ABSTRACT
Objective To study the photodynamic therapeutical efficacy of a novel photosensitizer DTP on sensitive gastric cancer cells (SGC7901) and vincristine-resistant gastric cancer cells (SGC7901/VCR).Methods The P-gp expression on the SGC7901 and SGC7901/VCR cell membrane was indirectly confirmed by fluorescence microscopy.The survival rates of SGC7901 and SGC7901/VCR cells were evaluated by cell counting kit (CCK-8) after photodynamic therapy with DTP.The intracellular DTP uptake levels of two types of cell were determined using a fluorescence spectrophotometer,and the intracellular DTP distributions were observed by laser scanning confocal microscopy.Results The novel photosensitizer DTP has considerable photodynamic cytotoxic effect on SGC7901 and SGC7901/VCR cells.However,this effect on the SGC7901NCR cells was relatively weak (P＜0.05),and could not be enhanced by P-gp inhibitor verapamil or cyclosporine A(P＞0.05).The DTP uptake level in SGC7901 cells was higher than that in SGC7901/VCR cells (P＜0.05),and could not be enhanced by P-gp inhibitor verapamil and cyclosporin A (P＞0.05).It was found that DTP distributed in the lysosomes of SGC7901 cells and in the lysosomes and mitochondria of SGC7901/VCR cells.Conclusions The novel photosensitizer DTP is not the substrate of multidrug transporter P-gp,and its weaker photodynamic cytotoxic effect on SGC7901/VCR cells is independent of the P-gp overexpression on its cell membrane,which may be related to the distribution of intracellular DTP in the two types of cell.
ABSTRACT
OBJECTIVE: To study the effect of different graft ratios of PEG on the toxicity in vitro and cellular uptake of PAMAM G5 dendrimers. METHODS: Nuclear magnetic resonance (1H-NMR) and Fourier transform infrared (FT-IR) spectroscopy were used to confirm the structure of PEG-PAMAM G5 dendrimers with four different graft ratios. The particle size and Zeta potential of the nanoparticles were determined by nanoparticle size-Zeta potential analyzer. The toxicity in vitro,cellular uptake, and intracellular localization were tested by hemolysis assay,cytotoxicity assay,cellular uptake test,and laser scanning confocal microscope images,respectively. RESULTS: The particle sizes of dendrimers with PEG graft ratios of 7.8%,14.1%, 20.3%,and 24.2% were (17.05 ± 1.77), (20.77 ± 1.02),(21.68 ± 1.04),and (23.19 ± 0.54) nm,respectively. The Zeta potential decreased from (25.57 ± 1.37) mV of PAMAM G5 to (9.27 ± 0.40) mV of PEG31-PAMAM G5. In addition, the hemolytic toxicity and cytotoxicity of PAMAM G5 dendrimers also markedly decreased especially at high concentrations because of PEG modification. Moreover, the PEG-PAMAM G5 dendrimers with particle diameter of nearly 20 nm not only could be taken in by HBMEC cells, but also accumulated in the cell nucleus. CONCLUSION: Modification of PEG can greatly reduce the toxicity of PAMAM G5 dendrimers in vitro, and the higher the degree of modification, the more obvious is the attenuated effect. The PEG-PAMAM G5 dendrimers with particle diameter larger than 20 nm still can be taken in by HBMEC cells and accumulate in the cell nucleus, which provide a foundation for the further research using modified PEG-PAMAM G5 as a basic carrier for genes and nuclear targeting agents in nano medicine.
ABSTRACT
The UL49.5 gene of most herpesviruses is conserved and encodes glycoprotein N. However, the UL49.5 protein of duck enteritis virus (DEV) (pUL49.5) has not been reported. In the current study, the DEV pUL49.5 gene was first subjected to molecular characterization. To verify the predicted intracellular localization of gene expression, the recombinant plasmid pEGFP-C1/pUL49.5 was constructed and used to transfect duck embryo fibroblasts. Next, the recombinant plasmid pDsRed1-N1/glycoprotein M (gM) was produced and used for co-transfection with the pEGFP-C1/pUL49.5 plasmid to determine whether DEV pUL49.5 and gM (a conserved protein in herpesviruses) colocalize. DEV pUL49.5 was thought to be an envelope glycoprotein with a signal peptide and two transmembrane domains. This protein was also predicted to localize in the cytoplasm and endoplasmic reticulum with a probability of 66.7%. Images taken by a fluorescence microscope at different time points revealed that the DEV pUL49.5 and gM proteins were both expressed in the cytoplasm. Overlap of the two different fluorescence signals appeared 12 h after transfection and continued to persist until the end of the experiment. These data indicate a possible interaction between DEV pUL49.5 and gM.
