Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines¹ support our understanding of invasion of the uterine wall² and remodeling of uterine spiral arteries^3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts^5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation^7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies^10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu¹³.We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues^7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models^10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.     

Comparison of the mutagenic potential of 17 physical and chemical agents analyzed by the flow cytometry mutation assay

Several methods to assess genotoxicity of physical and chemical agents have been developed, most of which depend on growing colonies in selective medium. We recently published a new method for detecting mutations in the CD59 gene in a Chinese hamster ovary cell line that contains a single copy of human chromosome 11 (CHO A_L). The assay is based on detecting the surface expression of CD59 with monoclonal antibodies using flow cytometry. The capabilities of this flow cytometry mutation assay (FCMA) to detect mutations from a wide variety of genotoxic agents are described here. There was a 400-fold separation between CD59⁻ and CD59⁺ populations based on fluorescence intensity. Small numbers of negative cells mixed in with positive cells were detected in a highly linear fashion. Mutation dose response curves over a dose range yielding 80% to 20% survival are shown for ethyl methane sulfonate (EMS), mitomycin C (MMC) and lead acetate. EMS and lead acetate exhibited a threshold in response while MMC had a linear dose response over the full dose range. The mutant fraction was measured over time periods ranging up to 35 days following treatment. The mutant fraction peaked at different times ranging from 6 to 12 days after treatment. An additional 14 chemical and physical agents including point mutagens, heavy metals, ionizing and UV radiation, and DNA intercalators and cross linkers, were analyzed for mutagenic potential after doses giving 80% to 20% survival. The results presented here demonstrate the sensitivity and broad-ranging capability of the FCMA to detect mutations induced by a variety of genotoxic agents.     

转录因子Sp1上调前列腺癌细胞中CD59的表达

目的:构建针对Sp1基因siRNA真核表达载体,转染前列腺癌细胞PC-3,研究反式作用因子Sp1时CD59表达的影响.方法:应用siRNA表达载体介导的RNAi技术,构建含特异性sp1基因的重组载体pSUPER-siSp1,脂质体法转染前列腺癌细胞,G418筛选建立稳定表达转染基因的细胞株,Western blotting检测转染细胞中sp1和CD59基因的表达,MTT和染料释放试验判断CD59基因抑制后对补体溶破的抵抗作用.结果:成功构建了Sp1基因siRNA真核表达载体,转染PC-3细胞可表达荧光蛋白,稳定转染的Pc-3细胞Sp1及CD59基因蛋白水平降低,MTT和染料释放实验表明CD59基因受抑制后对补体溶破的抵抗作用降低.结论:siRNA-Sp1重组载体有效地抑制了CD59的表达,降低CD59的抗补体活性,结果证明反式作用因子Sp1是CD59表达调控中重要的转录因子,为探讨CD59在肿瘤细胞中高表达的研究奠定了基础.     

Arabidopsis CURLY LEAF functions in leaf immunity against fungal pathogens by concomitantly repressing SEPALLATA3 and activating ORA59

Arabidopsis non-host resistance against non-adapted fungal pathogens including Colletotrichum fungi consists of pre-invasive and post-invasive immune responses. Here we report that non-host resistance against non-adapted Colletotrichum spp. in Arabidopsis leaves requires CURLY LEAF (CLF), which is critical for leaf development, flowering and growth. Microscopic analysis of pathogen behavior revealed a requirement for CLF in both pre- and post-invasive non-host resistance. The loss of a functional SEPALLATA3 (SEP3) gene, ectopically expressed in clf mutant leaves, suppressed not only the defect of the clf plants in growth and leaf development but also a defect in non-host resistance against the non-adapted Colletotrichum tropicale. However, the ectopic overexpression of SEP3 in Arabidopsis wild-type leaves did not disrupt the non-host resistance. The expression of multiple plant defensin (PDF) genes that are involved in non-host resistance against C. tropicale was repressed in clf leaves. Moreover, the Octadecanoid-responsive Arabidopsis 59 (ORA59) gene, which is required for PDF expression, was also repressed in clf leaves. Notably, when SEP3 was overexpressed in the ora59 mutant background, C. tropicale produced clear lesions in the inoculated leaves, indicating an impairment in non-host resistance. Furthermore, ora59 plants overexpressing SEP3 exhibited a defect in leaf immunity to the adapted Colletotrichum higginsianum. Since the ora59 plants overexpressing SEP3 did not display obvious leaf curling or reduced growth, in contrast to the clf mutants, these results strongly suggest that concomitant SEP3 repression and ORA59 induction via CLF are required for Arabidopsis leaf immunity to Colletotrichum fungi, uncoupled from CLF’s function in growth and leaf development.     

