ABSTRACT
Accumulating evidence has shown that the cell-penetrating peptide TAT can be applied to deliver different types of drug molecules, including nucleic acids, proteins and small molecule drugs. Usually TAT delivers cargoes on the basis of their covalent bonds or non-covalent interactions. However, there are few reports on the delivery of proteins by TAT in a non-covalent manner, and no quantitative comparisons have been made on the protein delivery ability of TAT in fusion and non-fusion manners. In order to explore the ability of TAT to deliver proteins in non-fusion manner, here we used fluorescence microscopy and flow cytometry to investigate the ability of TAT to deliver enhanced green fluorescent protein (EGFP) into non-small cell lung cancer cells A549 in a non-fusion manner. It was found that TAT could deliver EGFP into A549 cells, and its delivery ability was positively correlated with its concentration. In addition, the fusion protein TAT-EGFP was overexpressed and purified, and its permeability across cell membrane was also investigated. In this paper, based on quantitative comparison, we found that the delivery of EGFP by TAT in fusion manner is significantly efficient than that of TAT in non-fusion manner. This is the report that TAT can deliver EGFP in a non-fusion manner. Although its delivery efficiency remains to be improved as compared with the fusion manner, the non-fusion manner has shown incomparable advantages in ease of operation, suggesting that it is also a candidate for delivery strategy in the future.
ABSTRACT
OBJECTIVE:To provide ideas for revise and improve the standard and related method of the quality control of ben-zalkonium chloride in Chinese Pharmacopoeia (2015 edition,Ⅱ). METHODS:The standards and related methods of the quality control of benzalkonium chloride in Chinese Pharmacopoeia(2015 edition,Ⅱ),British Pharmacopoeia(2013 edition),European Pharmacopoeia (7.0 edition) and United States Pharmacopoeia (36 edition) were comprehensively compared. RESULTS:Com-pared with Chinese Pharmacopoeia(2015 edition,Ⅱ),the standards abroad provided the component and the ratio of the benzalko-nium chloride substituted homolog,the method for ammonia compound test had higher sensibility,it also added the test for benzyl alcohol,benzaldehyde and benzyl chloride impurity,as well as the component ratio test and average relative molecular mass calcu-lation. CONCLUSIONS:The standard and related method of the quality control of benzalkonium chloride in Chinese Pharmacopoe-ia(2015 edition,Ⅱ)still need to be further improved.
ABSTRACT
OBJECTIVE:To establish a method for the content determination of Aciclovir cream. METHODS:Centrifugal parti-tion chromatography(CPC)was conducted. The solvent system was hexane-acetonitrile-water(2∶1∶1,V/V/V),the injection vehicle was an aqueous solution of 5%Tween 80 and the volume was 1.0 ml;the flow rate was 5 ml/min;the wavelength was 254 nm. RE-SULTS:There was a good linear relationship between quality concentration and peak area in the range of 0.012 7-0.126 7 mg/ml (r=0.998 7). The RSD of precision,stability and reproducibility tests was all no more than 2.0% and the average recovery was 97.34%(RSD=0.90%,n=9). CONCLUSIONS:The method is with high precision and accuracy,and can be used for the content determination of principal components of Aciclovir cream.
ABSTRACT
Objective:To establish an HPLC method for the determination of gabapentin and the related substances in gabapentin capsules. Methods:A Kromasil C8 column (250 mm × 4. 6 mm,5μm)was used. The mobile phase was methanol-0. 01 mol·L-1 po-tassium dihydrogen phosphate solution (adjusting pH to 6. 9 with 2 mol·L-1 potassium hydroxide) (40∶60). The detection wave-length was 210 nm. The column temperature was 40℃ and the injection volume was 20μl. The main component and the known impuri-ty were determined by an external standard method. The unknown impurities were determined by a self-control method. Results: The calibration curve of gabapentin was linear within the range of 2.5-20.0 mg·ml-1(r=0.999 9). The average sample recovery was 100. 74% with RSD of 0. 24%(n=5). Conclusion:The method is simple, accurate,specific and applicable in the determination of gabapentin and the related substances in gabapentin capsules.
ABSTRACT
In order to confirm the role that the 49th amino acid residue plays in enzymatic inactivity of Glutamine 49 phospholipase A2(Gln49-PLA2),site-directed mutagenesis of its 49th amino acid gene codon was conducted using PCR.Aspartic acid 49 phospholipase A2(Asp49-PLA2-Q49D-PLA2),the mutant of Gln49-PLA2 was expressed in E.coli with pET32a+ vector.The fusion protein,expressed as inclusion body,after being denatured,was on-column refolded and purified by immobilized metal affinity chromatography(IMAC),and then cleaved by Factor Xa.The mature Q49D-PLA2 mutant was obtained by Hitrap SP cation exchange and Superdex 75 gel filtration chromatography,with the recovery rate of 1.3%,and the specific activity of the mature Q49D-PLA2 mutant was 72 U/mg.It has been demonstrated that the 49th glutamine amino acid residue is the main reason in enzymatic inactivity of Gln49-PLA2 and the results are helpful for denatured protein refolding,especially in rich disulfide bonds conditions.