ABSTRACT
We developed histone deacetylase inhibitor (HDACI) prodrugs to enhance the expression of the external genes transfected into human cells with cationic nanoparticles (NPs). We synthesized five kinds of lipid-linked HDACI prodrugs in which n-dodecanoic acid or cholesterol is linked with a potent HDACI, K-182, by an ester bond or a disulfide carbonate linker. The prodrugs were able to admix as a component of NPs, although the intact K-182 was not incorporated into NPs. Namely, NPs composed of cholesteryl-3beta-carboxyamidoethylene-N-hydroxyethylamine and Tween 80 with the 10 mol% K-182 prodrug were prepared as a DNA vector to transfect plasmid DNAs into human prostate cancer cells, PC-3, or human breast cancer cells, Sk-Br-3. The NPs containing K-182 prodrugs with n-dodecanoic acid exhibited two to four times higher the gene expression than the original NPs. The enhancement of the gene expression will be due to the hyperacetylation of core histones caused by intact K-182 degraded from the prodrug in the vector incorporated into the cells.
Subject(s)
Gene Expression Regulation, Neoplastic/drug effects , Histone Deacetylase Inhibitors/administration & dosage , Histone Deacetylase Inhibitors/pharmacology , Nanoparticles/chemistry , Prodrugs/administration & dosage , Prodrugs/pharmacology , Breast Neoplasms/drug therapy , Cell Line, Tumor , Cholesterol/chemistry , Female , Histones/metabolism , Humans , Male , Prostatic Neoplasms/drug therapyABSTRACT
Genetically modified (GM) papaya has not yet been approved for importation into, or cultivation in the European Union (EU) and Japan. A DNA extraction method using the Qiagen DNeasy Plant Mini Kit (PM method) and a method using a buffer containing cetyltrimethyl ammonium bromide (CTAB method) have been adopted as the official Japanese methods for detecting GM foods. However, the amounts of DNA extracted from papaya by these methods are very low. Therefore, we investigated an extraction method to obtain a high yield of DNA from raw or freeze-dried fresh papaya using the Promega Wizard DNA Clean-Up Resin System (WCR). The incubation for the extraction was carried out at 58 degrees C without proteinase K for 15 min. The extract was applied to a mini-column, then the column was washed with 80% isopropyl alcohol, and genomic DNA adsorbed on the column was eluted with TE buffer. The WCR method gave a higher yield of genomic DNA, and was simpler and faster than the PM method or CTAB method. In addition, it could be used to extract genomic DNA from fresh papaya at various stages of ripeness. Based on these results, we propose that the present method using WCR is the most practical and useful way to extract genomic DNA for the purpose of detecting GM papaya.
Subject(s)
Carica , DNA, Plant/isolation & purification , Food Analysis/methods , Food, Genetically Modified , Genome, Plant/genetics , Plants, Genetically Modified , Carica/genetics , Polymerase Chain Reaction/methods , Silica Gel , Silicon DioxideABSTRACT
The yield of genomic DNA extracted from corn-processed foods, such as corn flake and one of the corn snack what is called "Jumbo corn", using an ion-exchange resin type kit (Gtip) has been reported to be very low, and it is thought to be difficult to detect the intrinsic corn gene "Zein" in the foods. Therefore, we developed a new method using Gtip, which we called the "KNG-Gtip method," by modification of the Ministry of Health, Labour and Welfare (MHLW) method using Gtip (MHLW-Gtip method). We compared the KNG-Gtip method, MHLW-Gtip method, the Gtip method for detection of allergen (ALG-Gtip method), and the Gtip method according to the Ministry of Agriculture, Forestry and Fisheries (MAFF) (JAS-Gtip method) in terms of the yield and quality of genomic DNA and the detection probabilities of the PCR-amplified Zein gene. The concentrations of DNA and the detection probabilities of the PCR-amplified Zein gene of genomic DNA extracted from 4 g corn flake and 4 g Jumbo corn by the KNG-Gtip method were larger than those by using the conventional methods. In addition, the PCR-amplified Zein gene from 4 g of corn starch could be detected by the KNG-Gtip method. We propose that the KNG-Gtip method, in which requires sample weight of four grams, is practical and useful to extract genomic DNA from corn flake and Jumbo corn.
Subject(s)
DNA, Plant/isolation & purification , Food Analysis/methods , Zea mays/genetics , Ion Exchange Resins , Nucleic Acid Amplification TechniquesABSTRACT
A simple and rapid HPLC method was developed for the simultaneous determination of nine kinds of preservatives, benzoic acid (BA), sorbic acid (SOA), dehydroacetic acid (DHA), methyl p-hydroxybenzoate (PHBA-Me), ethyl p-hydroxybenzoate (PHBA-Et), isopropyl p-hydroxybenzoate (PHBA-isoPu), propyl p-hydroxybenzoate (PHBA-Pu), isobutyl p-hydroxybenzoate (PHBA-isoBu) and butyl p-hydroxybenzoate (PHBA-Bu), in foods. For solid foods, the preservatives were extracted with methanol. After addition of 5 mmol/L citrate buffer to the extract, the extract solution was cleaned up on an Oasis HLB cartridge. The cartridge was washed with 5 mmol/L citrate buffer and methanol-5 mmol/L citrate buffer (4:6). Then, nine kinds of preservatives were eluted with methanol. The eluent was used for BA, SOA and DHA determination by HPLC. Furthermore, a part of the eluent was cleaned up on a Bond Elut PSA cartridge for p-hydroxybenzoate esters determination by HPLC. Liquid foods were cleaned up after addition of 5 mmol/L citrate buffer without the extraction process, and the subsequent procedure was the same as for solid foods. The recoveries of p-hydroxybenzoate esters from ten kinds of foods fortified at levels of 0.01 and 0.10 g/kg each were 91.5 to 107.4%, and those of BA, SOA and DHA were 76.4 to 104.8%. The quantitation limits of the preservatives in foods were 0.005 g/kg. (Received March 20, 2006)
