Site-specific monoPEGylated interferon alpha2a mediated by microbial transglutaminase / 生物工程学报

PEGylation is considered one of the most successful techniques to improve the characteristics of protein drugs including to increase the circulating half-life of proteins in blood and to decrease their immunogenicity and antigenicity. One known PEG modification method is to attach PEG to the free amino group, typically at lysine residues or at the N-terminal amino acid with no selectivity, resulting in a heterogeneous product mixture. This lack of selectivity can present problems when a therapeutic PEGylated protein is being developed, because predictability of activity and manufacturing reproducibility are needed for regulatory approval. Enzymatic PEGylation of proteins is one route to overcome this limitation. Transglutaminases (TGase) are enzyme candidates for site-specific PEGylation. We use human interferon alpha 2a (IFN α2a) as a test case, and predict that the potential modification residues are Gln101 by computational approach as it contains 12 potential PEGylation sites. IFN α2a was PEGylated by Y shaped PEG40k-NH2 mediated by microbial transglutaminase. Our results show that the microbial transglutaminase mediated PEGylation of IFN α2a was site-specific only at the site of Gln101 in IFN α2a, yielding the single mono-conjugate PEG-Gln101-IFN α2a with a mass of 59 374.66 Da. Circular dichroism studies showed that PEG-Gln101-IFN α2a preserved the same secondary structures as native IFN α2a. As expected, the bioactivity and pharmacokinetic profile in rats of PEG-Gln101-IFN α2a revealed a significant improvement to unmodified IFN α2a, and better than PEGASYS.

Clinical Significance of Plasma Lipoprotein-Associated Phospholipase A2 Detecting in Patients with Ischemic Cerebral Stroke / 现代检验医学杂志

Objective To investigate the clinical significance of plasma lipoprotein-associated phospholipase A2 (LP-PLA2) determination in patients with ischemic cerebral stroke(ICS).Methods 124 patients with ICS (ICS group)admitted to Dongfeng Huaguo Hospital in Shiyan City from Jan.2011 to June.2014 and 50 patients with hemorrhagic stroke (hemor-rhagic group)were as the research objects,and 50 healthy controls at the same time were as the control group,compared the plasma Lp-PLA2 levels between the three groups,and compared carotid artery atherosclerotic plaque and plasma Lp-PLA2 levels in patients with different severity of ICS.Results The levels of plasma Lp-PLA2 of the patients of the ICS group,the hemorrhage group and the control group were 58.4±9.6 g/L,30.5±9.2 g/L and 18.7±8.3 g/L,respectively.There were significant differences between the groups (F = 16.741,P = 0.000).The level of plasma Lp-PLA2 of the patients of the plaque group was higher than that of the non-plaque group (66.3 ±9.7 g/L vs 54.1±10.3 g/L,t=5.775,P =0.000),and the unstable plaque group was higher than that of the stable plate group (72.4±9.5 g/L vs 62.8±10.1 g/L,t=4.797,P =0.000).The plasma Lp-PLA2 level of patients with light,medium,heavy ICS were 48.4± 9.2 g/L,60.9 ± 9.6 g/L and 74.5±10.3 g/L,respectively,and the difference was statistically significant between the three groups (F = 13.629,P =0.000).The plasma levels of Lp-PLA2 were positively correlated with NIHSS scores in patients with ICS (r=0.716,P <0.05).Lp-PLA2 in diagnosis of ICS area under the ROC curve was 0.904,the optimal critical value was 45.2 μg/L,the sen-sitivity and specificity were 81.5% and 80.2%.Conclusion The determination of plasma LP-PLA2 has a good effect in di-agnosis of ICS,the plasma level of ICS can be responsed the severity of the patients and the stability of atherosclerotic plaque.

Clinical significance of serum concentrations of Cystatin C in children with kidney disease / 中国医师进修杂志

Objective To explore the clinical significance of serum concentrations of Cystatin C (CysC) in children with kidney disease in different disease courses. Methods The serum concentrations of CysC, creatinine(Cr) and endogenous creatinine clearance rate(Ccr) were determined in 182 children with kidney disease. They were divided into five study groups:normal renal function group (73 cases), compensatory renal disfunction group (44 cases), non-compensatory renal disfunction group (35 cases), renal failure group(22 cases) and end stage of renal failure group(8 cases). Meanwhile 70 healthy children were involved in control group. Relationships among CysC, Cr and Ccr were calculated and the diagnostic efficiency was assessed by comparing the areas under the ROC curves. Results The serum concentrations of CysC increased in different courses of renal function impairment in children and were significantly related to the severity of impairment. CysC was positively correlated to Cr and negatively correlated to Ccr. The areas under the ROC curves of Cr and CysC were 0.764 and 0.725. Conclusions The serum concentrations of CysC can accurately identify different impairment grades of renal function and the function of glomerular filtration. As an indicator of early reduction of renal function, its sensitivities and specificities are superior to Cr and Ccr. CysC can be a sensitive endogenous marker for glomerular filtration rate determination. With simple, convenient and applicable assays methods, CysC is essential for early diagnosis of children kidney diseases.