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  • Human and mouse microglia express connexin36, and functional gap junctions are formed between rodent microglia and neurons. 16211561

    Microglia, the tissue macrophages of the central nervous system (CNS), intimately interact with neurons physically and through soluble factors that can affect microglial activation state and neuronal survival and physiology. We report here a new mechanism of interaction between these cells, provided by the formation of gap junctions composed of connexin (Cx) 36. Among eight Cxs tested, expression of Cx36 mRNA and protein was found in microglial cultures prepared from human and mouse, and Cx45 mRNA was found in mouse microglial cultures. Electrophysiological measurements found coupling between one-third of human or mouse microglial pairs that averaged below 30 pico-Siemens and displayed electrical properties consistent with Cx36 gap junctions. Importantly, similar frequency of low-strength electrical coupling was also obtained between microglia and neurons in cocultures prepared from neocortical or hippocampal rodent tissue. Lucifer yellow dye coupling between neurons and microglia was observed in 4% of pairs tested, consistent with the low strength and incidence of electrical coupling. Cx36 expression level and/or the degree of coupling between microglia did not significantly change in the presence of activating agents, including lipopolysaccharide, granulocyte-macrophage colony-stimulating factor, interferon-gamma, and tumor necrosis factor-alpha, except for some reduction of Cx36 protein when exposed to the latter two agents. Our findings that intercellular coupling occurs between neuronal and microglial populations through Cx36 gap junctions have potentially important implications for normal neural physiology and microglial responses in neuronopathology in the mammalian CNS.
    Document Type:
    Reference
    Product Catalog Number:
    MAB3045
    Product Catalog Name:
    Anti-Connexin 35/36 Antibody, clone 8F6.2

  • PP56 improves energy homeostasis in a mouse model of pancreatic cancer. 20422342

    In this study, we investigated whether the anti-inflammatory drug PP56 (alpha-trinositol) may improve cancer-induced metabolic disorders. We implanted human MiaPaCa2 pancreatic cancer cells in the pancreas of 14 athymic mice for 12 weeks, using six intact littermates as normal controls. During the 12 weeks, seven tumor-cell recipients were treated with PP56 by daily injection (PPT mice). The tumor-cell recipients that were otherwise untreated were used as tumor controls (TC mice). Impaired glucose tolerance and decreased body weight gain were seen in TC but not PPT mice. When an enzyme for fatty acid beta-oxidation namely medium-chain acyl-CoA dehydrogenase (MCAD) was determined in tumor grafts; tumors from PPT mice showed more MCAD than those from TC mice. This suggests that PP56 stimulated fatty acid beta-oxidation in MiaPaCa2 cells in vivo. In keeping with this notion, PPT mice had decreased plasma free fatty acids. In vitro, we demonstrated that MiaPaCa2 cells consumed more fatty acids in the presence of PP56. In another experiment, we infused PP56 or vehicle in normal mice and found that PP56 decreased circulating glucose in the animals. We also showed that PP56 increased glucose transport in L6 skeletal muscle cells in vitro. In conclusion, PP56 increases the turnover of circulating nutrients such as glucose and helps maintain energy homeostasis in mice with pancreatic cancer.
    Document Type:
    Reference
    Product Catalog Number:
    AP106P
    Product Catalog Name:
    Rabbit Anti-Goat IgG Antibody, HRP conjugate

  • Tetraploid cells from cytokinesis failure induce aneuploidy and spontaneous transformation of mouse ovarian surface epithelial cells. 22801546

    Most ovarian cancers originate from the ovarian surface epithelium and are characterized by aneuploid karyotypes. Aneuploidy, a consequence of chromosome instability, is an early event during the development of ovarian cancers. However, how aneuploid cells are evolved from normal diploid cells in ovarian cancers remains unknown. In the present study, cytogenetic analyses of a mouse syngeneic ovarian cancer model revealed that diploid mouse ovarian surface epithelial cells (MOSECs) experienced an intermediate tetraploid cell stage, before evolving to aneuploid (mainly near-tetraploid) cells. Using long-term live-cell imaging followed by fluorescence in situ hybridization (FISH), we demonstrated that tetraploid cells originally arose from cytokinesis failure of bipolar mitosis in diploid cells, and gave rise to aneuploid cells through chromosome mis-segregation during both bipolar and multipolar mitoses. Injection of the late passage aneuploid MOSECs resulted in tumor formation in C57BL/6 mice. Therefore, we reveal a pathway for the evolution of diploid to aneuploid MOSECs and elucidate a mechanism for the development of near-tetraploid ovarian cancer cells.
    Document Type:
    Reference
    Product Catalog Number:
    MAB374
    Product Catalog Name:
    Anti-Glyceraldehyde-3-Phosphate Dehydrogenase Antibody, clone 6C5

