The synthesized full-length hGLP-1 gene was cloned into pET-32a(+) to get the recombinant plasmid pET32-GLP-1, which could express a fusion protein containing thioredox, hexahistidine, and rhGLP-1 consecutively. The recombinant plasmid containing hGLP-1 was transformed into E.coli BL21 (DE3) and expressed by IPTG induction. The fusion protein was purified from lysates with Ni?IDA His?Bind affinity chromatography. rhGLP-1 with the purity of 90% was achieved after enterokinase digestion, Ni?IDA His?Bind affinity chromatography again, then was concentrated by ultrafiltration. The purified rhGLP-1 showed a single band on IEF gel with an isoelectric point between pH5.2 and pH5.85. ESI mass spectrometry showed that the molecular weight was 3355.0kDa as expected. rhGLP-1 was digested with trypsin followed by mass analysis and the peptide mapping produced two expected fragments with the molecular weights of 2097.7kDa and 1005.5kDa, respectively. The purified rhGLP-1 also showed obvious biological activity for both lowering plasma glucose and stimulating insulin secretion in mice.

To make a preliminary study on the mechanism of Coptidis Rhizoma(CR) and Rehmanniae Radix(RR) before and after the combined administration in treating type II diabetes mellitus. The type I diabetes animal model in rats was established by fat emulsion and intraperitoneal injection with streptozotocin, in order to compare the hpyerglycemic and hypolipidemic effects of CR, RR and their combined administration of different ratio. The urinary metabolic profiling in rats of Coptidis Rhizoma and Rehmanniae Radix before and after the combined administration was analyzed by using the gas chromatography-mass spectrometry. The differences among groups in metabolome were analyzed by the principal component analysis (PCA). The biochemical index results indicated that both CR and RR before and after the combined administration could lower high blood glucose, hypertriglyceride and high cholesterol. According to the analytical results of PCA of the rats' urine samples, the CR group was the most close to the normal group, with no significant difference in CR and RR group of different combination ratios. Twelve differentiated metabolites were identified to be related to type II diabetes. Compared with the normal group, the CR-treated group showed significant increase in seven differentiated metabolites. Among CR and RR drugs with different combination ratios, CR played a major role and thus acted as the monarch drug. Whereas RR served as the ministerial drug and assisted CR to show the efficacy. This study laid a foundation for the explanation of the combination mechanism of traditional Chinese medicines.
Animals ; Blood Glucose ; metabolism ; Diabetes Mellitus, Type 2 ; blood ; drug therapy ; urine ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Male ; Rats ; Rats, Wistar ; Rehmannia ; chemistry

OBJECTIVE To investigate the protective effect of total saponins from stems and leaves of Panax ginseng (GSLS) on cisplatin (CDDP)-induced kidney damage in mice and its possible mechanism. METHODS Thirty-two male ICR mice were randomly divided into normal control group, CDDP group, and GSLS(150 and 300)+CDDP groups. GSLS was administered to mice by oral gavage once a day for 7 d. On the 7th day, a single injection of CDDP 20 mg·kg-1 was given 1 h after GSLS 150 and 300 mg·kg-1 before GSLS 150 and 300 mg·kg-1 continued to be given for 3 d. Blood urea nitrogen (BUN) and creatinine (CRE) , catalase (CAT) in renal tissue, reduced glutathione (GSH), tumor necrosis factorα(TNF-α) and interleukin 1β(IL-1β) of cisplatin induced mice were detected after 72 h. HE and PAS staining were used to observe the renal histopathological changes;While TUNEL and Hoechst33258 staining were employed to observe apoptosis in kidney tissues. RESULTS Compared with normal control group, CDDP group had a significant reduction in relative body mass (P<0.05), and the level of GSH and CAT in kidney tissues (P<0.05). The level of CRE, BUN, TNF-α, and IL-1βin serum and renal indexes significantly increased (P<0.05, P<0.01), especially BUN and CRE that respectively doubled and quadrupled. CDDP group developed glomerulus swelling, renal tubular expansion and epithelial cell necrosis. Trans?parent tube type of tube cavity appeared, the nucleus pycnosis disappeared, but renal interstitial edema and inflammatory cell infiltration appeared. There was a large amount of glycogen deposition and high expressions of TUNEL positive cells and Hoechst33258 positive cells. Compared with CDDP group, the levels of BUN and CRE in GSLS treatment group significantly decreased (P<0.05, P<0.01) in serum, glycogen deposition was reducted and apoptosis of renal tubular epithelial cells decreased in kidney tissues (P<0.05). The level of TNF-α, IL-1β(P<0.05) and the degree of renal tissue necrosis were significantly reduced (P<0.05) in CDDP+GSLS 300 group, but there was a significant increase in the level of CAT and GSH (P<0.05). CONCLUSION GSLS can protect against mouse kidney injury induced by cisplatin. The mechanism may be related to oxidation, reduced inflammation reaction and resistance to apoptosis.