TMEM59 defines a novel ATG16L1‐binding motif that promotes local activation of LC3

Selective autophagy underlies many of the important physiological roles that autophagy plays in multicellular organisms, but the mechanisms involved in cargo selection are poorly understood. Here we describe a molecular mechanism that can target conventional endosomes for autophagic degradation. We show that the human transmembrane protein TMEM59 contains a minimal 19‐amino‐acid peptide in its intracellular domain that promotes LC3 labelling and lysosomal targeting of its own endosomal compartment. Interestingly, this peptide defines a novel protein motif that mediates interaction with the WD‐repeat domain of ATG16L1, thus providing a mechanistic basis for the activity. The motif is represented with the same ATG16L1‐binding ability in other molecules, suggesting a more general relevance. We propose that this motif may play an important role in targeting specific membranous compartments for autophagic degradation, and therefore it may facilitate the search for adaptor proteins that promote selective autophagy by engaging ATG16L1. Endogenous TMEM59 interacts with ATG16L1 and mediates autophagy in response to Staphylococcus aureus infection.     

Cloning and characterisation of a novel holotype crystal protein gene,cry59Ba1, from Bacillus thuringiensis strain Bm59-2, toxic to Spodoptera exigua (Lepidoptera)

Abstract

A novel cry59-type gene, cry59Ba1, was obtained from isolate Bm59-2 and identified from an assembled plasmid genome sequence. This gene was found to encode a polypeptide of 674 amino acid residues with a predicted molecular mass of 75.2 kDa. This polypeptide was 62.1% identical to cry59Aa1. The Cry59Ba1 protein was expressed in the acrystalliferous mutant strain HD73^? and tested against Culex quinquefasciatus (Diptera), Spodoptera exigua (Lepidoptera) and Helicoverpa armigera (Lepidoptera). The bioassay showed Cry59Ba1 protein to be highly toxic to S. exigua (Lepidoptera) (LC50 =26.2 µg/ml, 95% confidence limit, 16.2-75.3 µg/ml). The cloning of cry59Ba1 gene may provide a novel type insecticidal resource for resolving the problem of lepidopteran insects developing resistance to the Cry1 proteins.     

Effect of clotrimazole on cytosolic Ca2+ rise and viability in HA59T human hepatoma cells

Abstract

Clotrimazole is an antimycotic imidazole derivative that interferes with cellular Ca²⁺ homeostasis. This study examined the effect of clotrimazole on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) and viability in HA59T human hepatoma cells. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]_i. Clotrimazole induced [Ca²⁺]_i rises in a concentration-dependent manner. The response was reduced by removing extracellular Ca²⁺. Clotrimazole-evoked Ca²⁺ entry was suppressed by store-operated channel inhibitors (nifedipine, econazole and SK&F96365) and protein kinase C modulators (GF109203X and phorbol, 12-myristate, 13-acetate). In Ca²⁺-free medium, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-tert-butylhydroquinone abolished clotrimazole-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 abolished clotrimazole-induced [Ca²⁺]_i rise. At 10–40?µM, clotrimazole inhibited cell viability, which was not reversed by chelating cytosolic Ca²⁺. Clotrimazole at 10 and 30?µM also induced apoptosis. Collectively, in HA59T cells, clotrimazole-induced [Ca²⁺]_i rises by evoking phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via store-operated Ca²⁺ channels. Clotrimazole also caused apoptosis.     

Thermodynamic principles for the engineering of pH-driven conformational switches and acid insensitive proteins

The general thermodynamic principles behind pH driven conformational transitions of biological macromolecules are well understood. What is less obvious is how they can be used to engineer pH switches in proteins. The acid unfolding of staphylococcal nuclease (SNase) was used to illustrate different factors that can affect pH-driven conformational transitions. Acid unfolding is a structural transition driven by preferential H⁺ binding to the acid unfolded state (U) over the native (N) state of a protein. It is the result of carboxylic groups that titrate with more normal pK_a values in the U state than in the N state. Acid unfolding profiles of proteins reflect a balance between electrostatic and non-electrostatic contributions to stability. Several strategies were used in attempts to turn SNase into an acid insensitive protein: (1) enhancing global stability of the protein with mutagenesis or with osmolytes, (2) use of high salt concentrations to screen Coulomb interactions, (3) stabilizing the N state through specific anion effects, (4) removing Asp or Glu residues that titrate with depressed pK_a values in the N state, and (5) removing basic residues that might have strong repulsive interactions in the N state at low pH. The only effective way to engineer acid resistance in SNase is not through modulation of pK_a values of Asp/Glu but by enhancing the global stability of the protein. Modulation of pH-driven conformational transitions by selective manipulation of the electrostatic component of the switch is an extremely difficult undertaking.