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  • In vitro development of mouse embryonic stem cells lacking JNK/stress-activated protein kinase-associated protein 1 (JSAP1) scaffold protein revealed its requirement duri ... 12968026

    The Jsap1 gene encodes a scaffold protein for c-Jun N-terminal kinase cascades. We established c-Jun N-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1)-null mouse embryonic stem cell lines by homologous recombination. The JSAP1-null embryonic stem cells were viable, however, exhibited hyperplasia of the ectoderm during embryoid body formation, and spontaneously differentiated into neurons more efficiently than did wild type. The expression of components of c-Jun N-terminal kinase cascades and a subset of marker mRNAs during early embryogenesis was altered in the JSAP1-null mutants. Retinoic acid dramatically increased the expression of JSAP1 and JNK3, which were co-precipitated with anti-JNK3 in the neuroectoderm of wild type but not JSAP1-null embryoid bodies. In the neurons differentiated from the wild type embryoid bodies, JSAP1 was localized in the soma, neurites, and growth cone-like structure of the neurites, and neurite outgrowth from the JSAP1-null embryoid bodies was apparently less efficient than from wild type. JSAP1 and c-Jun N-terminal kinase 3 were coexpressed in the embryonic ectoderm of E7.5 mouse embryo, whereas Wnt1 and Pax2 were coexpressed with JSAP1 at the midbrain-hindbrain junction in E12.5 mouse embryo, thus suggesting that JSAP1 is required for early embryonic neurogenesis.
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  • In vitro engineering of human 3D chondrosarcoma: a preclinical model relevant for investigations of radiation quality impact. 26253487

    The benefit of better ballistic and higher efficiency of carbon ions for cancer treatment (hadron-therapy) is asserted since decades, especially for unresectable or resistant tumors like sarcomas. However, hadron-therapy with carbon ions stays underused and raises some concerns about potential side effects for patients. Chondrosarcoma is a cartilaginous tumor, chemo- and radiation-resistant, that lacks reference models for basic and pre-clinical studies in radiation-biology. Most studies about cellular effects of ionizing radiation, including hadrons, were performed under growth conditions dramatically different from human homeostasis. Tridimensional in vitro models are a fair alternative to animal models to approach tissue and tumors microenvironment.By using a collagen matrix, standardized culture conditions, physiological oxygen tension and a well defined chondrosarcoma cell line, we developed a pertinent in vitro 3D model for hadron-biology studies. Low- and high-Linear Energy Transfer (LET) ionizing radiations from GANIL facilities of ~1 keV/μm and 103 ± 4 keV/μm were used respectively, at 2 Gy single dose. The impact of radiation quality on chondrosarcoma cells cultivated in 3D was analyzed on cell death, cell proliferation and DNA repair.A fair distribution of chondrosarcoma cells was observed in the whole 3D scaffold. Moreover, LET distribution in depth, for ions, was calculated and found acceptable for radiation-biology studies using this kind of scaffold. No difference in cell toxicity was observed between low- and high-LET radiations but a higher rate of proliferation was displayed following high-LET irradiation. Furthermore, 3D models presented a higher and longer induction of H2AX phosphorylation after 2 Gy of high-LET compared to low-LET radiations.The presented results show the feasibility and usefulness of our 3D chondrosarcoma model in the study of the impact of radiation quality on cell fate. The observed changes in our tissue-like model after ionizing radiation exposure may explain some discrepancies between radiation-biology studies and clinical data.
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  • In vitro synthesis of tensioned synoviocyte bioscaffolds for meniscal fibrocartilage tissue engineering. 24299420