OBJECTIVE: To investigate the potential risk factors and management of excessive epistaxis after endoscopic endonasal surgery (EES). METHOD: Six hundred and forty-one patients who underwent EES in our hospital from December 2011 to December 2012 were reviewed retrospectively. Factors which potentially affect the incidence of excessive epistaxis after EES were analyzed with univariate and multivariate logistic regression model. RESULT: The incidence rate of excessive epistaxis after EES was 8.4% in our study. Multivariate logistic regression analysis revealed that history of previous EES, along with other four factors, correlated significantly with the occurrence of excessive epistaxis after EES. CONCLUSIONS Previous EES, along with other three factors, may increase the chance of excessive epistaxis after EES, while pre-operative corticosteroid therapy may reduce the risk to some extent.
Adolescent ; Adult ; Aged ; Child ; Endoscopy ; adverse effects ; Epistaxis ; etiology ; Female ; Humans ; Logistic Models ; Male ; Middle Aged ; Multivariate Analysis ; Nasal Surgical Procedures ; adverse effects ; Nose ; surgery ; Postoperative Complications ; etiology ; Retrospective Studies ; Risk Factors ; Young Adult

Objective To develop and optimize a module for the automatic production of N-succinimidyl-4-[18F] fluorobenzoate (18F-SFB) that is used for further 18F labelling C2A domain of Synaptotagmin Ⅰ . The conjugated compound was applied for detecting the tumor apoptosis in rabbit model after chemotherapy. Methods GE TRACERlab and TRACERlab FXF-N modules were modified and programmed to automatically produce 18 F-SFB which was further analyzed by high performance liquid chromatography (HPLC).C2A-glutathione S transferase (GST) was conjugated with 18F-SFB (18F-FB-C2A-GST) and subsequently purified by HPLC. Two rabbits grafted with VX2 lung cancer were first treated with chemotherapy and then,37 MBq of 18F-FB-C2A-GST was administered via the auricular vein. Serial PET/CT imagings were performed at 0.5, 1 and 2 h post-injection respectively. Tumor apoptosis was examined by pathological study. Results The TRACERlab FXFoG and TRACERlab FXF-N modules were successfully adapted to synthesize18F-SFB, with the radiochenmical yield (76.41 ±4.00)% (n = 10), the corrected yield (45.43 ±5.90 ) % and the radiochemical purity about 95%. The whole procedure for labeling 18 F-SFB was about 87 min.From PET/CT imagings, significant uptake was found in the tumor after chemotherapy, but no obvious up-take was found in heart, lungs and liver. HE staining demonstrated large number of apoptotic bodies within the tumor tissues. Conclusions 18 F-SFB can be automatically synthesized. 18F-FB-C2A-GST might be useful for the detection of apoptosis in tumor after chemotherapy.

OBJECTIVETo evaluate the value of FOXP1 and Cyclin E gene in primary central nervous system lymphoma(PCNSL) of immunocompetent patients on prognostic significance.

METHODSClinical data of 71 patients with newly diagnosed PCNSL from 2002 to 2007 was analyzed retrospectively. Immunohistochemistry method (HRP-EnVision(TM)) was performed to observe the expression of FOXP1 and Cyclin E gene in tumor tissue samples. The survival was analyzed by Kaplan-Meier survival curve, survival factors analysis by the Log-rank test and COX proportional hazards regression model.

