We describe a development of a novel high-throughput phagocytosis assay based on a pH-sensitive cyanine dye, CypHer5E, which is maximally fluorescent in an acidic environment. This dye is ideally suited for the study of phagocytosis because of the acidic conditions generated in the intracellular phagocytic vesicles after particle uptake. Use of CypHer5E-labeled particles results in greatly reduced background from noninternalized particles and makes the assay more robust. Additionally, CypHer5E-labeled particles are resistant to fluorescence quenching observed in the aggressive and acidic environment of the phagosome with traditional dyes. The CypHer5E-based assay has been shown to work reliably in a variety of cell types, including primary human monocytes, primary human dendritic cells, primary human endothelial cells, human monocytic THP-1 cell line, and human/mouse hybrid macrophage cell line WBC264-9C. Inhibition of CypHer5E bead uptake by cytochalasin D was studied, and the 50% inhibition concentration (IC50) was determined. The assay was performed in 96- and 384-well formats, and it is appropriate for high-throughput cellular screening of processes and compounds affecting phagocytosis. The CypHer5E phagocytosis assay is superior to existing protocols because it allows easy distinction of true phagocytosis from particle adherence and can be used in microscopy-based measurement of phagocytosis.
Intraperitoneal injection of an unmodified antisense peptide nucleic acid (PNA) complementary to mRNA of the rat neurotensin (NT) receptor (NTR1) was demonstrated by a gel shift assay to be present in brain, thus indicating that the PNA had in fact crossed the blood-brain barrier. An i.p. injection of this antisense PNA specifically inhibited the hypothermic and antinociceptive activities of NT microinjected into brain. These results were associated with a reduction in binding sites for NT both in brain and the small intestine. Additionally, the sense-NTR1 PNA, targeted to DNA, microinjected directly into the brain specifically reduced mRNA levels by 50% and caused a loss of response to NT. To demonstrate the specificity of changes in behavioral, binding, and mRNA studies, animals treated with NTR1 PNA were tested for behavioral responses to morphine and their mu receptor levels were determined. Both were found to be unaffected in these NTR1 PNA-treated animals. The effects of both the antisense and sense PNAs were completely reversible. This work provides evidence that any antisense strategy targeted to brain proteins can work through i.p. delivery by crossing the normal blood-brain barrier. Equally important was that an antigene strategy, the sense PNA, was shown in vivo to be a potentially effective therapeutic treatment.Peptide nucleic acids (PNAs), a new type of DNA analog ( Fig. 1), hold great promise as antisense or antigene drugs, because they are electrically neutral oligomers that are stable against nucleases and proteases, bind independently of salt concentration to their complementary nucleic acids, and have higher affinity for nucleic acids than do DNA͞DNA duplexes (1, 2). Additionally, PNA͞DNA duplexes are much more gene specific, because they are less tolerant of mismatches than are DNA͞DNA duplexes (3). Initial enthusiasm for their use as antisense or antigene drugs was dampened by the fact that these molecules pass poorly into cells (4, 5). Our laboratory reported that unmodified (carrier-free) PNAs, on their direct injection into rat brain, enter neuronal cells and inhibit protein synthesis in a gene-specific manner (6).To determine both the mechanism of action of PNAs and whether PNAs could pass the blood-brain barrier (BBB), brain neurotensin (NT) receptors (NTR1) again were targeted. After a single i.p. injection of antisense PNA to NTR1 (targeted to mRNA) behavioral and physiological responses to NT (antinociception and hypothermia) were specifically and almost completely lost. These results were accompanied by specific reductions in receptor sites as determined by radioligand binding assays. However, there were no changes in mRNA levels. A sensitive assay developed to detect the amount of PNAs in tissue (gel shift assay) confirmed the presence of PNA in brain after i.p. injection. Therefore, these results provided evidence that any antisense strategy targeted to brain proteins can work by i.p. delivery and by crossing the normal (i.e., not compromised by malignancy) BBB. Also, of ...
miRNAs are~22-nt RNAs that bind to the Argonaute family of proteins and have important regulatory roles in plants and animals. Here, we show that miRNAs exhibit targeting activity in cells when delivered as single strands that are 59-phosphorylated and that contain 29-fluoro ribose modifications. Length preferences, chemical modification sensitivity, and genome-wide seed-based targeting all suggest that this activity is Ago-based. Activity could be enhanced by annealing of segmented passenger strands containing non-nucleic acid spacers. Furthermore, screening of randomly generated sequences identified pyrimidine rich 39 cassette sequences that increased single strand activity. These results provide an initial step in the development of single-stranded miRNA mimics for therapeutic use.
market share delegation, sales delegation, asymmetric cost, export subsidy, import tariff, D21, D42, F12, F13, L13,
The purpose of this paper is to explore how strategic tariff policy and welfare are affected by the consumer-friendly initiative of foreign exporting firms. We define a firm that is consumer-friendly or non-profit-based if it considers both its own profit and consumer surplus. This paper extends Brander and Spencer by taking the consideration of consumer-friendly firms into an international duopoly, and within such context examining the tariff policy and welfare. The consumer-friendly initiative that leads to trade liberalization is a 'Win-Win-Win' solution in the sense that it is not only beneficial for foreign exporting firms, but also for the government and consumers of the importing country.
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