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  • Ubiquitination regulates PSD-95 degradation and AMPA receptor surface expression. 14642282

    PSD-95 is a major scaffolding protein of the postsynaptic density, tethering NMDA- and AMPA-type glutamate receptors to signaling proteins and the neuronal cytoskeleton. Here we show that PSD-95 is regulated by the ubiquitin-proteasome pathway. PSD-95 interacts with and is ubiquitinated by the E3 ligase Mdm2. In response to NMDA receptor activation, PSD-95 is ubiquitinated and rapidly removed from synaptic sites by proteasome-dependent degradation. Mutations that block PSD-95 ubiquitination prevent NMDA-induced AMPA receptor endocytosis. Likewise, proteasome inhibitors prevent NMDA-induced AMPA receptor internalization and synaptically induced long-term depression. This is consistent with the notion that PSD-95 levels are an important determinant of AMPA receptor number at the synapse. These data suggest that ubiquitination of PSD-95 through an Mdm2-mediated pathway is critical in regulating AMPA receptor surface expression during synaptic plasticity.
    Document Type:
    Reference
    Product Catalog Number:
    MAB397
    Product Catalog Name:
    Anti-Glutamate Receptor 2 Antibody, extracellular, clone 6C4
  • Ultrasound induces hypoxia-inducible factor-1 activation and inducible nitric-oxide synthase expression through the integrin/integrin-linked kinase/Akt/mammalian target o ... 17588951

    It has been shown that ultrasound (US) stimulation accelerates fracture healing in the animal models and clinical studies. Nitric oxide (NO) is a crucial early mediator in mechanically induced bone formation. Here we found that US stimulation increased NO formation and the protein level of inducible nitric-oxide synthase (iNOS). US-mediated iNOS expression was attenuated by anti-integrin alpha5beta1 or beta1 antibodies but not anti-integrin alphavbeta3 or beta3 antibodies or focal adhesion kinase mutant. Integrin-linked kinase (ILK) inhibitor (KP-392), Akt inhibitor (1L-6-hydroxymethyl-chiro-inositol-2-[(R)-2-O-methyl-3-O-octadecylcarbonate]) or mammalian target of rapamycin (mTOR) inhibitor (rapamycin) also inhibited the potentiating action of US. US stimulation increased the kinase activity of ILK and phosphorylation of Akt and mTOR. Furthermore, US stimulation also increased the stability and activity of HIF-1 protein. The binding of HIF-1alpha to the HRE elements on the iNOS promoter was enhanced by US stimulation. Moreover, the use of pharmacological inhibitors or genetic inhibition revealed that both ILK/Akt and mTOR signaling pathway were potentially required for US-induced HIF-1alpha activation and subsequent iNOS up-regulation. Taken together, our results provide evidence that US stimulation up-regulates iNOS expression in osteoblasts by an HIF-1alpha-dependent mechanism involving the activation of ILK/Akt and mTOR pathways via integrin receptor.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
  • Unique and highly selective anticytomegalovirus activities of artemisinin-derived dimer diphenyl phosphate stem from combination of dimer unit and a diphenyl phosphate mo ... 23774439

    We report that the artemisinin-derived dimer diphenyl phosphate (DPP; dimer 838) is the most selective inhibitor of human cytomegalovirus (CMV) replication among a series of artemisinin-derived monomers and dimers. Dimer 838 was also unique in being an irreversible CMV inhibitor. The peroxide unit within artemisinins' chemical structures is critical to their activities, and its absence results in loss of anti-CMV activities. Surprisingly, the deoxy dimer of 838 retained modest anti-CMV activity, suggesting that the DPP moiety of dimer 838 contributes to its anti-CMV activities. DPP alone did not inhibit CMV replication, but triphenyl phosphate (TPP) had modest CMV inhibition, although its selectivity index was low. Artemisinin DPP derivatives dimer 838 and monomer diphenyl phosphate (compound 558) showed stronger CMV inhibition and a higher selectivity index than their analogs lacking the DPP unit. An add-on and removal assay revealed that removing DPP derivatives (compounds 558 and 838) but not the non-DPP backbones (artesunate and compound 606) at 24 h postinfection (hpi) already resulted in dominant CMV inhibition. CMV inhibition was fully irreversible with 838 and partially irreversible with 558, while non-DPP artemisinins were reversible inhibitors. While all artemisinin derivatives and TPP reduced the expression of the CMV immediate early 2 (IE2), UL44, and pp65 proteins at or after 48 hpi, only TPP inhibited the expression of both IE1 and IE2. Combination of a non-DPP dimer (compound 606) with TPP was synergistic in CMV inhibition, while ganciclovir and TPP were additive. Although TPP shared structural similarity with monomer DPP (compound 558) and dimer DPP (compound 838), its pattern of CMV inhibition was significantly different from the patterns of the artemisinins. These findings demonstrate that the DPP group contributes to the unique activities of compound 838.
    Document Type:
    Reference
    Product Catalog Number:
    MAB810
  • Upregulation of acid-sensing ion channel ASIC1a in spinal dorsal horn neurons contributes to inflammatory pain hypersensitivity. 17928456