  • Cyclic mechanical strain maintains Nanog expression through PI3K/Akt signaling in mouse embryonic stem cells. 22683858

    Mechanical strain has been reported to affect the proliferation/differentiation of many cell types; however, the effects of mechanotransduction on self-renewal as well as pluripotency of embryonic stem (ES) cells remains unknown. To investigate the effects of mechanical strain on mouse ES cell fate, we examined the expression of Nanog, which is an essential regulator of self-renewal and pluripotency as well as Nanog-associated intracellular signaling during uniaxial cyclic mechanical strain. The mouse ES cell line, CCE was plated onto elastic membranes, and we applied 10% strain at 0.17Hz. The expression of Nanog was reduced during ES cell differentiation in response to the withdrawal of leukemia inhibitory factor (LIF); however, two days of cyclic mechanical strain attenuated this reduction of Nanog expression. On the other hand, the cyclic mechanical strain promoted PI3K-Akt signaling, which is reported as an upstream of Nanog transcription. The cyclic mechanical strain-induced Akt phosphorylation was blunted by the PI3K inhibitor wortmannin. Furthermore, cytochalasin D, an inhibitor of actin polymerization, also inhibited the mechanical strain-induced increase in phospho-Akt. These findings imply that mechanical force plays a role in regulating Nanog expression in ES cells through the actin cytoskeleton-PI3K-Akt signaling.
    Document Type:
    Reference
    Product Catalog Number:
    MAB374
    Product Catalog Name:
    Anti-Glyceraldehyde-3-Phosphate Dehydrogenase Antibody, clone 6C5

  • Angiotensin II-inhibiting drugs have no effect on intraneuronal Aβ or oligomeric Aβ levels in a triple transgenic mouse model of Alzheimer's disease. 21416061

    Reducing the excessive accumulation of amyloid β-protein (Aβ) in Alzheimer's disease (AD) is a key objective of most AD therapies. Several studies suggest that pharmacological inhibition of angiotensin-converting enzyme (ACE) or its by-product angiotensin II may delay onset or progression of dementia and it has been suggested that this occurs via regulation of Aβ. Intraneuronal oligomeric accumulation of Aβ is postulated to be one of the earliest pathological events. Thus this study investigated the effect of an ACE-inhibitor, captopril, and two angiotensin II receptor blockers (ARBs), eprosartan and valsartan, on intraneuronal Aβ pathology and oligomeric Aβ levels in a triple transgenic (3xTGAD) mouse model of AD.Male, adult (3-4 month old) 3xTgAD mice (n=39) were randomly assigned to 4 treatment groups: valsartan (0.17g/l), eprosartan (0.8g/l), captopril (5g/l) or normal drinking water and the drugs given ad libitum for 2 months. Mean arterial blood pressure (MABP) was measured at baseline, at 2 weeks and at 2 months when the mice were sacrificed and the brains hemisected for analysis. One hemisphere was processed for Aβ and amyloid precursor protein (APP) immunohistochemistry and the other for biochemical measurement of oligomeric Aβ and APP. ACE activity was measured in the brain and kidney.MABP was significantly reduced at 2 weeks and 2 months in the ACE-I group (p=0.0006) but was unaltered in the ARB groups compared to vehicle. Neither ACE-I nor ARB treatment altered Aβ and APP immunolabelling or the level of Aβ or APP in brain tissue homogenates. Similarly neither ACE-I nor ARB treatment altered ACE activity in either brain or kidney compared to control tissue.ACE-I or ARB administration over 2 months did not affect APP levels or either intraneuronal Aβ or oligomeric Aβ levels in 3xTGAD mice. While ARBs did not alter MABP, captopril did mediate reductions in MABP in the 3xTGAD mice which appeared to be independent of ACE activity. Further studies are needed to examine the effects of these drugs over a longer term and in older mice (i.e. when AD-like changes are more pronounced).
    Document Type:
    Reference
    Product Catalog Number:
    MAB348
    Product Catalog Name:
    Anti-APP A4 Antibody, a.a. 66-81 of APP {NT}, clone 22C11

