Proteolytic cleavage is one of the post-translational modifications and plays important roles in many biological processes, such as apoptosis and tumor cell metastasis. The identification of the cleavage events can improve our understanding of their biological functions in these processes. Although proteomic approaches using N-terminal labeling have resulted in the discovery of many proteolytic cleavages, this strategy has its own inherent drawbacks. Labeling of protein C-termini is an alternative approach. Here, we optimized the labeling procedure in the profiling protein C-termini by enzymatic labeling (ProC-TEL) and improved the labeling efficiency for the positive isolation of protein C-terminal peptides and mass spectrometric identification. We applied this approach to a complex protein mixture from Escherichia coli and identified many C-terminal peptides and internal cleaved peptides from more than 120 proteins. From the identified cleavages, we found several previously known internal proteolytic cleavage sites and many novel ones which may play roles in regulating normal biological processes. This work provides a potential new way, complementary to the N-terminomics, for the identification of proteolytic cleavages in complex biological systems.
Cathepsin A ; chemistry ; Protein C ; chemistry ; Protein Processing, Post-Translational ; Proteolysis ; Proteomics

Objective To investigate the abnormality of parameters of thyroid function and incidence of autoimmune diseases in children with vitiligo.Methods A total of 363 children with vitiligo,including 198 males and 165 females were recruited into this study together with 93 normal human controls(55 males and 38 females).The serum levels of free tetraiodothyronine,free triiodothyronine,thyroid stimulating hormone,antithyroperoxidase antibody and thyroglobulin antibody were determined by chemiluminescent immunoassay in these subiects.Results The abnormality of parameters of thyroid function was observed in 43 out of 363(11.8%)patients affected by vitiligo and in 4 out of 93(4.3%)normal human controls;a significant difference was observed between the two groups (P＜0.05).Of the 43 patients wim abnormality of parameters of thyroid function,39 were diagnosed as vitiligo vulgaris,4 as segmental vitiligo.A significant increase Was observed in the incidence of abnormality of parameters of thyroid function in patients with vitiligo vulgaris compared with those with segmental vitiligo(P＜0.05).Conclusion There is an increase in tbe abnormality of parameters of thyroid function in children with vitiligo.

Objective To analyze the clinical features of Hashimoto thyroiditis with thyroid cancer, and provide scientific basis for clinical diagnosis and treatment. Methods The clinical data of 87 patients of Hashimoto thyroiditis with thyroid cancer and 105 patients of Hashimoto thyroiditis were retrospectively analyzed. Results The rates of Hashimoto thyroiditis with thyroid cancer in age <30, 30-39, 40-49, 50-59 and≥60 years were 1/3, 47.5%(29/61), 51.4%(38/74), 36.0%(18/50) and 1/4. The rates of Hashimoto thyroiditis with thyroid cancer in patients of age 30-39 years and 40-49 years were higher than that in patients of age ≥60 years, but there were no statistical differences (χ2=0.327 and 0.418, P>0.05). There were statistical differences in total thyroxine (TT4), thyroid stimulating hormone (TSH), thyroglobulin antibody (TgAb) and thyroid peroxidase antibody (TPOAb) levels between Hashimoto thyroiditis with thyroid cancer and Hashimoto thyroiditis, there were statistical differences (P<0.05 or <0.01). Multiariable Logistic regression analysis result showed that the decreased serum level of TT4 and the increased levels of TSH, TgAb and TPOAb were the correlated factors of Hashimoto thyroiditis with thyroid cancer (P<0.05 or <0.01). Conclusions Low level of TT4 and high levels of TSH, TgAb and TPOAb may increase the risk of Hashimoto thyroiditis with thyroid cancer. The 30-49 years male patients with Hashimoto thyroiditis should be highly suspected of the possibility of merger thyroid cancer.

Hyperglycemia has been identified as one of the important factors involved in the microvascular complications of diabetes, and has been related to increased cardiovascular mortality. Endothelial damage and dysfunction result from diabetes; therefore, the aim of this study was to determine the response of endothelial cells to stressful stimuli, modelled in normal and high glucose concentrations in vitro. Eahy 926 endothelial cells were cultured in 5 mmol/L or 30 mmol/L glucose conditions for a 24 hour period and oxidative stress was induced by exposure to hydrogen peroxide (H2O2) or tumour necrosis factor- α (TNF- α ), following which the protective effect of the glucocorticoid dexamethasone was assessed. Apoptosis, necrosis and cell viability were determined using an ELISA for DNA fragmentation, an enzymatic lactate dehydrogenase assay and an MTT assay, respectively. High glucose significantly increased the susceptibility of Eahy 926 cells to apoptosis in the presence of 500 μmol/L H2O2, above that induced in normal glucose (P<0.02). A reduction of H2O2- and TNF- α -induced apoptosis occurred in both high and low glucose after treatment with dexametha-sone (P<0.05). Conclusion high glucose is effective in significantly augmenting stress caused by H2O2, but not in causing stress alone. These findings suggest a mechanism by which short term hyperglycemia may facilitate and augment endothelial damage.