Subject(s)
Animals , Ducks/virology , Genes, Viral/genetics , Mardivirus/genetics , Membrane Glycoproteins/genetics , Microscopy, Fluorescence , Phylogeny , Polymerase Chain Reaction/veterinary , Viral Envelope Proteins/geneticsABSTRACT
Triptofano (TRP) é metabolizado por duas vias, a via serotonérgica e a via das quinureninas. Na via serotonérgica, TRP é metabolizado a serotonina (5-HT) e, em algumas células, à melatonina (MLT) que pode ser oxidada à N1-acetil-N2-formil-5- metoxiquinuramina (AFMK) e N1-acetil-5-metoxiquinuramina (AMK) por ação de peroxidases. Na via das quinureninas o TRP é diretamente metabolizado à N formilquinurenina (NFK) e em seguida a quinurenina (QUIN). A enzima indolamina 2, 3 dioxigenase (IDO) é uma das responsáveis por esta reação. Dada a importância da IDO na tolerância imunológica e pelo fato desta enzima ser induzível nos propusemos a avaliar a existência de uma regulação cruzada entre esta enzima e a via serotonérgica. Avaliando a interferência de AMK sobre a ação de IDO e a interferência de QUIN sobre a formação de AFMK por peroxidases, observamos uma possível interação entre as vias. AMK é um inibidor competitivo clássico de IDO e o Ki encontrado foi de 0,98 mM. QUIN é um inibidor acompetitivo linear simples da formação de AFMK e o Ki encontrado foi de 0,1 mM. A inibição da formação de AFMK também ocorre para a peroxidase humana (mieloperoxidase, MPO). Além de representarem uma regulação cruzada utilizada in vivo, as inibições encontradas podem ser relevantes para a proposta de novos inibidores de IDO e MPO na terapia imunomodulatória. Dado o nosso interesse pelas enzimas IDO e MPO, avaliamos ainda a localização intracelular destas enzimas em células de peritônio de camundongo, tanto residente como ativada com concanavalina A (Con A). O estímulo com Con A representa uma ativação de linfócitos T mediado por interferon gama (IFN-γ) e foi usado como modelo experimental para avaliar condições de localização em células ativadas. Por imunocitoquímica verificamos que IDO e MPO localizam-se próxima à membrana plasmática sendo que uma leve dispersão apenas de MPO foi observada em células ativadas com Con A. A localização intracelular das duas enzimas é no...
Tryptophan (TRP) is metabolized by two mains pathways, the serotoninergic pathway and the kynurenine pathway. In the serotoninergic pathway, TRP is metabolized to serotonin (5-HT) and, in some cells, to melatonin (MLT). The later can even be oxidized to acetyl-N1-N2-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5 -methoxykynuramine (AMK) by peroxidases. In the kynurenine pathway, TRP is metabolized to N-formylkynurenine (NFK) and to kynurenine (KYN). Indoleamine 2, 3 dioxygenase (IDO) is one of those responsible for this reaction. Since IDO is importat in immune tolerance and the fact that this enzyme is inducible by cytokines we proposed whether there is a cross regulation between this enzyme and the serotoninergic pathway. A possible interaction between MLT and TRP oxidation pathways was shown by the AMK influence on IDO activity and QUIN interference on AFMK formation by peroxidases. AMK was shown to be an IDO classical competitive inhibitor with a Ki of 0.98 mM. QUIN was a peroxidase (horseradish peroxidase, HRP) classical uncompetitive inhibitor and Ki was found to be 0,1 mM. AFMK formation inhibition was also found in human peroxidase (myeloperoxidase, MPO). Beyond the in vivo crosstalk, new IDO and MPO inhibitors in immunomodulatory therapy would be proposed by the compounds shown in this study. Given our interest in IDO and MPO, we also evaluated their intracellular localization in both resident and concanavalin A (Con A) activated mice peritoneum cells. Con A stimulation is a IFN-γ mediated T lymphocytes activation and was our experimental model to evaluate activated cells. In light microscopy we observed IDO and MPO localization near the membrane and MPO only had a dispersed localization in Con A activated cells. Cytoplasm, nucleus and vesicles were the intracellular localization of both enzymes. Interestingly, we found MPO in isolated cells and in cell clusters of two or more cells. MPO was founded on macrophages, B1 cells and cell clusters by...