RESULTSFOXP1 positive was observed in 35 of 51 patients (68.63%) and Cyclin E staining was present in 29 of 50 cases (58.00%). FOXP1(+) patients had a shorter overall survival (OS) than FOXP1(-) ones. 2-year OS rate in FOXP1(+) and FOXP1(-) patients were 23.33% and 73.56%, respectively(P = 0.0015). Cyclin E(+) patients had a shorter overall survival(OS) than cyclinE(-) ones. 2-year OS rate in Cyclin E(+) and Cyclin E(-) patients were 17.56% and 69.76%, respectively (P = 0.0017). Multivariate analysis showed that Cyclin E expression was an independent prognostic factor for shorter OS (P = 0.048). FOXP1 expression might be an important prognostic factor for shorter OS (P = 0.065).

CONCLUSIONCyclin E expression is an independent prognostic factor and FOXP1 expression is a possible prognostic factor for poor clinical outcome in patients with PCNSL.

Aged ; Aged, 80 and over ; Central Nervous System Neoplasms ; genetics ; metabolism ; Cyclin E ; genetics ; metabolism ; Female ; Forkhead Transcription Factors ; genetics ; metabolism ; Humans ; Lymphoma, Non-Hodgkin ; genetics ; metabolism ; Male ; Middle Aged ; Prognosis ; Repressor Proteins ; genetics ; metabolism ; Retrospective Studies ; Survival Rate

The present study was aimed at investigating the expression of calbindin-D28k (CaBP-D28k) in human fallopian tube, which were collected from 33 childbearing age women undergoing abdominal hysterectomy with adnexectomy for benign disease in the pelvic cavity. These women had normal menstrual cycle and history of normal pregnancy. Isthmus, ampullary and umbrella segments of fallopian tubes were respectively collected. These specimens were divided into 6 groups based on their menstrual cycles: early-proliferative stage (n=6), mid-proliferative stage (n=5), late-proliferative stage (n=5), early-secretory stage (n=7), mid-secretory stage (n=5) and late-secretory stage (n=5). The expressions of CaBP-D28k protein and mRNA in fallopian tubes were determined by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) methods. Positive expressions of CaBP-D28k protein and mRNA were observed in human fallopian tubes. There was no significant difference in the expression of CaBP-D28k protein among the isthmus, ampulla and umbrella segments in the same phase of menstrual cycle (P>0.05). However, in the menstrual cycle, the expression level of CaBP-D28k protein in the epithelium was the lowest during the early- and mid-proliferative stages and increased in both the late-proliferative and early-secretory stages (P<0.05), and then decreased in the mid- and late-secretory stages (P<0.05). The expressed CaBP-D28k protein was disposed to gobbets or dispersed sheets in cytoplasm in the early- and mid- proliferative stages, and showed concentrated granules on the top of cells in the late-proliferative and early-, mid-secretory stages. Then in the late-secretory stage redistribution renewed as in the early- and mid-proliferative stages. The CaBP-D28k mRNA obviously increased in the late-proliferative and early-secretory stages (P<0.05). These findings indicate that the expressions of CaBP-D28k protein and mRNA exist in human fallopian tubes and exhibit a cyclic change.
Calbindin 1 ; metabolism ; Fallopian Tubes ; metabolism ; Female ; Gene Expression ; Humans ; Menstrual Cycle

