=3) to determine differential gene expression at both mRNA and protein levels. Finally, TUNEL staining was performed on PFD 14 samples (N=2) to elucidate differential apoptosis. RESULTS: Surprisingly, fracture calluses from HIF-1alpha+/- mice exhibited greater mineralization and were larger, stronger, and stiffer. Microarray analyses focused on hypoxia-induced genes revealed differential expression (between genotypes) of several genes associated with the apoptotic pathway. Real-time PCR confirmed these results, showing higher expression of proapoptotic protein phosphatase 2a (PP2A) and lower expression of anti-apoptotic B-cell leukemia/lymphoma 2 (BCL2) in HIF-1alpha+/+ calluses. Subsequent TUNEL staining showed that HIF-1alpha+/+ calluses contained larger numbers of TUNEL+ chondrocytes and osteoblasts than HIF-1alpha+/- calluses. CONCLUSIONS: We conclude that partial HIF-1alpha deficiency results in decreased chondrocytic and osteoblastic apoptosis, thereby allowing the development of larger, stiffer calluses and enhancing bone regeneration. Furthermore, apoptosis inhibition may be a promising target for developing new treatments to accelerate bone regeneration.
    Document Type:
    Reference
    Product Catalog Number:
    AP308P
    Product Catalog Name:
    Goat Anti-Mouse IgG Antibody, (H+L) HRP conjugate

  • Resveratrol helps recovery from fatty liver and protects against hepatocellular carcinoma induced by hepatitis B virus X protein in a mouse model. 22659145

    Resveratrol is a natural polyphenol that has beneficial effects across species and various disease models. Here, we investigate whether resveratrol is effective against hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) using HBV X protein (HBx) transgenic mice. We found that resveratrol (30 mg/kg/d) has a therapeutic effect on HBx-induced fatty liver and the early stages of liver damage. Resveratrol decreased intracellular reactive oxygen species and transiently stimulated hepatocyte proliferation. Interestingly, resveratrol inhibited LXRα and downregulated the expression of the lipogenic genes, Srebp1-c and PPARγ. The decrease in Srebp1-c seems to further downregulate the expression of its target genes, Acc and Fas. In addition, resveratrol stimulated the activity of Ampk and SirT1. Thus, resveratrol has a pleiotropic effect on HBx transgenic mice in terms of the downregulation of lipogenesis, the promotion of transient liver regeneration, and the stimulation of antioxidant activity. Furthermore, at the later precancerous stages, resveratrol delayed HBx-mediated hepatocarcinogenesis and reduced HCC incidence from 80% to 15%, a 5.3-fold reduction. Resveratrol should be considered as a potential chemopreventive agent for HBV-associated HCC.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • H2O2 production downstream of FLT3 is mediated by p22phox in the endoplasmic reticulum and is required for STAT5 signalling. 22807997

    The internal tandem duplication (ITD) of the juxtamembrane region of the FLT3 receptor has been associated with increased reactive oxygen species (ROS) generation in acute myeloid leukemia (AML). How this elevated level of ROS contributes to the leukemic phenotype, however, remains poorly understood. In this work we show that ROS in the FLT3-ITD expressing AML cell line MV4-11 is reduced by treatment with PKC412, an inhibitor of FLT3, DPI, a flavoprotein inhibitor, and VAS2870, a Nox specific inhibitor, suggesting that ROS production is both FLT3 and NADPH oxidase dependent. The majority of these ROS co-localize to the endoplasmic reticulum (ER), as determined with the H(2)O(2)-specific aryl-boronate dye Peroxyorange 1, which also corresponds to co-localization of p22phox. Moreover, knocking down p22phox dramatically reduces H(2)O(2) after 24 hours in the ER, without affecting mitochondrial ROS. Significantly, the FLT3 inhibitor PKC412 reduces H(2)O(2) in FLT3-ITD expressing cell lines (MV4-11, MOLM-13) through reduction of p22phox over 24 hours. Reduced p22phox is achieved by proteasomal degradation and is prevented upon GSK3-β inhibition. Knockdown of p22phox resulted in reduced STAT5 signalling and reduced Pim-1 levels in the cells after 24 hours. Thus, we have shown that FLT3 driven H(2)O(2) production in AML cells is mediated by p22phox and is critical for STAT5 signalling.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple