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On-Demand Webinar Available: Cell Freezing Technologies and Disposable Bioreactors Towards a USP Process
Develop a Fully-Closed USP Process: Use Cell Freezing in Bags and SU Bioreactors
  • Recorded on May 22, 2014
  • Duration: 50 minutes
  • Gamma-diketone axonopathy: analyses of cytoskeletal motors and highways in CNS myelinated axons. 20554699

    2,5-Hexanedione (HD) intoxication is associated with axon atrophy that might be responsible for the characteristic gait abnormalities, hindlimb skeletal muscle weakness and other neurological deficits that accompany neurotoxicity. Although previous mechanistic research focused on neurofilament triplet proteins (NFL, NFM, NFH), other cytoskeletal targets are possible. Therefore, to identify potential non-NF protein targets, we characterized the effects of HD on protein-protein interactions in cosedimentation assays using microtubules and NFs prepared from spinal cord of rats intoxicated at different daily dose rates (175 and 400 mg/kg/day). Results indicate that HD did not alter the presence of alpha- or beta-tubulins in these preparations, nor were changes noted in the distribution of either anterograde (KIF1A, KIF3, KIF5) or retrograde (dynein) molecular motors. The cosedimentation of dynactin, a dynein-associated protein, also was not affected. Immunoblot analysis of microtubule-associated proteins (MAPs) in microtubule preparations revealed substantial reductions (45-80%) in MAP1A, MAP1B heavy chain, MAP2, and tau regardless of HD dose rate. MAP1B light chain content was not altered. Finally, HD intoxication did not influence native NF protein content in either preparation. As per previous research, microtubule and NF preparations were enriched in high-molecular weight NF species. However, these NF derivatives were common to both HD and control samples, suggesting a lack of pathognomonic relevance. These data indicate that, although motor proteins were not affected, HD selectively impaired MAP-microtubule binding, presumably through adduction of lysine residues that mediate such interactions. Given their critical role in cytoskeletal physiology, MAPs could represent a relevant target for the induction of gamma-diketone axonopathy.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
  • TWIST is expressed in human gliomas and promotes invasion. 16229805

    TWIST, a basic helix-loop-helix (bHLH) transcription factor that regulates mesodermal development, has been shown to promote tumor cell metastasis and to enhance survival in response to cytotoxic stress. Our analysis of rat C6 glioma cell-derived cDNA revealed TWIST expression, suggesting that the gene may play a role in the genesis and physiology of primary brain tumors. To further delineate a possible oncogenic role for TWIST in the central nervous system (CNS), we analyzed TWIST expression in human gliomas and normal brain by using reverse transcription polymerase chain reaction, Northern blot analysis, in situ hybridization, and immunohistochemistry. TWIST expression was detected in the large majority of human glioma-derived cell lines and human gliomas examined. Levels of TWIST mRNA were associated with the highest grade gliomas, and increased TWIST expression accompanied transition from low grade to high grade in vivo, suggesting a role for TWIST in promoting malignant progression. In accord, elevated TWIST mRNA abundance preceded the spontaneous malignant transformation of cultured mouse astrocytes hemizygous for p53. Overexpression of TWIST protein in a human glioma cell line significantly enhanced tumor cell invasion, a hallmark of high-grade gliomas. These findings support roles for TWIST both in early glial tumorigenesis and subsequent malignant progression. TWIST was also expressed in embryonic and fetal human brain, and in neurons, but not glia, of mature brain, indicating that, in gliomas, TWIST may promote the functions also critical for CNS development or normal neuronal physiology.
    Document Type:
    Reference
    Product Catalog Number:
    MAB377
    Product Catalog Name:
    Anti-NeuN Antibody, clone A60
  • Differential miRNA expression profiles in proliferating or differentiated keratinocytes in response to gamma irradiation. 23496899

    MicroRNAs (miRNAs), a group of short non-coding RNAs that negatively regulate gene expression, have recently emerged as potential modulators of cellular response to ionizing radiations both in vitro and in vivo in various cell types and tissues. However, in epidermal cells, the involvement of the miRNA machinery in the cellular response to ionizing radiations remains to be clarified. Indeed, understanding the mechanisms of cutaneous radiosensitivity is an important issue since skin is the most exposed organ to ionizing radiations and among the most sensitive.We settled up an expression study of miRNAs in primary human skin keratinocytes using a microfluidic system of qPCR assay, which permits to assess the expression of almost 700 annotated miRNAs. The keratinocytes were cultured to a proliferative or a differentiated state mimicking basal or suprabasal layers of human epidermis. These cells were irradiated at 10 mGy or 6 Gy and RNA was extracted 3 hours after irradiation. We found that proliferative cells irradiated at 6 Gy display a global fall of miRNA expression whereas differentiated cells exposed to the same dose display a global increase of miRNAs expression. We identified twenty miRNAs weakly but significantly modulated after 6 Gy irradiation, whereas only 2 miRNAs were modulated after low-dose irradiation in proliferating cells. To go further into the biological meaning of this miRNA response, we over-expressed some of the responding miRNA in proliferating cells: we observed a significant decrease of cell viability 72 hours after irradiation. Functional annotation of their predicted targets revealed that G-protein related pathways might be regulated by these responding miRNAs.Our results reveal that human primary keratinocytes exposed to ionizing irradiation expressed a miRNA pattern strongly related to the differentiation status of irradiated cells. We also demonstrate that some miRNAs play a role in the radiation response to ensure the short-term survival of irradiated keratinocytes.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1501
    Product Catalog Name:
    Anti-Actin Antibody, clone C4
  • Polycystin-1 is a microtubule-driven desmosome-associated component in polarized epithelial cells. 20211617

    In this study, we have analyzed the expression and localization of polycystin-1 in intestinal epithelial cells, a system lacking primary cilia. Polycystin-1 was found to be expressed in the epithelium of the small intestine during development and levels remained elevated in the adult. Dual-labelling indirect immunofluorescence revealed polycystin-1 at sites of cell-cell contact co-localizing with the desmosomes both in situ as well as in polarized Caco-2/15 cells. In unpolarized cultures of Caco-2/15 cells, polycystin-1 was recruited to the cell surface early during initiation of cell junction assembly. In isolated Caco-2/15 cells and HIEC-6 cell cultures, where junctional complexes are absent, polycystin-1 was found predominantly associated with the cytoskeletal elements of the intermediate filaments and microtubule networks. More precisely, polycystin-1 was seen as brightly labelled puncta decorating the keratin-18 positive filaments as well as the beta-tubulin positive microtubules, which was particularly obvious in the lamellipodia. Treatment with the microtubule-disrupting agent, nocodazole, eliminated the microtubule association of polycystin-1 but did not seem to affect its association with keratin or the desmosomes. Taken together these data suggest that polycystin-1 is involved with the establishment of cell-cell junctions in absorptive intestinal epithelial cells and exploits the microtubule-based machinery in order to be transported to the plasma membrane.
    Document Type:
    Reference
    Product Catalog Number:
    MAB3408
    Product Catalog Name:
    Anti-Tubulin Antibody, beta, clone KMX-1
  • Regulation and regulatory role of WNT signaling in potentiating FSH action during bovine dominant follicle selection. 24936794

    Follicular development occurs in wave like patterns in monotocous species such as cattle and humans and is regulated by a complex interaction of gonadotropins with local intrafollicular regulatory molecules. To further elucidate potential mechanisms controlling dominant follicle selection, granulosa cell RNA harvested from F1 (largest) and F2 (second largest) follicles isolated at predeviation (PD) and onset of diameter deviation (OD) stages of the first follicular wave was subjected to preliminary RNA transcriptome analysis. Expression of numerous WNT system components was observed. Hence experiments were performed to test the hypothesis that WNT signaling modulates FSH action on granulosa cells during follicular waves. Abundance of mRNA for WNT pathway members was evaluated in granulosa cells harvested from follicles at emergence (EM), PD, OD and early dominance (ED) stages of the first follicular wave. In F1 follicles, abundance of CTNNB1 and DVL1 mRNAs was higher and AXIN2 mRNA was lower at ED versus EM stages and DVL1 and FZD6 mRNAs were higher and AXIN2 mRNA was lower in F1 versus F2 follicle at the ED stage. Bovine granulosa cells were treated in vitro with increasing doses of the WNT inhibitor IWR-1+/- maximal stimulatory dose of FSH. IWR-1 treatment blocked the FSH-induced increase in granulosa cell numbers and reduced the FSH-induced increase in estradiol. Granulosa cells were also cultured in the presence or absence of FSH +/- IWR-1 and hormonal regulation of mRNA for WNT pathway members and known FSH targets determined. FSH treatment increased CYP19A1, CCND2, CTNNB1, AXIN2 and FZD6 mRNAs and the stimulatory effect on CYP19A1 mRNA was reduced by IWR-1. In contrast, FSH reduced CARTPT mRNA and IWR-1 partially reversed the inhibitory effect of FSH. Results support temporal and hormonal regulation and a potential role for WNT signaling in potentiating FSH action during dominant follicle selection.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1501
    Product Catalog Name:
    Anti-Actin Antibody, clone C4
  • Expansion of vitiligo lesions is associated with reduced epidermal CDw60 expression and increased expression of HLA-DR in perilesional skin. 14616364

