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  • DHT selectively reverses Smad3-mediated/TGF-beta-induced responses through transcriptional down-regulation of Smad3 in prostate epithelial cells. 20739403

    Androgens suppress TGF-β responses in the prostate through mechanisms that are not fully explored. We have recently reported that 5α-dihydrotestosterone (DHT) suppresses the ability of TGF-β to inhibit proliferation and induce apoptosis of prostatic epithelial cells and provided evidence that such suppression was fueled by transcriptional down-regulation of TGF-β receptor II (ΤβRII). We now show that androgen receptor (AR) activated by DHT suppresses the TGF-β-induced phosphorylation of Sma- and Mad-related protein (Smad)3 in LNCaP cells overexpressing TβRII under the control of a cytomegalovirus promoter, which is not regulated by DHT, suggesting that transcriptional repression of TβRII alone does not fully account for the impact of DHT on TGF-β responses. Instead, we demonstrate that such suppression occurs through loss of total Smad3, resulting from transcriptional suppression of Smad3. We provide evidence that DHT down-regulates the promoter activity of Smad3 in various prostate cancer cell lines, including NRP-154+AR, DU145+AR, LNCaP, and VCaP, at least partly through androgen-dependent inactivation of Sp1. Moreover, we show that overexpression of Smad3 reverses the ability of DHT to protect against TGF-β-induced apoptosis in NRP-154+AR, supporting our model that loss of Smad3 by DHT is involved in the protection against TGF-β-induced apoptosis. Together, these findings suggest that deregulated/enhanced expression and activation of AR in prostate carcinomas may intercept the tumor suppressor function of TGF-β through transcriptional suppression of Smad3, thereby providing new mechanistic insight into the development of castration-resistant prostate cancer.
    Document Type:
    Reference
    Product Catalog Number:
    07-645
    Product Catalog Name:
    Anti-Sp1 Antibody

  • Short and long term fate of human AMSC subcutaneously injected in mice. 21860670

    To study the ability of human adipose-derived mesenchymal stem cells (AMSCs) to survive over the short and long term, their biodistribution and their biosafety in vivo in tumor-prone environments.We subcutaneously injected human AMSCs from different human donors into immunodeficient SCID mice over both short- (2 and 4 mo) and long- (17 mo) term in young, and aged tumor-prone mice. Presence of human cells was studied by immunohistochemistry and polymerase chain reaction analysis in all organs of injected mice.Subcutaneously injected AMSCs did not form teratomas at any time point. They did not migrate but remained at the site of injection regardless of animal age, and did not fuse with host cells in any organ examined. AMSCs survived in vivo for at least 17 mo after injection, and differentiated into fibroblasts of the subdermic connective tissue and into mature adipocytes of fat tissue, exclusively at the site of injection.Our results support the assertion that AMSC may be safe candidates for therapy when injected subcutaneously because of their long term inability to form teratomas.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Light-activated nanotube-porphyrin conjugates as effective antiviral agents. 22361811

    Porphyrins have been used for photodynamic therapy (PDT) against a wide range of targets like bacteria, viruses and tumor cells. In this work, we report porphyrin-conjugated multi-walled carbon nanotubes (NT-P) as potent antiviral agents. Specifically, we used Protoporphyrin IX (PPIX), which we attached to acid-functionalized multi-walled carbon nanotubes (MWNTs). We decided to use carbon nanotubes as scaffolds because of their ease of recovery from a solution through filtration. In the presence of visible light, NT-P was found to significantly reduce the ability of Influenza A virus to infect mammalian cells. NT-P may be used effectively against influenza viruses with little or no chance of them developing resistance to the treatment. Furthermore, NT-P can be easily recovered through filtration which offers a facile strategy to reuse the active porphyrin moiety to its fullest extent. Thus NT-P conjugates represent a new approach for preparing ex vivo reusable antiviral agents.
    Document Type:
    Reference
    Product Catalog Number:
    MAB8800

  • Transduction of brain dopamine neurons by adenoviral vectors is modulated by CAR expression: rationale for tropism modified vectors in PD gene therapy. 20862245

