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51.
Canopy and substratum heterogeneity influence recruitment of the mangrove Avicennia marina 总被引:3,自引:0,他引:3
Todd E. Minchinton 《Journal of Ecology》2001,89(5):888-902
52.
Characterization of three-dimensional tissue cultures using electrical impedance spectroscopy
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Electrical impedance spectroscopy was used to characterize the cell environment of multilayered cell cultures (MCCs), a culture system in which cells are grown on a permeable support membrane to form a thick disc of cells with tumor-like properties. Cultures were grown using SiHa tumor cells as well as V79 wild-type cells and V79/DOX cells cultivated to exhibit multidrug resistance. Electrical impedance measurements were made on MCCs over a frequency range of 0. 1 kHz to 1 MHz. Data analysis using a simple electrical model for the cell environment yielded estimates for parameters related to the intra- and extracellular resistance and net membrane capacitance, which were then related to MCC thickness. The extracellular fraction and tortuosity of the MCCs were determined in separate experiments where the rate of diffusion and the equilibrium level of C14-inulin, which does not penetrate the cell membrane, was measured within MCCs. Impedance measurements predicted the barrier to diffusion posed by the extracellular space of MCCs to be roughly two times greater than that inferred from the C14-inulin experiments. However, the relative ranking of the three cell types used to grow MCCs was similar for the two methods. Results indicate that impedance spectroscopy is well suited for use in characterizing the MCC cell environment, offering a fast, nondestructive method for monitoring cell culture growth and integrity. 相似文献
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HER-2/neu overexpression increases the viable hypoxic cell population within solid tumors without causing changes in tumor vascularization 总被引:4,自引:0,他引:4
Dragowska WH Warburton C Yapp DT Minchinton AI Hu Y Waterhouse DN Gelmon K Skov K Woo J Masin D Huxham LA Kyle AH Bally MB 《Molecular cancer research : MCR》2004,2(11):606-619
The effects of HER-2/neu overexpression on the tumor microenvironment in an aggressive breast cancer xenograft model were investigated. These studies focused on tumors derived following the subcutaneous injection of MDA-MB-435/LCC6 cells transfected with human c-erbB2 (LCC6(HER-2)) into SCID-Rag2M mice. LCC6(HER-2) tumors were more viable (H&E-stained tumor sections) than isogenic vector control tumors (LCC6(Vector)). Correspondingly, a 2.7-fold increase in trypan blue-excluding cells (P = 0.00056) and a 4.8-fold increase in clonogenic cells (P = 0.00146) were noted in cell suspensions derived from disaggregated LCC6(HER-2) versus LCC6(Vector) tumors. Tumor sections stained with the antibody detecting 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide (EF5), a marker of hypoxia, showed a greater fraction of hypoxic tissue in LCC6(HER-2) tumors compared with control tumors. Flow cytometric analyses based on viable tumor cells (DNA content >/= 2N) in cell suspensions from disaggregated tumors confirmed that there were significantly more EF5-positive cells (i.e., hypoxic) in LCC6(HER-2) than in LCC6(Vector) tumors (16.41 +/- 8.1% and 5.96 +/- 4.1%, respectively; P = 0.0015). Protein levels of phosphorylated (Ser(536)) nuclear factor-kappaB p65 were significantly elevated in LCC6(HER-2) tumors (P = 0.00048), and a trend in increased hypoxia-inducible factor-1alpha protein levels was observed in LCC6(HER-2) compared with LCC6(Vector) tumors. Despite the substantial viable hypoxic cell fraction and a 1.7-fold increase of vascular endothelial growth factor protein (P = 0.05) in LCC6(HER-2) tumors, no significant differences were found (P > 0.05) between LCC6(HER-2) and LCC6(Vector) vasculature (CD31 staining and Hoechst 33342 perfusion). These results suggest that HER-2/neu overexpression may be linked with overall increased tumor viability and a significant increase in the population of viable hypoxic cells, which is not due to differences in tumor vascularization. 相似文献
54.