To develop an analytic method for qualitative and quantitative analysis of dodecatetraenamides A, B in 42 samples of two official species of Asari Radix et Rhizoma( ARR) (37 samples of Asarum heterotropoides var. mandshuricum with different collection time and 5 samples of Asarum sieboldiivar. seoulense). The HPLC-IT-TOF-MS/MS methods for the qualitative and UPLC-PDA methods for the quantitative analysis were established. Dodecatetraenamides A, B were identified by comparing the retention time, UV absorption spectrum and quasi-molecular ion peak [ M + H]+ with the reference compound using HPLC-IT-TOF-MS/MS. The content of dodecatetraenamides A and B in ARR were determined by UPLC-PDA. The separation was successfully carried out on a ACQUITY UPLC BEH C18 (2.1 mm x 100 mm, 1.7 µm) column eluted with mobile phases of water (A) and acetonitrile (B) in gradient program (0-3 min, 35% B; 3-5 min, 35%-36% B; 5-6 min, 36%-43% B; 6 min-11 min 43% B; 11-12 min, 43%-100% B). The column temperature was 45 °C, and the detection wavelength was set at 254 nm. The flow rate was 0.6 mL · min(-1). On one level mass spectrometry scanning, the results showed that the quasi-molecular ion [M + H] + of both dodecatetraenamides A and B were m/z 248.20. The quantitative method with UPLC-PDA has made the baseline separation of the constituents, which were reported as mixtures in the most literatures. The average recovery of dodecatetraenamides A and B were 97.90% and 99.86%, the relative standard deviation were 0.4% and 1.1%, respectively. The contents of dodecatetraenamides A, B in all ARR samples was in the range of 0.11-3.89 and 0.24-6.65 mg · g(-1). Their contents reduced with the extension of storage time. Compared with the samples of 2013, the average content of the two constituents in the samples collected in year 2002-2003 reduced 34% and 36%, respectively (P < 0.05). Compared the A. sieboldii var. seoulense and A. heterotropoides var. mandshuricum with the same collective time and production area, the average contents of the two constituents in latter were up to (1.59 ± 0.75) mg · g(-1) and (2.90 ± 1.17) mg · g(-1), respectively, significantly higher than that in A. sieboldii var. seoulense (dodecatetraenamide A were (0.78 ± 0.52) mg · g(-1), dodecatetraenamide B were (1.69 ± 0.83) mg · g(-1)) (P < 0.05). The content of the dodecatetraenamide A in overground part was in the range of 0.11-0.33 mg · g(-1), dodecatetraenamide B was 0. 24-0.60 mg · g(-1), which were much lower than that of the underground part of ARR (dodecatetraenamide A was in the range of 0.73-3.89 mg · g(-1), dodecatetraenamide B was 2.11-6.24 mg · g(-1)). The method was certified to be simple, accurate and reliable and could be used for qualitative and quantitative analysis of dodecatetraenamide A and B in different species of ARR, also can be used for the comprehensive quality control of traditional Chinese medicine, Asari Radix et Rhizoma.
Amides ; chemistry ; Asarum ; chemistry ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Mass Spectrometry ; Molecular Structure ; Rhizome ; chemistry

As one of the major sources of infection, viruses could infect all organisms including bacteria, plants, animals, and humans. Infectious diseases caused by viruses pose a great threat and damage to human health and economic activities all over the world. Adaptor-associated protein kinase 1 (AAK1) is a member of the Ark1/Prk1 family of serine/threonine kinases and a specific key kinase regulating the phosphorylation of AP-2 protein μ2 subunit T156. In the past, AAK1 has been regarded as a feasible biological target for the treatment of nerve pain. Recently, scientists have found that inhibiting AAK1 can regulate endocytosis and inhibit virus invasion into cells. Therefore, AAK1 could be the potential target of anti-virus therapy. This paper reviews the research progress of small molecule AAK1 inhibitors in the field of antiviral, analyzes the future research directions and challenges, and provides new ideas for the development of antiviral drugs targeting AAK1.

AIM To establish an HPLC method for the simultaneous content determination of echinacoside,verbascoside,aucubin,pinoresinol diglucoside,epimedin A,epimedin B,epimedin C,icariin and baohuoside Ⅰ in Jingang Tablets (Cistanches Herba,Eucommiae Cortex,Epimedii Folium,etc.).METHODS The analysis of 50％ methanol extract of this drug was performed on a 30 ℃ thermostatic Agilent TC-C18 column (250 mm × 4.6 mm,5 μm),with the mobile phase comprising of (methanol-acetonitrile)-0.2％ phosphoric acid flowing at 1.0 mL/min in a gradient elution manner,and the detection wavelengths were set at 330,208 and 270 nm.RESULTS Nine constituents showed good linear relationships within their own ranges (r ＞ 0.999 0),whose average recoveries were 96.64％-100.08％ with the RSDs of 0.71％-1.76％.CONCLUSION This sensitive and accurate method can be used for the quality control of Jingang Tablets.