Decreased mitochondrial deoxyribonucleic acid and increased oxidative damage in chronic hepatitis C

AIM： To determine whether alteration of the mito- chondria DNA （mtDNA） copy number and its oxidative damage index （mtDNA△CT） can be detected by analysis of peripheral blood cells in hepatitis C virus （HCV）- infected patients. METHODS： This study enrolled two groups of pa- tients aged 40-60 years： a control group and an HCV- infected group in Department of Gastroenterology and Hepatology in Changhua Christian Hospital. Patients with co-infection with hepatitis B virus or human im- munodeficiency virus, autoimmune disease, malignant neoplasia, pregnancy, thyroid disease, or alcohol con- sumption 〉 40 g/d were excluded. HCV-infected pa- tients who met the following criteria were included： （1） positive HCV antibodies for 〉 6 mo; （2） alanine aminotransferase （ALT） levels more than twice the upper lim- it of normal on at least two occasions during the past 6 mo; and （3） histological fibrosis stage higher than F1. The mtDNA copy number and oxidative damage index of HCV mtDNA （mtDNA△CT） were measured in periph- eral blood leukocytes. The association between mtDNA copy number and mtDNA△CT was further analyzed using clinical data. RESULTS： Forty-seven normal controls （male/female： 26/21, mean age 50.51 ± 6.15 years） and 132 HCV- infected patients （male/female： 76/61, mean age 51.65 ± 5.50 years） were included in the study. The geno- types of HCV-infected patients include type 1a （n = 3）, type 1b （n = 83）, type 2a （n = 32）, and type 2b （n = 14）. Liver fibrosis stages were distributed as follows： F1/F2/F3/F4 = 1/61/45/25 and activity scores were A0/ A1/A2/A3 = 7/45/55/25. There were no age or gender differences between the two groups. HCV-infected pa- tients had higher hepatitis activity （aspartate transami- nase levels 108.77 ± 60.73 vs 23.19 ± 5.47, P 〈 0.01; ALT levels 168.69 ± 93.12 vs 23.15 ± 9.45, P 〈 0.01） and lower platelet count （170.40±58.00 vs 251.24 ± 63.42, P 〈 0.01） than controls. The mtDNA copy num- ber was lower in HCV-infected patients than in controls （173.49 vs 247.93, P 〈 0.05）. The mtDNA△CT was higher in HCV-infected patients than in controls （2.92 vs 0.64, P 〈 0.05）. To clarify the clinical significance of these results in HCV-infected patients, their association with different clinical parameters among HCV-infected pa- tients was analyzed. A negative association was found between mtDNA copy number and elevated aspartate transaminase levels （r = -0.17, P 〈 0.05）. Changes in mtDNA copy number were not associated with HCV RNA levels, HCV genotypes, liver fibrosis severity, or inflammatory activity in the liver biopsy specimen. How- ever, a correlation was observed between mtDNA△CT and platelet count （r = -0.22, P 〈 0.01）, HCV RNA level （r = 0.36, P 〈 0.01）, and hepatitis activity （r = 0.20, P = 0.02）. However, no difference in the change in mtDNA△CT was observed between different fibrosis stages or HCV CONCLUSION： Oxidative stress and mtDNA dam- age are detectable in patient＇s peripheral leukocytes. Increased leukocyte mtDNA△CT correlates with higher HCV viremia, increased hepatitis activity, and lower platelet count.

The Influence of Magnetite Nanoparticles on Human Leukocyte Activity

In this contribution the influence of chemically synthesized magnetite particles coated by sodium oleate and PEG （MPEG）, and magnetosomes （MS） was gradually tested on the process of phagocytosis and the metabolic activity （lysozyme and peroxidase activity） in leukocyte. Lysozyme activity is oxygen-independent liquidation mechanisms of engulfed microorganism, peroxidase activity is oxygen-dependent one. The both tested samples MS and MPEG lysed leukocyte cells during incubation. MPEG with concentration 10 and 20 μg/mL lysed almost all leukocytes and their cell viability was in the 14 ± 0.05% range. On the other hand, MS begin to influence leukocytes activity at the concentration of 1 μg/mL and this influence grows with increasing concentration up to 20 μg/mL. MS are more suitable for biological applications than MPEG which are more aggressive material than MS and their using is unavailable for these types of the test mainly for the concentration 10 - 20 μg/mL.

Functional study of p38 mitogen-activated protein kinase based on cell-penetrating peptide delivery system

Objective p38 Mitogen-activated protein kinase （MAPK） is a crossing center of various pathways. In this study, protein transduction system based on human immunodeficiency virus （HIV）-1 transactivator of transcription （TAT）, which is an efficient delivery peptide of the foreign proteins into cells, was employed to study p38 MAPK functions in eukaryotic cells. Methods p38 And its dominant negative form, p38AF, were constructed into pET-His-TAT vector correctly to verify that the recombinant plasmids were well-founded through restriction enzyme digestion and DNA sequencing. The two proteins, His-TAT-p38 and His-TAT-p38AF, were expressed and purified in Escherichia coli by SDS-PAGE. Then they were incubated with ECV304 cells respectively and readily transduced into cells in a time-dependent and dose-dependent manner. The cells were stimulated by sorbitol. Activating transcription factor （ATF） 2 phosphorylation level was checked using Western blot to assess the activity of endogenous p38. Results Compared with controls, it was found that His-TAT-p38 increased the level ofATF2 phosphorylation in sorbitol-stimulated ECV304 cells, while His-TAT-p38AF inhibited it, indicating p38 MAPK protein delivery system based on TAT was constructed successfully. TAT-p38 and its dominant negative form possessed high biological activity after transduction into ECV304 cells by TAT protein delivery system. The results showed that p38AF fused with TAT could inhibit the transduction of endogenous p38 signal pathway in part, and other pathway might regulate p38 phosphorylation. Conclusions Our study provides a novel pathway to inhibit p38 signal pathway and establish a new method to study p38 function.