Fortunato Ferrara Sara D’Angelo Tiziano Gaiotto Leslie Naranjo Hongzhao Tian Susanne Gr?slund Elena Dobrovetsky Peter Hraber Fridtjof Lund-Johansen Silvia Saragozza Daniele Sblattero Csaba Kiss Andrew RM Bradbury 《MABS-AUSTIN》2015,7(1):32-41
Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products. 相似文献
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Lúcia de Paula Célio L Silva Daniela Carlos Camila Matias-Peres Carlos A Sorgi Edson G Soares Patrícia RM Souza Carlos RZ Bladés Fábio CS Galleti Vânia LD Bonato Eduardo DC Gonçalves Érika VG Silva Lúcia H Faccioli 《Genetic vaccines and therapy》2007,5(1):1-7
The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly (DL-lactide-co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in mice as well as in guinea pigs by comparison with the efficacy and toxicity induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in both mice and guinea pigs. 相似文献
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Aileen?G?RowanEmail author Koichiro?Suemori Hiroshi?Fujiwara Masaki?Yasukawa Yuetsu?Tanaka Graham?P?Taylor Charles?RM?Bangham 《Retrovirology》2014,11(1):116
Background
Immunogenetic evidence indicates that cytotoxic T lymphocytes (CTLs) specific for the weak CTL antigen HBZ limit HTLV-1 proviral load in vivo, whereas there is no clear relationship between the proviral load and the frequency of CTLs specific for the immunodominant antigen Tax. In vivo, circulating HTLV-1-infected cells express HBZ mRNA in contrast, Tax expression is typically low or undetectable. To elucidate the virus-suppressing potential of CTLs targeting HBZ, we compared the ability of HBZ- and Tax-specific CTLs to lyse naturally-infected cells, by co-incubating HBZ- and Tax-specific CTL clones with primary CD4+ T cells from HLA-matched HTLV-1-infected donors. We quantified lysis of infected cells, and tested whether specific virus-induced host cell surface molecules determine the susceptibility of infected cells to CTL-mediated lysis.Results
Primary infected cells upregulated HLA-A*02, ICAM-1, Fas and TRAIL-R1/2 in concert with Tax expression, forming efficient targets for both HTLV-1-specific CTLs and CTLs specific for an unrelated virus. We detected expression of HBZ mRNA (spliced isoform) in both Tax-expressing and non-expressing infected cells, and the HBZ26–34 epitope was processed and presented by cells transfected with an HBZ expression plasmid. However, when coincubated with primary cells, a high-avidity HBZ-specific CTL clone killed significantly fewer infected cells than were killed by a Tax-specific CTL clone. Finally, incubation with Tax- or HBZ-specific CTLs resulted in a significant decrease in the frequency of cells expressing high levels of HLA-A*02.Conclusions
HTLV-1 gene expression in primary CD4+ T cells non-specifically increases susceptibility to CTL lysis. Despite the presence of HBZ spliced-isoform mRNA, HBZ epitope presentation by primary cells is significantly less efficient than that of Tax.60.
A molecular and evolutionary study of the beta-globin gene family of the Australian marsupial Sminthopsis crassicaudata 总被引:1,自引:0,他引:1
Cooper SJ; Murphy R; Dolman G; Hussey D; Hope RM 《Molecular biology and evolution》1996,13(7):1012-1022
Beta-globin gene families in eutherians (placental mammals) consist of a
set of four or more developmentally regulated genes which are closely
linked and, in general, arranged in the order 5'-embryonic/fetal genes-
adult genes-3'. This cluster of genes is proposed to have arisen by tandem
duplication of ancestral beta-globin genes, with the first duplication
occurring 200 to 155 MYBP just prior to a period in mammalian evolution
when eutherians and marsupials diverged from a common ancestor. In this
paper we trace the evolutionary history of the beta-globin gene family back
to the origins of these mammals by molecular characterization of the
beta-globin gene family of the Australian marsupial Sminthopsis
crassicaudata. Using Southern and restriction analysis of total genomic DNA
and bacteriophage clones of beta-like globin genes, we provide evidence
that just two functional beta-like globin genes exist in this marsupial,
including one embryonic- expressed gene (S.c-epsilon) and one
adult-expressed gene (S.c-beta), linked in the order 5'-epsilon-beta-3'.
The entire DNA sequence of the adult beta-globin gene is reported and shown
to be orthologous to the adult beta-globin genes of the North American
marsupial Didelphis virginiana and eutherian mammals. These results,
together with results from a phylogenetic analysis of mammalian beta-like
globin genes, confirm the hypothesis that a two-gene cluster, containing an
embryonic- and an adult-expressed beta-like globin gene, existed in the
most recent common ancester of marsupials and eutherians. Northern analysis
of total RNA isolated from embryos and neonatals indicates that a switch
from embryonic to adult gene expression occurs at the time of birth,
coinciding with the transfer of the marsupial from a uterus to a pouch
environment.
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