    Development of chronic pain involves alterations in peripheral nociceptors as well as elevated neuronal activity in multiple regions of the CNS. Previous pharmacological and behavioral studies suggest that peripheral acid-sensing ion channels (ASICs) contribute to pain sensation, and the expression of ASIC subunits is elevated in the rat spinal dorsal horn (SDH) in an inflammatory pain model. However, the cellular distribution and the functional consequence of increased ASIC subunit expression in the SDH remain unclear. Here, we identify the Ca2+-permeable, homomeric ASIC1a channels as the predominant ASICs in rat SDH neurons and downregulation of ASIC1a by local rat spinal infusion with specific inhibitors or antisense oligonucleotides markedly attenuated complete Freund's adjuvant (CFA)-induced thermal and mechanical hypersensitivity. Moreover, in vivo electrophysiological recording showed that the elevated ASIC1a activity is required for two forms of central sensitization: C-fiber-induced wind-up and CFA-induced hypersensitivity of SDH nociceptive neurons. Together, our results reveal that increased ASIC activity in SDH neurons promotes pain by central sensitization. Specific blockade of Ca2+-permeable ASIC1a channels thus may have antinociceptive effect by reducing or preventing the development of central sensitization induced by inflammation.
    Document Type:
    Reference
    Product Catalog Number:
    3301
    Product Catalog Name:
    LIGHT DIAGNOSTICS™ Coxsackievirus A9 Reagent, ~25 tests, included in kit #3350
  • Upregulation of AKT1 protein expression in forskolin-stimulated macrophage: evidence from ChIP analysis that CREB binds to and activates the AKT1 promoter. 17152074

    Recently, we reported that silencing CREB gene expression by RNAi significantly attenuates forskolin-induced activation of Akt1. We now provide evidence that forskolin-treatment causes transcriptional and translational upregulation of Akt1 in macrophages. Akt synthesis was demonstrated by [(14)C]leucine or [(35)S] incorporation into newly synthesized Akt1 protein. Akt protein levels increased by approximately 1.5-fold after only a 5 min exposure of macrophages to forskolin. Akt1 levels thereafter rapidly returned to basal values (t(1/2) approximately 15 min). Maximal upregulation of Akt1 occurred in cells treated with 10 microM forskolin. Forskolin-dependent Akt1 synthesis was abolished by pretreating the cells with CREB-directed dsRNA as demonstrated at both the message and protein level, as well as by determining the synthesis of [(35)S]-labeled Akt1 protein. The PKA inhibitor H-89, greatly attenuated forskolin-induced Akt1 synthesis. Transcriptional and translational inhibitors also greatly reduced Akt1 synthesis in forskolin-stimulated [(14)C]leucine-labeled macrophages. Using a chromatin immunoprecipitation assay, we demonstrate that CREB binds to a CRE binding domain of the Akt1 gene promoter. In conclusion, we show here for the first time transcriptional upregulation of Akt1 by CREB, based upon Akt1 protein synthesis and its modulation by transitional and translational inhibitors in forskolin-stimulated cells, Akt1 protein. and mRNA levels upon silencing CREB gene expression, and binding of CREB to the Akt1 gene promoter.
    Document Type:
    Reference
    Product Catalog Number:
    17-295
    Product Catalog Name:
    Chromatin Immunoprecipitation (ChIP) Assay Kit
  • Upregulation of CREB-mediated transcription enhances both short- and long-term memory. 21677163