    Meniscal injury is a common cause of lameness in the dog. Tissue engineered bioscaffolds may be a treatment option for meniscal incompetency, and ideally would possess meniscus- like extracellular matrix (ECM) and withstand meniscal tensile hoop strains. Synovium may be a useful cell source for meniscal tissue engineering because of its natural role in meniscal deficiency and its in vitro chondrogenic potential. The objective of this study is to compare meniscal -like extracellular matrix content of hyperconfluent synoviocyte cell sheets ("HCS") and hyperconfluent synoviocyte sheets which have been tensioned over wire hoops (tensioned synoviocyte bioscaffolds, "TSB") and cultured for 1 month.Long term culture with tension resulted in higher GAG concentration, higher chondrogenic index, higher collagen concentration, and type II collagen immunoreactivity in TSB versus HCS. Both HCS and TSB were immunoreactive for type I collagen, however, HCS had mild, patchy intracellular immunoreactivity while TSB had diffuse moderate immunoreactivity over the entire bisocaffold. The tissue architecture was markedly different between TSB and HCS, with TSB containing collagen organized in bands and sheets. Both HCS and TSB expressed alpha smooth muscle actin and displayed active contractile behavior. Double stranded DNA content was not different between TSB and HCS, while cell viability decreased in TSB.Long term culture of synoviocytes with tension improved meniscal- like extra cellular matrix components, specifically, the total collagen content, including type I and II collagen, and increased GAG content relative to HCS. Future research is warranted to investigate the potential of TSB for meniscal tissue engineering.
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  • In vitro modeling of the neurovascular environment by coculturing adult human brain endothelial cells with human neural stem cells. 25187991

    Brain and vascular cells form a functionally integrated signalling network that is known as the neurovascular unit (NVU). The signalling (autocrine, paracrine and juxtacrine) between different elements of this unit, especially in humans, is difficult to disentangle in vivo. Developing representative in vitro models is therefore essential to better understand the cellular interactions that govern the neurovascular environment. We here describe a novel approach to assay these cellular interactions by combining a human adult cerebral microvascular endothelial cell line (hCMEC/D3) with a fetal ganglionic eminence-derived neural stem cell (hNSC) line. These cell lines provide abundant homogeneous populations of cells to produce a consistently reproducible in vitro model of endothelial morphogenesis and the ensuing NVU. Vasculature-like structures (VLS) interspersed with patches of differentiating neural cells only occurred when hNSCs were seeded onto a differentiated endothelium. These VLS emerged within 3 days of coculture and by day 6 were stabilizing. After 7 days of coculture, neuronal differentiation of hNSCs was increased 3-fold, but had no significant effect on astrocyte or oligodendrocyte differentiation. ZO1, a marker of adherens and tight junctions, was highly expressed in both undifferentiated and differentiated endothelial cells, but the adherens junction markers CD31 and VE-cadherin were significantly reduced in coculture by approximately 20%. A basement membrane, consisting of laminin, vitronectin, and collagen I and IV, separated the VLS from neural patches. This simple assay can assist in elucidating the cellular and molecular signaling involved in the formation of VLS, as well as the enhancement of neuronal differentiation through endothelial signaling.
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    Reference
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  • In vitro Pb exposure disturbs the balance between Aβ production and elimination: the role of AβPP and neprilysin. 21315759

    Metabolism of β-amyloid peptide (Aβ) is closely associated with the pathology and etiology of Alzheimer's disease (AD). Our previous studies on aging primates and rodents have revealed that early life lead exposure increases the expression of the β-amyloid precursor protein (AβPP), elevates Aβ levels, and promotes neurodegeneration in old age. These effects were attributed to de novo synthetic pathways; however, the impact on Aβ degradation was not explored. Neprilysin (NEP), a rate-limiting catabolic peptidase is involved in Aβ metabolism in vivo. In the present study we sought to investigate whether accumulation of Aβ induced by Pb exposure is partially due to its ability to subdue NEP expression and consequently NEP activity. SH-SY5Y cells were exposed to Pb concentrations of 0, 5, 10, 20, and 50 μM for 48 h and AβPP, NEP protein and mRNA levels were measured. Additionally, NEP enzymatic activity and Aβ levels were also assessed. Western blot and RT-PCR analysis indicated significant increases in the protein and mRNA expression of AβPP, which appeared to be concentration and time-dependent, while the protein and mRNA expression of NEP as well as NEP activity declined. These actions of Pb were specific and were not observed when substituted by another metal. These results suggest that Pb causes both the overexpression of AβPP and repression of NEP resulting in the buildup of Aβ.
    Document Type:
    Reference
    Product Catalog Number:
    MAB348
    Product Catalog Name:
    Anti-APP A4 Antibody, a.a. 66-81 of APP {NT}, clone 22C11