    Gene-based therapy is a new paradigm for the treatment of Parkinson disease (PD) and offers considerable promise for precise targeting and flexibility to impact multiple pathobiological processes for which small molecule agents are not available. Some success has been achieved utilizing adeno-associated virus for this approach, but it is likely that the characteristics of this vector system will ultimately create barriers to progress in clinical therapy. Adenovirus (Ad) vector overcomes limitations in payload size and targeting. The cellular tropism of Ad serotype 5 (Ad5)-based vectors is regulated by the Ad attachment protein binding to its primary cellular receptor, the coxsackie and adenovirus receptor (CAR). Many clinically relevant tissues are refractory to Ad5 infection due to negligible CAR levels but can be targeted by tropism-modified, CAR-independent forms of Ad. Our objective was to evaluate the role of CAR protein in transduction of dopamine (DA) neurons in vivo.Ad5 was delivered to the substantia nigra (SN) in wild type (wt) and CAR transgenic animals. Cellular tropism was assessed by immunohistochemistry (IHC) in the SN and striatal terminals. CAR expression was assessed by western blot and IHC. We found in wt animals, Ad5 results in robust transgene expression in astrocytes and other non-neuronal cells but poor infection of DA neurons. In contrast, in transgenic animals, Ad5 infects SNc neurons resulting in expression of transduced protein in their striatal terminals. Western blot showed low CAR expression in the ventral midbrain of wt animals compared to transgenic animals. Interestingly, hCAR protein localizes with markers of post-synaptic structures, suggesting synapses are the point of entry into dopaminergic neurons in transgenic animals.These findings demonstrate that CAR deficiency limits infection of wild type DA neurons by Ad5 and provide a rationale for the development of tropism-modified, CAR-independent Ad-vectors for use in gene therapy of human PD.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • European journal of immunology 12209638

    CD223 (LAG-3) is an activation-induced cell surface molecule, structurally similar to the T cell coreceptor CD4, that binds MHC class II molecules with high affinity. Little is known about the expression and function of murine CD223. Here, we show that mRNA expression is restricted to the thymic medulla, splenic red pulp and sparse cells in the adult brain cortex. In contrast, surprisingly high expression was seen in defined tracts at the base of the cerebellum and in the choroid plexus of day 7 postnatal brain. mCD223:Ig, but not CD4:Ig, fusion proteins stained cells expressing MHC class II molecules. Analysis of mCD223 cell surface expression was performed with a new monoclonal antibody (mAb) that recognizes an epitope in the D2 domain. Although it blocked mCD223 function in vitro, it did not block binding of mCD223 to MHC class II molecules. While very few TCRalpha beta T cells in the spleen and thymus of naive mice express surface mCD223 (<3 %), approximately 18 % TCR gamma delta T cells and approximately 10 % NK cells are positive. This small population of TCRalpha beta T cells are cycling memory T cells (BrdU(+), CD44(hi), CD62L(lo)). In contrast, all T cells express mCD223 2-3 days post activation. This study and the anti-CD223 mAb should greatly assist in the elucidation of CD223 function.
    Document Type:
    Reference
    Product Catalog Number:
    MABF846
    Product Catalog Name:
    Anti-LAG3 Antibody, clone C9B7W (Azide Free)

  • Ethanol alters endosomal recycling of human dopamine transporters. 20133946

    Dynamic membrane trafficking of the monoamine dopamine transporter (DAT) regulates dopaminergic signaling. Various intrinsic and pharmacological modulators can alter this trafficking. Previously we have shown ethanol potentiates in vitro DAT function and increases surface expression. However, the mechanism underlying these changes is unclear. In the present study, we found ethanol directly regulates DAT function by altering endosomal recycling of the transporter. We defined ethanol action on transporter regulation by [(3)H]DA uptake functional analysis combined with biochemical and immunological assays in stably expressing DAT HEK-293 cells. Short-term ethanol exposure potentiated DAT function in a concentration-, but not time-dependent manner. This potentiation was accompanied by a parallel increase in DAT surface expression. Ethanol had no effect on function or surface localization of the ethanol-insensitive mutant (G130T DAT), suggesting a trafficking-dependent mechanism in mediating the ethanol sensitivity of the transporter. The ethanol-induced increase in DAT surface expression occurred without altering the overall size of DAT endosomal recycling pools. We found ethanol increased the DAT membrane insertion rate while having no effect on internalization of the transporter. Ethanol had no effect on the surface expression or trafficking of the endogenously expressing transferrin receptor, suggesting ethanol does not have a nonspecific effect on endosomal recycling. These results define a novel trafficking mechanism by which ethanol regulates DAT function.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Hypoxia-induced inhibition of lung development is attenuated by the peroxisome proliferator-activated receptor-γ agonist rosiglitazone. 21531777

    Hypoxia enhances transforming growth factor-β (TGF-β) signaling, inhibiting alveolar development and causing abnormal pulmonary arterial remodeling in the newborn lung. We hypothesized that, during chronic hypoxia, reduced peroxisome proliferator-activated receptor-γ (PPAR-γ) signaling may contribute to, or be caused by, excessive TGF-β signaling. To determine whether PPAR-γ was reduced during hypoxia, C57BL/6 mice were exposed to hypoxia from birth to 2 wk and evaluated for PPAR-γ mRNA and protein. To determine whether rosiglitazone (RGZ, a PPAR-γ agonist) supplementation attenuated the effects of hypoxia, mice were exposed to air or hypoxia from birth to 2 wk in combination with either RGZ or vehicle, and measurements of lung histology, function, parameters related to TGF-β signaling, and collagen content were made. To determine whether excessive TGF-β signaling reduced PPAR-γ, mice were exposed to air or hypoxia from birth to 2 wk in combination with either TGF-β-neutralizing antibody or vehicle, and PPAR-γ signaling was evaluated. We observed that hypoxia reduced PPAR-γ mRNA and protein, in association with impaired alveolarization, increased TGF-β signaling, reduced lung compliance, and increased collagen. RGZ increased PPAR-γ signaling, with improved lung development and compliance in association with reduced collagen and TGF-β signaling. However, no reduction was noted in hypoxia-induced pulmonary vascular remodeling. Inhibition of hypoxia-enhanced TGF-β signaling increased PPAR-γ signaling. These results suggest that hypoxia-induced inhibition of lung development is associated with a mutually antagonistic relationship between reduced PPAR-γ and increased TGF-β signaling. PPAR-γ agonists may be of potential therapeutic significance in attenuating TGF-β signaling and improving alveolar development.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Cloning and characterization of the murine Nek3 protein kinase, a novel member of the NIMA family of putative cell cycle regulators. 10224116

    We have cloned and characterized murine Nek3 (NIMA-related kinase 3), a novel mammalian gene product structurally related to the cell cycle-regulatory kinase NIMA of Aspergillus nidulans. By RNase protection, low levels of Nek3 expression could be detected in all organs examined, regardless of proliferative index. In contrast to Nek1 and Nek2, Nek3 levels were not particularly elevated in either the male or the female germ line. Nek3 levels showed at most marginal variations through the cell cycle, but they were elevated in G0-arrested, quiescent fibroblasts. Furthermore, no cell cycle-dependent changes in Nek3 activity could be detected, and no effects upon cell cycle progression could be observed upon antibody microinjection or overexpression of either wild-type or catalytically inactive Nek3. Finally, Nek3 was found to be a predominantly cytoplasmic enzyme. These data indicate that Nek3 differs from previously characterized Neks with regard to all parameters investigated, including organ specificity of expression, cell cycle dependence of expression and activity, and subcellular localization. Hence, the structural similarity between mammalian Neks may not necessarily be indicative of a common function, and it is possible that some members of this kinase family may perform functions that are not directly related to cell cycle control.
    Document Type:
    Reference
    Product Catalog Number:
    14-694
    Product Catalog Name:
    NEK3 Protein, active, 10 µg

  • Variable RXR requirements for thyroid hormone responsiveness of endogenous genes. 17161906

    Thyroid hormone receptors heterodimerize with retinoid X receptors in vitro and it is widely assumed that these heterodimers mediate the T3 induction of target genes. However, the importance of RXR for the T3 induction of endogenous genes has not been assessed. We used cDNA microarrays to identify 54 genes induced by T3 in Neuro2a cells that express thyroid hormone receptor beta. RNA interference-mediated knock down of endogenous RXRs showed that these genes vary from being highly dependent on RXR for T3 induction to being independent of RXR. Thus, the availability of RXR may differentially regulate the T3 induction of subsets of genes within a cell. Furthermore, coregulatory proteins that preferentially interact with TR homodimers or RXR-TR heterodimers may further expand the range of T3 response for genes within the same cell.
    Document Type:
    Reference
    Product Catalog Number:
    06-527

  • Activation of group II metabotropic glutamate receptors reduces directional selectivity in retinal ganglion cells. 17010323

    In the mammalian retina, high levels of the group II metabotropic glutamate receptor (mGluR) subtype are expressed in starburst amacrine cells. A prominent role of starburst amacrine cells is the generation of directional selectivity in ON-OFF directionally selective retinal ganglion cells (DS RGCs). Extracellular microelectrodes were used to study the effects of activation of group II mGluRs on the responses of rabbit ON-OFF DS RGCs to a moving light stimulus. Directionally selective responses in these RGCs were substantially reduced by the selective group II mGluR agonist DCG-IV. DCG-IV brought out a response to movement in the null direction that was similar in magnitude and time course to the response to movement in the preferred direction. This effect of DCG-IV was reversed by the group II mGluR antagonist EGLU. Application of EGLU alone failed to alter directional selectivity in the RGCs but did reduce the response to movement in the preferred direction. To determine whether group II mGluRs modulate the release of the neurotransmitter acetylcholine from starburst amacrine cells, the effect of DCG-IV on ON-OFF DS RGCs was examined in the presence of ambenonium, an acetylcholinesterase inhibitor. When applied alone, ambenonium greatly prolonged the responses of ON-OFF DS RGCs to a light stimulus. This effect of ambenonium was completely abolished upon application of DCG-IV. Overall, the results suggest that postsynaptic group II mGluRs have the potential to influence directional selectivity in RGCs by inhibiting transmitter release from starburst amacrine cells.
    Document Type:
    Reference
    Product Catalog Number:
    AB1553
    Product Catalog Name:
    Anti-Metabotropic Glutamate Receptor 2/3 Antibody