    Unraveling the mechanisms by which the molecular manipulation of genes of interest enhances cognitive function is important to establish genetic therapies for cognitive disorders. Although CREB is thought to positively regulate formation of long-term memory (LTM), gain-of-function effects of CREB remain poorly understood, especially at the behavioral level. To address this, we generated four lines of transgenic mice expressing dominant active CREB mutants (CREB-Y134F or CREB-DIEDML) in the forebrain that exhibited moderate upregulation of CREB activity. These transgenic lines improved not only LTM but also long-lasting long-term potentiation in the CA1 area in the hippocampus. However, we also observed enhanced short-term memory (STM) in contextual fear-conditioning and social recognition tasks. Enhanced LTM and STM could be dissociated behaviorally in these four lines of transgenic mice, suggesting that the underlying mechanism for enhanced STM and LTM are distinct. LTM enhancement seems to be attributable to the improvement of memory consolidation by the upregulation of CREB transcriptional activity, whereas higher basal levels of BDNF, a CREB target gene, predicted enhanced shorter-term memory. The importance of BDNF in STM was verified by microinfusing BDNF or BDNF inhibitors into the hippocampus of wild-type or transgenic mice. Additionally, increasing BDNF further enhanced LTM in one of the lines of transgenic mice that displayed a normal BDNF level but enhanced LTM, suggesting that upregulation of BDNF and CREB activity cooperatively enhances LTM formation. Our findings suggest that CREB positively regulates memory consolidation and affects memory performance by regulating BDNF expression.
    Document Type:
    Reference
    Product Catalog Number:
    AB1513P
    Product Catalog Name:
    Anti-Brain Derived Neurotrophic Factor Antibody
  • Upregulation of p21 activates the intrinsic apoptotic pathway in β-cells. 23592481

    Diabetes manifests from a loss in functional β-cell mass, which is regulated by a dynamic balance of various cellular processes, including β-cell growth, proliferation, and death as well as secretory function. The cell cycle machinery comprised of cyclins, kinases, and inhibitors regulates proliferation. However, their involvement during β-cell stress during the development of diabetes is not well understood. Interestingly, in a screen of multiple cell cycle inhibitors, p21 was dramatically upregulated in INS-1-derived 832/13 cells and rodent islets by two pharmacological inducers of β-cell stress, dexamethasone and thapsigargin. We hypothesized that β-cell stress upregulates p21 to activate the apoptotic pathway and suppress cell survival signaling. To this end, p21 was adenovirally overexpressed in pancreatic rat islets and 832/13 cells. As expected, p21 overexpression resulted in decreased [(3)H]thymidine incorporation. Flow cytometry analysis in p21-transduced 832/13 cells verified lower replication, as indicated by a decreased cell population in the S phase and a block in G2/M transition. The sub-G0 cell population was higher with p21 overexpression and was attributable to apoptosis, as demonstrated by increased annexin-positive stained cells and cleaved caspase-3 protein. p21-mediated caspase-3 cleavage was inhibited by either overexpression of the antiapoptotic mitochondrial protein Bcl-2 or siRNA-mediated suppression of the proapoptotic proteins Bax and Bak. Therefore, an intact intrinsic apoptotic pathway is central for p21-mediated cell death. In summary, our findings indicate that β-cell apoptosis can be triggered by p21 during stress and is thus a potential target to inhibit for protection of functional β-cell mass.
    Document Type:
    Reference
    Product Catalog Number:
    06-536
    Product Catalog Name:
    Anti-Bak Antibody, NT
  • Upregulation of SOCS-1 by Nutlin-3 in acute myeloid leukemia cells but not in primary normal cells. 24473562

