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  • Identification of the cannabinoid receptor type 1 in serotonergic cells of raphe nuclei in mice. 17383106

    The endocannabinoid system (ECS) possesses neuromodulatory functions by influencing the release of various neurotransmitters, including GABA, noradrenaline, dopamine, glutamate and acetylcholine. Even though there are studies indicating similar interactions between the ECS and the serotonergic system, there are no results showing clear evidence for type 1 cannabinoid receptor (CB1) location on serotonergic neurons. In this study, we show by in situ hybridization that a low but significant fraction of serotonergic neurons in the raphe nuclei of mice contains CB1 mRNA as illustrated by the coexpression with the serotonergic marker gene tryptophane hydroxylase 2, the rate limiting enzyme for the serotonin synthesis. Furthermore, by double immunohistochemistry and confocal microscopy, we were able to detect CB1 protein on serotonergic fibers and synapses expressing the serotonin uptake transporter in the hippocampus and the amygdala. Our findings indicate that the CB1-mediated regulation of serotonin release can depend in part on a direct cross-talk between the two systems at single cell level, which might lead to functional implications in the modulation of emotional states.
    Document Type:
    Reference
    Product Catalog Number:
    AB1772
    Product Catalog Name:

  • In situ adenovirus vaccination engages T effector cells against cancer. 19481312

    The efficacy of cancer immunotherapy is limited because of central and peripheral immune tolerance towards tumor-antigens. We propose a novel approach based on the fact that the immune system has not evolved tolerance towards adenoviruses (Ads) and that Ads have not evolved efficient mechanisms for immune-escape. The host-response to intratumoral Ad-vector injection in mice that were immunologically tolerant to neu-positive syngeneic mammary-cancer (MMC) was investigated. Intratumoral injection with replication-deficient, transgene-devoid Ad induced immune responses at two different anatomical sites: the tumor-draining lymph nodes and the tumor microenvironment. The lymph nodes supported the generation of both neu- and Ad-specific T effector cells, while inside the tumor microenvironment only Ad-specific T cells expanded. Importantly, Ad-specific T cells were anti-tumor-reactive despite the presence of active regulatory T cell-mediated immune tolerance inside MMC tumors and anti-tumor efficacy of Ad was increased by pre-immunization against Ad despite the production of Ad-neutralizing antibodies.
    Document Type:
    Reference
    Product Catalog Number:
    AB1056F
    Product Catalog Name:
    Anti-Adenovirus Antibody, FITC-conjugated

  • Visualization of dopamine transporter trafficking in live neurons by use of fluorescent cocaine analogs. 19474307

    The dopamine transporter (DAT) mediates reuptake of dopamine from the synaptic cleft and is a target for widely abused psychostimulants such as cocaine and amphetamine. Nonetheless, little is known about the cellular distribution and trafficking of natively expressed DAT. Here we use novel fluorescently tagged cocaine analogs to visualize DAT and DAT trafficking in cultured live midbrain dopaminergic neurons. The fluorescent tags were extended from the tropane N-position of 2beta-carbomethoxy-3beta-(3,4-dichlorophenyl)tropane using an ethylamino-linker. The rhodamine-, OR Green-, or Cy3-labeled ligands had high binding affinity for DAT and enabled specific labeling of DAT in live neurons and visualization by confocal imaging. In the dopaminergic neurons, DAT was uniformly distributed in the plasma membrane of the soma, the neuronal extensions, and varicosities along these extensions. FRAP (fluorescence recovery after photobleaching) experiments demonstrated bidirectional movement of DAT in the extensions and indicated that DAT is highly mobile both in the extensions and in the varicosities (immobile fraction less than approximately 30%). DAT was constitutively internalized into vesicular structures likely representing intracellular transporter pools. The internalization was blocked by lentiviral-mediated expression of dominant-negative dynamin and internalized DAT displayed partial colocalization with the early endosomal marker EGFP-Rab5 and with the transferrin receptor. DAT internalization and function was not affected by activation of protein kinase C (PKC) with phorbol-12-myristate-13-acetate (PMA) or by inhibition with staurosporine or GF109203X. These data are in contrast to findings for DAT in transfected heterologous cells and challenge the paradigm that trafficking and cellular distribution of endogenous DAT is subject to regulation by PKC.
    Document Type:
    Reference
    Product Catalog Number:
    MAB369
    Product Catalog Name:
    Anti-Dopamine Transporter Antibody, NT, clone DAT-Nt

  • Glutamatergic neuronal populations in the forebrain of the sea lamprey, petromyzon Marinus: An in situ hybridization and immunocytochemical study. 21452205

