Objective. To establish a PCR- SSP method for discriminating as many HLA- A* 02 alleles, which could easily be introduced into a routine laboratory. Methods. In this study we typed HLA- A* 02 polymorphisms by a sequen...Objective. To establish a PCR- SSP method for discriminating as many HLA- A* 02 alleles, which could easily be introduced into a routine laboratory. Methods. In this study we typed HLA- A* 02 polymorphisms by a sequence- specific primer (SSP) method, which involved round 1 and round 2 PCR reactions to detect 17 HLA- A* 02 alleles (they are HLA- A* 0201- 0217 alleles) covering exon 2 and exon 3. Results. We have found that DNA sample concentration and purity were the most important variables in determining the quality of the results. For identifying correct band size, the size marker used was important. We noticed that different PCR machines performed differently. By this method, we detected 20 HLA- A* 02 positive genomic DNA samples and found 4 kinds of HLA- A* 02 alleles. They were HLA- A* 0201, 0203, 0206 and 0210. Conclusion. The HLA- A* 02 PCR- SSP method was proven to be a reliable and easily applicable typing method. Our results suggest that the SSP described here provides an optimal HLA- A* 02 typing technique that may be useful in selecting donor- recipient pairs in bone marrow transplantation between unrelated individuals.展开更多
Ginsenosides, the main active constituents of Panax ginseng Meyer （P. ginseng）, have potential therapeutic effects. All tested ginsenosides except gin- senoside F1 have previously been reported in inflammation studi...Ginsenosides, the main active constituents of Panax ginseng Meyer （P. ginseng）, have potential therapeutic effects. All tested ginsenosides except gin- senoside F1 have previously been reported in inflammation studies using the RAW 264.7 macrophage cell line. We ex- amined the anti-inflammatory effects of single sugar moiety ginsenosides such as compound K （CK）, Rh2, Rhl, and F1 that were isolated from P. ginseng through in silico docking studies. We investigated their biological activity predictions, including absorption, distribution, metabolism, excretion, and toxicity and PASS properties, on the suppression of NF- κB, followed by in vitro validation in lipopolysaccharide （LPS）-stimulated RAW 264.7 macrophage cells. The molecular docking results of our study showed that all treated ginsenosides are non-toxic and may be drug-like molecules. The molecular binding interactions of these ginsenosides with the active residues of NF-κB noticeably support their anti-inflammatory activity. CK and Rhl sig- nificantly reduced the production of nitric oxide, cyclooxy- genase-2 （COX-2）, and pro-inflammatory cytokines such as prostaglandin E2 and tumor necrosis factor alpha （TNF-α） in a dose-dependent manner. Real-time PCR and Western blot analyses further confirmed that protopanaxadiols （PPDs） and protopanaxatriols （PPTs） inhibitory effects may have been due to the down-regulation of TNF-α, inducible nitric oxide synthase, COX2, nuclear factor kappa B （NF-κB）, and I kappa B kinase. The expression of co-stimulatory molecules such as ROS was also inhibited by CK and Rhl in a dose- dependent manner. Furthermore, activation of NF-κB in LPS-stimulated RAW 264.7 macrophages was significantly suppressed by CK and Rhl. Taken together, these results provide evidence that PPD- and PPT-type ginsenosides in- cluding CK and Rhl may exhibit strong anti-inflammatory effects by inhibiting pro-inflammatory mediators through down-regulation of NF-κB.展开更多
文摘Objective. To establish a PCR- SSP method for discriminating as many HLA- A* 02 alleles, which could easily be introduced into a routine laboratory. Methods. In this study we typed HLA- A* 02 polymorphisms by a sequence- specific primer (SSP) method, which involved round 1 and round 2 PCR reactions to detect 17 HLA- A* 02 alleles (they are HLA- A* 0201- 0217 alleles) covering exon 2 and exon 3. Results. We have found that DNA sample concentration and purity were the most important variables in determining the quality of the results. For identifying correct band size, the size marker used was important. We noticed that different PCR machines performed differently. By this method, we detected 20 HLA- A* 02 positive genomic DNA samples and found 4 kinds of HLA- A* 02 alleles. They were HLA- A* 0201, 0203, 0206 and 0210. Conclusion. The HLA- A* 02 PCR- SSP method was proven to be a reliable and easily applicable typing method. Our results suggest that the SSP described here provides an optimal HLA- A* 02 typing technique that may be useful in selecting donor- recipient pairs in bone marrow transplantation between unrelated individuals.
基金supported by a post-doctoral fellowship grant from the Kyung Hee University in 20120351
文摘Ginsenosides, the main active constituents of Panax ginseng Meyer （P. ginseng）, have potential therapeutic effects. All tested ginsenosides except gin- senoside F1 have previously been reported in inflammation studies using the RAW 264.7 macrophage cell line. We ex- amined the anti-inflammatory effects of single sugar moiety ginsenosides such as compound K （CK）, Rh2, Rhl, and F1 that were isolated from P. ginseng through in silico docking studies. We investigated their biological activity predictions, including absorption, distribution, metabolism, excretion, and toxicity and PASS properties, on the suppression of NF- κB, followed by in vitro validation in lipopolysaccharide （LPS）-stimulated RAW 264.7 macrophage cells. The molecular docking results of our study showed that all treated ginsenosides are non-toxic and may be drug-like molecules. The molecular binding interactions of these ginsenosides with the active residues of NF-κB noticeably support their anti-inflammatory activity. CK and Rhl sig- nificantly reduced the production of nitric oxide, cyclooxy- genase-2 （COX-2）, and pro-inflammatory cytokines such as prostaglandin E2 and tumor necrosis factor alpha （TNF-α） in a dose-dependent manner. Real-time PCR and Western blot analyses further confirmed that protopanaxadiols （PPDs） and protopanaxatriols （PPTs） inhibitory effects may have been due to the down-regulation of TNF-α, inducible nitric oxide synthase, COX2, nuclear factor kappa B （NF-κB）, and I kappa B kinase. The expression of co-stimulatory molecules such as ROS was also inhibited by CK and Rhl in a dose- dependent manner. Furthermore, activation of NF-κB in LPS-stimulated RAW 264.7 macrophages was significantly suppressed by CK and Rhl. Taken together, these results provide evidence that PPD- and PPT-type ginsenosides in- cluding CK and Rhl may exhibit strong anti-inflammatory effects by inhibiting pro-inflammatory mediators through down-regulation of NF-κB.