    It has been shown that SOCS-1 plays an important role in the proper control of cytokine/growth factor responses and acts as a tumor suppressor in acute myeloid leukemias. Therefore, the objective of the present study was to evaluate the in vitro effect of treatment with Nutlin-3, a small molecule inhibitor of the MDM2/p53 interaction, on the expression of the suppressor of cytokine signaling 1 in primary acute myeloid leukemia cells and in myeloid cell lines with differential p53 status.The expression of the suppressor of cytokine signaling 1 was quantitatively analyzed by real-time PCR in myeloid p53wild-type (OCI and MOLM) and p53null HL-60, leukemic cell lines, in patient-derived acute myeloid leukemia blasts, and in primary normal cell types, such as macrophages, endothelial cells, and bone marrow mesenchymal stem cells. The p53-dependence of the suppressor of cytokine signaling 1 upregulation that is induced by Nutlin-3 was analyzed in experiments performed using siRNA for p53, while the functional upregulation of the suppressor of cytokine signaling 1 was analyzed by assessing the levels of phosphorylated STAT-3.Nutlin-3 significantly upregulated the transcription of the suppressor of cytokine signaling 1 in p53wild-type OCI and MOLM but not in p53deleted p53null HL60, myeloid leukemic cell lines, as well as in primary acute myeloid leukemia blasts. Conversely, and somewhat unexpectedly, Nutlin-3 did not modulate the suppressor of cytokine signaling 1 expression in primary normal macrophages, endothelial cells, and bone marrow mesenchymal stem cells. The p53-dependent upregulation of the suppressor of cytokine signaling 1 by Nutlin-3 was associated with the downregulation of phosphorylated STAT-3, a major molecular target of the suppressor of cytokine signaling 1.Overall, our data suggest a potential role for the suppressor of cytokine signaling 1 as a therapeutic target of Nutlin-3 in p53 wild-type acute myeloid leukemias.
    Document Type:
    Reference
    Product Catalog Number:
    48-680MAG
    Product Catalog Name:
    MILLIPLEX Multi-Pathway Magnetic Bead 9-Plex - Cell Signaling Multiplex Assay
  • Uptake of the dopaminergic neurotoxin, norsalsolinol, into PC12 cells via dopamine transporter. 11482518

    The uptake of norsalsolinol, a neurotoxin candidate causing parkinsonism-like symptoms, was studied in PC12 cells. The compound was actively taken up by the PC12 cells, with a Km value of 176.24 +/-9.1 microM and a maximum velocity of 55.6 +/- 7.0 pmol/min per mg protein; norsalsolinol uptake was dependent on the presence of extracellular Na+. The uptake of norsalsolinol was sensitive to two dopamine transporter inhibitors, GBR-12909 and reserpine, but was less sensitive to desipramine, a noradrenaline transporter inhibitor. Dopamine competitively inhibited norsalsolinol uptake into PC12 cells with a Ki value of 271.2 +/- 61.6 microM. These results suggest that norsalsolinol is taken up into PC12 cells mainly by the dopamine transporter.
    Document Type:
    Reference
    Product Catalog Number:
    01-125
    Product Catalog Name:
    NGF 2.5S Protein, mouse
  • Urinary matrix metalloproteinases and their endogenous inhibitors predict hepatic regeneration after murine partial hepatectomy. 15502710

    BACKGROUND: Matrix metalloproteinases (MMPs) play a key role in extracellular matrix remodeling events associated with hepatic regeneration after partial hepatectomy. We therefore hypothesized that urinary MMPs and their endogenous tissue inhibitors of matrix metalloproteinases (TIMPs) might also provide important information regarding initiation and progression of liver regeneration. METHODS: Groups of 20 mice underwent sham operations, two-thirds hepatectomy, or treatment with the angiogenesis inhibitor, AGM-1470,O-chloroacetyl-carbamoyl-fumagillol (TNP-470), after two-thirds hepatectomy to prevent hepatic regeneration. Urine was collected preoperatively and for 24 days after surgery and tested for MMP-2, MMP-9, TIMP-1, and TIMP-2 using substrate gel electrophoresis (zymography) and Western blot analysis. RESULTS: During hepatic regeneration, MMP-9 was detected in the urine at significantly lower levels on postoperative day 8 when the liver returned to its preoperative mass. In contrast, urine from mice whose livers were inhibited from regenerating (TNP-treated groups) contained increased levels of the gelatinases MMP-2 and MMP-9. The MMP inhibitors, TIMP-1 and TIMP-2, were significantly reduced in the urine of mice with normally regenerating livers but were increased in the urine of mice treated with TNP-470 on day 8. CONCLUSIONS: We demonstrate that (1) urinary MMPs and their cognate inhibitors, the TIMPs, can be detected in the urine of mice undergoing partial hepatectomy, (2) the presence of these remodeling proteins in the urine may predict the progressive return of the partially resected liver to its preoperative mass, and (3) analysis of urinary MMPs and TIMPs may someday provide a noninvasive means of monitoring the status of patients undergoing hepatic resection and transplantation.
    Document Type:
    Reference
    Product Catalog Number:
    MAB13405
    Product Catalog Name:
    Anti-MMP-2 Proform Antibody, NT, clone CA-4001