  • In vitro motility of liver connexin vesicles along microtubules utilizes kinesin motors. 21536677

    Trafficking of the proteins that form gap junctions (connexins) from the site of synthesis to the junctional domain appears to require cytoskeletal delivery mechanisms. Although many cell types exhibit specific delivery of connexins to polarized cell sites, such as connexin32 (Cx32) gap junctions specifically localized to basolateral membrane domains of hepatocytes, the precise roles of actin- and tubulin-based systems remain unclear. We have observed fluorescently tagged Cx32 trafficking linearly at speeds averaging 0.25 μm/s in a polarized hepatocyte cell line (WIF-B9), which is abolished by 50 μM of the microtubule-disrupting agent nocodazole. To explore the involvement of cytoskeletal components in the delivery of connexins, we have used a preparation of isolated Cx32-containing vesicles from rat hepatocytes and assayed their ATP-driven motility along stabilized rhodamine-labeled microtubules in vitro. These assays revealed the presence of Cx32 and kinesin motor proteins in the same vesicles. The addition of 50 μM ATP stimulated vesicle motility along linear microtubule tracks with velocities of 0.4-0.5 μm/s, which was inhibited with 1 mM of the kinesin inhibitor AMP-PNP (adenylyl-imidodiphosphate) and by anti-kinesin antibody but only minimally affected by 5 μM vanadate, a dynein inhibitor, or by anti-dynein antibody. These studies provide evidence that Cx32 can be transported intracellularly along microtubules and presumably to junctional domains in cells and highlight an important role of kinesin motor proteins in microtubule-dependent motility of Cx32.
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    Multiple

  • In vitro and in vivo reversal of multidrug resistance in a human leukemia-resistant cell line by mdr1 antisense oligodeoxynucleotides. 8813118

    A major obstacle to successful cancer chemotherapy is the development of multidrug resistance (MDR) by tumor cells. Overexpression of the mdrl gene product P-glycoprotein (P-170) is characteristic of such cells. In this study, in vitro and in vivo reversion of MDR was attempted in a human leukemia cell line resistant to vincristine (HL-60/Vinc) using an 18-mer mdr1 antisense phosphorothioate oligodeoxynucleotide ([S]ODN) in combination with vincristine. As control of sequence specificity, both sense and scrambled [S]ODNs were used. The ability of these [S]ODNs to reverse MDR was studied in vitro and in severe combined immunodeficient (SCID) mice. In vitro treatment with antisense [S]ODNs restored vincristine sensitivity of HL-60/Vinc cells, whereas no changes in drug sensitivity were observed upon treatment with the sense or scrambled sequence. The in vitro effects correlated with inhibition of P-170 expression in HL-60/Vinc cells exposed to the mdr1 antisense [S]ODNs. In vivo reversal of MDR was obtained in SCID mice given injections of HL-60/Vinc cells and systemically treated with [S]ODNs plus vincristine, as indicated by a significantly prolonged survival of SCID mice that received the combination therapy of mdr1 antisense [S]ODNs + vincristine. Treatments with mdr1 antisense or scrambled [S]ODNs, vincristine, or scrambled [S]ODNs + vincristine had no effect on survival. These results suggest that the use of mdr1 antisense ODNs in combination with standard antineoplastic drugs might be useful in reversing MDR in vitro and in vivo.
    Document Type:
    Reference
    Product Catalog Number:
    05-175
    Product Catalog Name:
    Anti-Myb Antibody, clone 1-1