The pharmacokinetics of 16-dehydropregnenolone （16-DHP）, a sterols compound isolated from Solanum lyratum Thunb., was investigated in rats following a single intramuscular administration （40 mg/kg）. The concentration of 16-DHP in rat plasma was determined by a high performance liquid chromatography （HPLC） method with UV detection. Levonorgestrel was used as the internal standard （IS）. The pharmacokinetic parameters of 16-DHP were derived by non-compartmental method. After a single intramuscular administration, the maximum plasma concentration （Cmax） was （289±25）ng/mL, time to reach Cmax（tmax） was （0.38±0.14） h, the elimination half-life （t1/z） was （2.5±1.1）h, the area under the plasma concentration-time curve from time zero to the time of the last measurable concentration （AUC（0-t）） was （544± 73）ng· h/mL. The results indicated that 16-DHP was absorbed quickly and eliminated rapidly in rats after the intramuscular injection.

The pharmacokinetics of 16-dehydropregnenolone (16-DHP),a sterols compound isolated from Solanum lyratum Thunb.,was investigated in rats following a single intramuscular administration (40 mg/kg).The concentration of 16-DHP in rat plasma was determined by a high performance liquid chromatography (HPLC) method with UV detection.Levonorgestrel was used as the internal standard (IS).The pharmacokinetic parameters of 16-DHP were derived by non-compartmental method.After a single intramuscular administration,the maximum plasma concentration (Cmax,) was (289 ±25)ng/mL,time to reach Cmax(tmax) was (0.38±0.14) h,the elimination half-life (t1/2) was (2.5±1.1)h,the area under the plasma concentration-time curve from time zero to the time of the last measurable concentration (AUC10-t)) was (544 ± 73 )ng· h/mL.The results indicated that 16-DHP was alsorbed quickly and eliminated rapidly in rats after the intramuscular injection.

Objective The aim of this study was to determine if isoflavone genistien has protective effects against high glucose-induced cell apoptosis in human aortic endlthelial cells,and investigate the possible mechanism for this protection.Methods Human aortic endothelial cells subjected to normal (5mmol/L) or high glucose (25mmol/L) were treated with genistein at 0,50,100nmol/L.Parallel experiments were performed with 100nM 17b-estradiol,and also in the presence and absence of the pure anti-estrogen ICI-182,780 (100nmol/L).The effects on cell apoptotic DNA fragmentation were determined using cell death ELISA,and the effects on cellular proliferation were determined using tritiated thymidine incorporation assay.Estrogen receptor expression was detected by Taqman quantitative PCR.Results Genistein at 100nmol/L significantly reduced high glucose-induced DNA fragmentation,and reversed cell DNA synthesis inhibition (P＜0.001) after 24 hours' incubation.The effect of genistein was completely blocked by ICI-182,780administration.Estrogen receptor beta,but not alpha was found to be expressed in these cells.Conclusion Isoflavone genistein shows protection against high glucose-induced cell damage through estrogen receptor beta,reducing apoptotic DNA damage and protecting from the inhibition of cell proliferation.

Fasting plasma preptin level was assayed by RIA in the patients with different glucose tolerances. Regarding fasting plasma preptin levels, it was higher in females than in males. Fasting plasma preptin level was increased in patients with type 2 diabetes as compared with subjects of impaired glucose tolerance and normal controls, and there were positive correlations with diastolic blood pressure, blood triglyceride, total cholesterol, high-density lipoprotein-cholesterol, free fatty acids, 2 h plasma glucose after glucose load, HbA1C and HOMA-IR, suggesting a potential link between preptin and glucose-lipid metabolism and insulin resistance.

A rapid method for the simultaneous determination of daidzein, genistein and formonetin in solanum Lyratum Thunb by high performance liquid chromatography (HPLC) was developed. Separation was achieved on a Diamonsil C18 column (250 mm×4.6 mm, 5 μm) with isocratic elution, using a mobile phase of methanol-tetrahydrofuran-water (44∶3∶53, v/v). The wavelength was set at 260 nm and column was maintained at 35 ℃. The linear ranges of daidzein, genistein and formonetin were 1.0-40.0, 0.1-4.0 and 0.1-4.0 μg/mL, respectively. The average recoveries were between 98.4% and 101.3%. This method could be used for the quality control of Solanum lyratum Thunb due to its simplification, reliability, rapidity and excellent precision.

Objective To study the protective effect of fluvastatin,one of the HMG-CoA reductase inhibitors (statins),against oxygen radical-induced oxidative damages in human aortic endothelial cell,and the role of Bcl-2 in this protection.Methods Human aortic endothelial cells with or without Bcl-2 siRNA transfection were subjected to 1-100 nM of fluvastatin and 100 la hydrogen peroxide for 24 hours.Bcl-2 mRNA and protein expression were measured by Taqman quantitative PCR and Western blotting.Cell apoptosis was measured by normal and fluorescent microscopy and Cell Death Detection ELISA.Results In the Bcl-2-expressed cells,fluvastatin significantly reversed hydrogen peroxide-induced microscopic apoptosis and apoptotic DNA fragmentation,which were accompanied by a markedly upregulation of Bcl-2 expression by fluvastatin.However,the endothelial protection by fluvastatin was completely lost in Bcl-2 siRNA transfected cells.Conclusion Fluvastatin protects human endothelial cells against oxygen radical-induced cell apoptosis in vitro,and this protection seemed to be mediated in a Bcl-2 dependent pathway.(J Geriatr Cardil 12008;5:33-38)