  • The glycine transporter GlyT1 controls N-methyl-D-aspartic acid receptor coagonist occupancy in the mouse retina. 20092573

    We examined the role of GlyT1, the high-affinity glycine transporter, in the mouse retina with an emphasis on the role of glycine as a coagonist of N-methyl-D-aspartic acid (NMDA) receptors. We pursued this objective by studying heterozygote mice deficient in the GlyT1 transporter (GlyT1(-/+)) and compared those results with wild-type (WT) littermate controls (GlyT1(+/+)). Capillary electrophoresis was used to separate and quantitatively measure glycine release from isolated retina preparations; pharmacologically blocking GlyT1 with N-[3-([1,1-biphenyl]-4-yloxy)-3-(4-fluorophenyl)propyl]-N-methylglycine in the WT retina generated a significantly larger accumulation of glycine into the bathing environment when compared with the GlyT1(-/+) retinas. The relative occupancy state of the NMDA receptor coagonist sites was tested using whole-cell recordings from ganglion cells while bath applying D-serine or D-serine + NMDA. The interpretation of these studies was simplified by blocking post-synaptic inhibition with picrotoxinin and strychnine. NMDA receptor coagonist sites were more saturated and less enhanced by D-serine in the GlyT1(-/+) mice compared with the WT controls. Immunoblots of NMDA receptor subunits (NR1, NR2A and NR2B) in WT and GlyT1(-/+) animals showed that the NR1 subunits were identical. These observations are discussed in view of contemporary issues about NMDA receptor coagonist function in the vertebrate retina and the role of glycine vs. D-serine as the endogenous coagonist.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Targeting human epidermal growth factor receptor 2 by a cell-penetrating peptide-affibody bioconjugate. 22192536

    Cell-penetrating peptide (CPP)-based delivery systems represent a strategy that facilitates DNA import efficiently and non-specifically into cells. To introduce specificity, we devised an approach that combines a cell-penetrating peptide, TAT-Mu (TM) and a targeting ligand, an HER2 antibody mimetic-affibody (AF), designated as TMAF to deliver nucleic acids into the cells. In this study, we synthesized TMAF protein and its truncated versions, i.e. MAF and AF, by expressing the corresponding plasmids in Escherichia coli BL21(DE3)pLysS cells. Purified TMAF binds DNA efficiently and protects plasmid DNA from DNaseI action. Transfection of HER2+ breast cancer cell lines MDA-MB-453, SK-OV-3, SK-BR-3 and an ovarian cancer cell line with plasmid DNA pCMV?-gal, resulted in enhanced ?-galactosidase activity when compared to control MDA-MB-231 cells. Maximal activity observed in MDA-MB-453 cells at DNA:TMAF:Protamine sulphate (PS) corresponding to 1:8:2 charge ratios. Further the observed gene transfection was resistant to serum, sensitive to the presence of free AF and non-toxic. Variants of TMAF although non-toxic, were far less efficient indicating the effective role of the TAT and Mu domains. The observed DNA uptake and reporter gene activity mediated by TMAFin vitro could be linked with the cell-surface density of tyrosine kinase receptor HER2 (ErbB2) levels estimated by Western blot. Further, we confirmed the efficacy of DNA transfer by TMAF protein in xenograft mouse models using MDA-MB-453 cells. Expression of ?-galactosidase as the reporter gene, upon intratumoral injection of DNA, in complex with TMAF, lends credence to specific DNA import and distribution within the tumor tissue that was attributed to high HER2 receptor overexpression in MDA-MB-453 cells. Through delivery of anti-TF hshRNA: TMAF: PS complex, we demonstrate specific knockdown of tissue factor (TF) in MDA-MB-453 cells in vitro. Most importantly, in a xenograft mouse model, we observe significant (P<0.05) and specific reduction of tumor volume when anti-TF hshRNA: TMAF: PS complex was injected intratumorally. Collectively our data indicate that AF-based chimeric peptides with nucleic acid binding properties may provide an effective tumor specific strategy to deliver therapeutic nucleic acids.
    Document Type:
    Reference
    Product Catalog Number:
    MAB374
    Product Catalog Name:
    Anti-Glyceraldehyde-3-Phosphate Dehydrogenase Antibody, clone 6C5

  • Cholinergic neuronal defect without cell loss in Huntington's disease. 16987871

    Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG-repeat expansion in the huntingtin (IT15) gene. The striatum is one of the regions most affected by neurodegeneration, resulting in the loss of the medium-sized spiny neurons. Traditionally, the large cholinergic striatal interneurons are believed to be spared. Recent studies demonstrate that neuronal dysfunction without cell death also plays an important role in early and mid-stages of the disease. Here, we report that cholinergic transmission is affected in a HD transgenic mouse model (R6/1) and in tissues from HD patients. Stereological analysis shows no loss of cholinergic neurons in the striatum or septum in R6/1 mice. In contrast, the levels of mRNA and protein for vesicular acetylcholine transporter (VAChT) and choline acetyltransferase (ChAT) are decreased in the striatum and cortex, and acetylcholine esterase activity is lowered in the striatum of R6/1 mice already at young ages. Accordingly, VAChT is also reduced in striatal tissue from patients with HD. The decrease of VAChT in the patient samples studied is restricted to the striatum and does not occur in the hippocampus or the spinal cord. The expression and localization of REST/NRSF, a transcriptional regulator for the VAChT and ChAT genes, are not altered in cholinergic neurons. We show that the R6/1 mice exhibit severe deficits in learning and reference memory. Taken together, our data show that the cholinergic system is dysfunctional in R6/1 and HD patients. Consequently, they provide a rationale for testing of pro-cholinergic drugs in this disease.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Fibroblast growth factor 2 regulates dopaminergic neuron development in vivo. 22537018

    Fibroblast growth factor 2 (FGF-2) is a neurotrophic factor participating in regulation of proliferation, differentiation, apoptosis and neuroprotection in the central nervous system. With regard to dopaminergic (DA) neurons of substantia nigra pars compacta (SNpc), which degenerate in Parkinson's disease, FGF-2 improves survival of mature DA neurons in vivo and regulates expansion of DA progenitors in vitro. To address the physiological role of FGF-2 in SNpc development, embryonic (E14.5), newborn (P0) and juvenile (P28) FGF-2-deficient mice were investigated. Stereological quantification of DA neurons identified normal numbers in the ventral tegmental area, whereas the SNpc of FGF-2-deficient mice displayed a 35% increase of DA neurons at P0 and P28, but not at earlier stage E14.5. Examination of DA marker gene expression by quantitative RT-PCR and in situ hybridization revealed a normal patterning of embryonic ventral mesencephalon. However, an increase of proliferating Lmx1a DA progenitors in the subventricular zone of the ventral mesencephalon of FGF-2-deficient embryos indicated altered cell cycle progression of neuronal progenitors. Increased levels of nuclear FgfR1 in E14.5 FGF-2-deficient mice suggest alterations of integrative nuclear FgfR1 signaling (INFS). In summary, FGF-2 restricts SNpc DA neurogenesis in vivo during late stages of embryonic development.
    Document Type:
    Reference
    Product Catalog Number:
    05-499
    Product Catalog Name:
    Anti-Histone H3 Antibody, clone 6.6.2

  • Molecular chaperone Hsp90 regulates REV1-mediated mutagenesis. 21690293

    REV1 is a Y-family polymerase that plays a central role in mutagenic translesion DNA synthesis (TLS), contributing to tumor initiation and progression. In a current model, a monoubiquitinated form of the replication accessory protein, proliferating cell nuclear antigen (PCNA), serves as a platform to recruit REV1 to damaged sites on the DNA template. Emerging evidence indicates that posttranslational mechanisms regulate REV1 in yeast; however, the regulation of REV1 in higher eukaryotes is poorly understood. Here we show that the molecular chaperone Hsp90 is a critical regulator of REV1 in human cells. Hsp90 specifically binds REV1 in vivo and in vitro. Treatment with a specific inhibitor of Hsp90 reduces REV1 protein levels in several cell types through proteasomal degradation. This is associated with suppression of UV-induced mutagenesis. Furthermore, Hsp90 inhibition disrupts the interaction between REV1 and monoubiquitinated PCNA and suppresses UV-induced focus formation. These results indicate that Hsp90 promotes folding of REV1 into a stable and/or functional form(s) to bind to monoubiquitinated PCNA. The present findings reveal a novel role of Hsp90 in the regulation of TLS-mediated mutagenesis.
    Document Type:
    Reference
    Product Catalog Number:
    05-724
    Product Catalog Name:
    Anti-Myc Tag Antibody, clone 4A6