    Detection of CDw60 in skin is representative of ganglioside D3 expression. This ganglioside is expressed primarily by melanocytes, and is of interest as a membrane antigen targeted by immunotherapy for melanoma patients. Expression of CDw60 by keratinocytes is defined by the presence of T-helper cell (Th)1 vs. Th2 cytokines, and can serve as a sentinel molecule to characterize an ongoing skin immune response.These immunobiological characteristics have provided the incentive to study the expression of CDw60 in the context of progressive vitiligo.Frozen sections were obtained from control skin and from vitiligo lesions and immunostained to show CDw60. Cells were cultured, their CDw60 expression studied and ribonuclease protection assays run to detect cytokine mRNA.Resistance to cytokine-mediated regulation of CDw60 expression was demonstrated in vitro by melanocytes, which appeared capable of generating autocrine and paracrine regulatory molecules supporting CDw60 expression. Induction of CDw60 expression was inhibited by antibodies to interleukin (IL)-4, suggesting that this cytokine was responsible, at least in part, for melanocyte-induced CDw60 expression. Marginal skin from patients with progressive generalized vitiligo consistently showed a reduction in epidermal CDw60 expression alongside elevated human leucocyte associated antigen (HLA)-DR expression at the margin. It thus appears that inflammatory infiltrates present in marginal skin generate type 1 rather than type 2 cytokines, supportive of a cell-mediated autoimmune response.These results support an active role of melanocytes within the skin immune system, and associate their loss in generalized vitiligo with a cell-mediated immune response mediated by type 1 cytokines.
    Document Type:
    Reference
    Product Catalog Number:
    MABF978
    Product Catalog Name:
    Anti-GD3, 9-O-acetyl Antibody, clone UM4D4
  • Detection of toxin translocation into the host cytosol by surface plasmon resonance. 22231143

    AB toxins consist of an enzymatic A subunit and a cell-binding B subunit(1). These toxins are secreted into the extracellular milieu, but they act upon targets within the eukaryotic cytosol. Some AB toxins travel by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) before entering the cytosol(2-4). In the ER, the catalytic A chain dissociates from the rest of the toxin and moves through a protein-conducting channel to reach its cytosolic target(5). The translocated, cytosolic A chain is difficult to detect because toxin trafficking to the ER is an extremely inefficient process: most internalized toxin is routed to the lysosomes for degradation, so only a small fraction of surface-bound toxin reaches the Golgi apparatus and ER(6-12). To monitor toxin translocation from the ER to the cytosol in cultured cells, we combined a subcellular fractionation protocol with the highly sensitive detection method of surface plasmon resonance (SPR)(13-15). The plasma membrane of toxin-treated cells is selectively permeabilized with digitonin, allowing collection of a cytosolic fraction which is subsequently perfused over an SPR sensor coated with an anti-toxin A chain antibody. The antibody-coated sensor can capture and detect pg/mL quantities of cytosolic toxin. With this protocol, it is possible to follow the kinetics of toxin entry into the cytosol and to characterize inhibitory effects on the translocation event. The concentration of cytosolic toxin can also be calculated from a standard curve generated with known quantities of A chain standards that have been perfused over the sensor. Our method represents a rapid, sensitive, and quantitative detection system that does not require radiolabeling or other modifications to the target toxin.
    Document Type:
    Reference
    Product Catalog Number:
    09-100
  • Mcl-1 is a key regulator of apoptosis during CNS development and after DNA damage. 18550749