    Despite the importance of glutamate as a major excitatory neurotransmitter in the brain, the distribution of glutamatergic populations in the brain of most vertebrates is still unknown. Here, we studied for the first time the distribution of glutamatergic neurons in the forebrain of the sea lamprey (Petromyzon marinus), belonging to the most ancient group of vertebrates (agnathans). For this, we used in situ hybridization with probes for a lamprey vesicular glutamate transporter (VGLUT) in larvae and immunofluorescence with antiglutamate antibodies in both larvae and adults. We also compared glutamate and γ-aminobutyric acid (GABA) immunoreactivities in sections using double-immunofluorescence methods. VGLUT-expressing neurons were observed in the olfactory bulb, pallium, septum, subhippocampal lobe, preoptic region, thalamic eminence, prethalamus, thalamus, epithalamus, pretectum, hypothalamus, posterior tubercle, and nucleus of the medial longitudinal fascicle. Comparison of VGLUT signal and glutamate immunoreactivity in larval forebrain revealed a consistent distribution of positive cells, which were numerous in most regions. Glutamate-immunoreactive cell populations were also found in similar regions of the adult forebrain. These include mitral-like cells of the olfactory bulbs and abundant cells in the lateral pallium, septum, and various diencephalic regions, mainly in the prethalamus, thalamus, habenula, pineal complex, and pretectum. Only a small portion of the glutamate-immunoreactive cells showed colocalization with GABA, which was observed mainly in the olfactory bulb, telencephalon, hypothalamus, ventral thalamus, and pretectum. Comparison with glutamatergic cells observed in rodent forebrains suggests that the regional distribution of glutamatergic cells does not differ greatly in lampreys and mammals.Copyright © 2011 Wiley-Liss, Inc.
    Document Type:
    Reference
    Product Catalog Number:
    AP132C
    Product Catalog Name:
    Goat Anti-Rabbit IgG Antibody, Cy3 conjugate

  • Expression of epithelial cadherin in the human male reproductive tract and gametes and evidence of its participation in fertilization. 18829448

    Epithelial cadherin (E-cadherin) has been involved in several calcium-dependent cell-cell adhesion events; however, its participation in gamete interaction has not been fully investigated. Our results have demonstrated expression of E-cadherin mRNA in the human male reproductive tract showing higher levels in the caput, corpus and cauda epididymis than in the testis. The mature 122 kDa E-cadherin was detected in epididymal protein extracts and was localized in the epithelial cells from the three epididymal regions. Moreover, the 86 kDa E-cadherin ectodomain was found in cauda epididymal and seminal plasma. Western immunoblotting of human sperm protein extracts allowed the identification of four E-cadherin forms (122, 105, 97 and 86 kDa). The protein was localized in the acrosomal region of intact spermatozoa, remained associated with the head of acrosome-reacted cells and was also detected on the oocyte surface. A similar localization was determined for other proteins of the adhesion complex (beta-catenin and actin). Spermatozoa incubated with anti-E-cadherin antibodies showed impaired binding to homologous zona pellucida (ZP); in addition, presence of these antibodies inhibited the penetration of human spermatozoa to ZP-free hamster oocytes. The results presented here describe the expression of E-cadherin in the male reproductive tract and gametes and strongly suggest its involvement in adhesion events during human fertilization. The identification of proteins involved in gamete interaction will contribute to the understanding of the molecular basis of fertilization and help in the diagnosis and treatment of infertility.
    Document Type:
    Reference
    Product Catalog Number:
    AB19022

  • Expression and localization of mesothelin in developing rat pancreas. 18505465

    To define a genetic network that regulates development of the pancreas, we used high-density microarray (Affymetrix) to generate transcriptional profiles of rat pancreas from five biologically significant stages of development: embryonic day 12.5 (E12.5), E15.5, E18.5, postnatal day 0 (P0) and adult. Many genes were notably highly expressed in the later gestation when islet architecture and function are gradually forming. The expression and localization of mesothelin, one of these genes, was further examined. Reverse transcription-polymerase chain reaction and Western blot analysis revealed that mRNA and protein levels of mesothelin were high from later gestation to 2-3 weeks after birth, and with relatively low but detectable expression levels in adult rat pancreas. Immunolocalization indicated that mesothelin localized not only in islet beta-cells but also in the mesenchyme of developing rat pancreas. Transient mesothelin expression was concomitant with the development of islets architecture formation, remodeling and maturation. These findings indicate that mesothelin is dynamically expressed in the developing rat pancreas and that mesothelin might be involved in some developmental events during development of rat pancreas.
    Document Type:
    Reference
    Product Catalog Number:
    CBL202
    Product Catalog Name:
    Anti-Vimentin Antibody, clone VIM 3B4

  • Phosphorylated Smad2/3 immunoreactivity in sporadic and familial amyotrophic lateral sclerosis and its mouse model. 18210139

    Phosphorylated Smad2/3 (pSmad2/3), the central mediators of transforming growth factor (TGF)-beta signaling, were recently identified in tau-positive inclusions in certain neurodegenerative disorders. To clarify whether the localization of pSmad2/3 is altered in amyotrophic lateral sclerosis (ALS), we immunohistochemically examined spinal cords from sporadic ALS (SALS), from familial ALS (FALS) patients with the A4V mutation in their Cu/Zn superoxide dismutase (SOD1) gene, and from G93A mutant SOD1 transgenic (mSOD1 Tg) mice. In control spinal cords, pSmad2/3 immunoreactivity was observed exclusively in neuronal and glial nuclei. In SALS and FALS patients the nuclei showed increased immunoreactivity for pSmad2/3. Noticeably, round hyaline inclusions (RHIs) and skein-like inclusions of SALS patients were immunoreactive for pSmad2/3. Double immunofluorescence staining for pSmad2/3 and transactive response-DNA-binding protein (TDP)-43 revealed co-localization of these proteins within RHIs. In contrast, Bunina bodies in SALS and Lewy body-like hyaline inclusions (LBHIs) in FALS were devoid of labeling for pSmad2/3. Similarly, in the mSOD1 Tg mice pSmad2/3 immunoreactivity was increased in the nuclei, while LBHIs were not labeled. These findings suggest increased TGF-beta-Smad signaling in SALS, FALS, and mSOD1 Tg mice, as well as impaired TGF-beta signal transduction in RHI-bearing neurons of SALS patients, presumably at the step of pSmad2/3 translocation into the nucleus. The pathomechanisms, including the process of inclusion development, appears to be different between SALS and mSOD1-related FALS or Tg mice.
    Document Type:
    Reference
    Product Catalog Number:
    AP180C
    Product Catalog Name:
    Donkey Anti-Goat IgG Antibody, Cy3 conjugate, Species Adsorbed

  • ERK5/BMK1 is indispensable for optimal colony-stimulating factor 1 (CSF-1)-induced proliferation in macrophages in a Src-dependent fashion. 18322228

    CSF-1, by binding to its high-affinity receptor CSF-1R, sustains the survival and proliferation of monocyte/macrophages, which are central cells of innate immunity and inflammation. The MAPK ERK5 (also known as big MAPK-1, BMK1, or MAPK7) is a 98-kDa molecule sharing high homology with ERK1/2. ERK5 is activated by oxidative stress or growth factor stimulation. This study was undertaken to characterize ERK5 involvement in macrophage signaling that is elicited by CSF-1. Exposure to the CSF-1 of primary human macrophages or murine macrophage cell lines, as well as murine fibroblasts expressing ectopic CSF-1R, resulted in a rapid and sustained increase of ERK5 phosphorylation on activation-specific residues. In the BAC1.2F5 macrophage cell line, ERK5 was also activated by another mitogen, GM-CSF, while macrophage activators such as LPS or IFN-gamma and a number of nonproliferative cytokines failed. Src family kinases were found to link the activation of CSF-1R to that of ERK5, whereas protein kinase C or the serine phosphatases PP1 and PP2A seem not to be involved in the process. Treatment of macrophages with ERK5-specific small interfering RNA markedly reduced CSF-1-induced DNA synthesis and total c-Jun phosphorylation and expression, while increasing the expression of the cyclin-dependent kinase inhibitor p27. Following CSF-1 treatment, the active form of ERK5 rapidly translocated from cytosol to nucleus. Taken together, the results reported in this study show that ERK5 is indispensable for optimal CSF-1-induced proliferation and indicate a novel target for its control.
    Document Type:
    Reference
    Product Catalog Number:
    AP180C
    Product Catalog Name:
    Donkey Anti-Goat IgG Antibody, Cy3 conjugate, Species Adsorbed

  • Expression and role of PDGF-BB and PDGFR-beta during testis morphogenesis in the mouse embryo. 14996938

    The role played by PDGF in testis morphogenesis is still incompletely understood. The present study investigates the expression and potential role of platelet-derived growth factor-BB (PDGF-BB) and its receptor, PDGF receptor beta (PDGFR-beta), during mouse testis cord formation, and the possibility that the growth factor may be involved in the migration to the gonad of mesenchymal cells of mesonephric origin. Studies from this laboratory have previously shown that mesenchymal cells that migrate from the mesonephros into the gonad, to form peritubular myoid cells and most of the intertubular cells, can be identified by the presence on their surface of the p75 neurotrophin receptor (p75NTR), and can be isolated to near-purity by immunomagnetic selection with anti-p75NTR antibody. We show here that mesonephric p75NTR(+) cells also bear the PDGFR-beta, and are able to migrate and proliferate in vitro in response to PDGF-BB. PDGF-BB is expressed at higher levels in male than female developing gonads, suggesting a role for this factor in testis development. Such a role is further supported by the observation that addition of PDGF-BB to serum-free medium is sufficient to allow organ-cultured male 11.5 days post-coitum urogenital ridges to form testis cords. Finally, we show that mesonephric cell motility and growth induced by exposure to PDGF-BB involve mitogen-activated protein kinases (MAPK) and phosphatidylinositol-3 kinase (PI3-K) pathways, as MAPK inhibitor U0126 and PI3K inhibitor Ly294002 inhibit migration and proliferation in vitro assays. The present findings support the hypothesis that the PDGF/PDGFR system plays a key role in testis morphogenesis in the mouse embryo.
    Document Type:
    Reference
    Product Catalog Number:
    AB1554
    Product Catalog Name:
    Anti-Nerve Growth Factor Receptor Antibody, p75