    Despite the importance of Mcl-1, an anti-apoptotic Bcl-2 family member, in the regulation of apoptosis, little is known regarding its role in nervous system development and injury-induced neuronal cell death. Because germline deletion of Mcl-1 results in peri-implantation lethality, we address the function of Mcl-1 in the nervous system using two different conditional Mcl-1 mouse mutants in the developing nervous system. Here, we show for the first time that Mcl-1 is required for neuronal development. Neural precursors within the ventricular zone and newly committed neurons in the cortical plate express high levels of Mcl-1 throughout cortical neurogenesis. Loss of Mcl-1 in neuronal progenitors results in widespread apoptosis. Double labeling with active caspase 3 and Tuj1 reveals that newly committed Mcl1 deficient neurons undergo apoptosis as they commence migration away from the ventricular zone. Examination of neural progenitor differentiation in vitro demonstrated that cell death in the absence of Mcl1 is cell autonomous. Although conditional deletion of Mcl-1 in cultured neurons does not trigger apoptosis, loss of Mcl-1 sensitizes neurons to an acute DNA damaging insult. Indeed, the rapid reduction of Mcl-1 mRNA and protein levels are early events after DNA damage in neurons, and maintaining high Mcl-1 levels can protect neurons against death. Together, our results are the first to demonstrate the requirement of Mcl-1, an anti-apoptotic Bcl-2 family protein, for cortical neurogenesis and the survival of neurons after DNA damage.
    Document Type:
    Reference
    Product Catalog Number:
    06-570
    Product Catalog Name:
    Anti-phospho-Histone H3 (Ser10) Antibody, Mitosis Marker
  • Knockdown of p53 combined with expression of the catalytic subunit of telomerase is sufficient to immortalize primary human ovarian surface epithelial cells. 16829690

    Ovarian cancer is developed from a single layer of thin epithelial cells covering the surface of ovary, named human ovarian surface epithelial cells. Like all primary human cells, human ovarian surface epithelial cells have a finite life span and will go into senescence and eventually die when cultured in vitro. Immortalized human ovarian surface epithelial cells will provide an important model system with which to study ovarian cancer initiation and progression. Here, we show that silencing p53 expression with retrovirus-mediated small interfering RNA can delay the senescence and extend cell passage number, but is not sufficient to immortalize normal ovarian surface epithelial cells. Introduction of the catalytic subunit of telomerase is similarly insufficient to achieve immortalization. However, concurrent disruption of p53 expression with small interfering RNA retroviral constructs and ectopic expression of the catalytic subunit of telomerase was sufficient to induce cellular immortalization in 3 of 3 human ovarian surface epithelial cell cultures tested. The immortalization is associated with increased telomerase activity and telomere length, and attenuated response of cell-cycle regulatory proteins to irradiation. The resultant immortal cells continued to express the same specific cytokeratins 8 and 18 as parental cells did, indicating that the epithelial characters are still maintained in the immortal cells. In addition, the immortalized cells are non-tumorigenic and nearly diploid, which is in constrast with one immortalized by SV40 T/t antigens and hTERT. As both p53 pathway dysfunction and activation of telomerase are commonly present in human ovarian cancer, these immortal cells provide an authetic cell model system for the study of the human ovarian cancer initiation, progression, differentiation and chemoprevention.
    Document Type:
    Reference
    Product Catalog Number:
    S7700
    Product Catalog Name:
    TRAPeze® Telomerase Detection Kit
  • A New Orbivirus Isolated from Mosquitoes in North-Western Australia Shows Antigenic and Genetic Similarity to Corriparta Virus but Does Not Replicate in Vertebrate Cells 27213426

    The discovery and characterisation of new mosquito-borne viruses provides valuable information on the biodiversity of vector-borne viruses and important insights into their evolution. In this study, a broad-spectrum virus screening system, based on the detection of long double-stranded RNA in inoculated cell cultures, was used to investigate the presence of novel viruses in mosquito populations of northern Australia. We detected and isolated a new virus (tentatively named Parry's Lagoon virus, PLV) from Culex annulirostris, Culex pullus, Mansonia uniformis and Aedes normanensis mosquitoes that shares genomic sequence similarities to Corriparta virus (CORV), a member of the Orbivirus genus of the family Reoviridae. Despite moderate to high (72.2% to 92.2%) amino acid identity across all proteins when compared to CORV, and demonstration of antigenic relatedness, PLV did not replicate in several vertebrate cell lines that were permissive to CORV. This striking phenotypic difference suggests that PLV has evolved to have a very restricted host range, indicative of a mosquito-only life cycle.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple