Two lipase active fractions Lip1 and Lip2 were purified from the cell extract of Rhizopus chinensis CCTCCM201021.Both gave a single band on SDS-PAGE after using ammonium sulfate precipitation、Phenyl-Sepharose FF、DEAE-Sepharose FF and Sephadex G100 gel filtration chromatographies.The molecular masses of two lipases were 59.2kD and 39.4kD respectively.Lip1 and Lip2 showed optimal pH at 8.0 and 8.5 and their optimal temperatures were 40℃ and 35℃ respectively.The substrate specificity of the two lipases was obviously different.Lip1 was more specific to long chain fatty acid of p-nitrophenyl esters while Lip2 had a preference for the hydrolysis of short chain fatty acid of p-nitrophenyl esters.Lip1 had 1,3-position specificity for triacylglycerols hydrolysis while Lip2 had nonspecific position.Both lipases were stimulated by Ca 2+、Mg 2+ while SDS had strong inhibition on their activities.Lip1 and Lip2 had good stability in cyclohexane、hexane、heptane and isooctane(30% V/V).

Child ; Electroacupuncture ; adverse effects ; instrumentation ; Electrolysis ; Equipment Failure ; Equipment and Supplies ; adverse effects ; Humans ; Male

Objective: To observe the effect of calcium on biological characteristics (proliferation, apoptosis and cell cycle) of ALC ameloblasts. . Methods: ALC cell lines were cultured in vitro in DMEM medium with high glucose at different concentrations (0, 2.0, 2.5, 3.0 and 3.5 mmol/L CaCl2 aqueous solution) for 24 h and 48 h, respectively. Changes of ALC cells under two kinds of incubation time were observed with an inverted microscope. CCK-8 method was used to analyze the effect of calcium ion on ALC cell proliferation. Hoechst staining was used to observe the effect of calcium ion on ALC cell apoptosis. PI staining and FCM method were used to analyze the effect of calcium ions on the growth cycle of ALC cells. Western blot was used to detect the effect of calcium ions on the expression of Cyclin A, Cyclin B and Cyclin D in ALC cells Results: In the 0 mmol/L CaCl2 group, ALC cells were oval or polygonal in shape, and the cells were closely connected and grew like paving stones. In other concentration groups, the morphology of ALC cells did not change significantly after calcium intervention for 24 h and 48 h. Results of CCK-8 method showed that the survival rate of ALC cells slightly decreased with increasing calcium ions concentration after calcium intervention for 24 h and 48 h. However, there was no significant differences in this trend. Results of Hoechst staining showed that the number of ALC cell apoptosis did not increase significantly after different concentrations of calcium intervention for 24 h and 48 h. With the increase of calcium ion concentration, results of PI staining and FCM method showed that the cell cycle of ALC cells gradually increased in S phase and decreased in G1 and G2 phase gradually. Western blot results showed that the expression of Cyclin A and Cyclin B in ALC cells decreased and the expression of Cyclin D increased after different concentrations of calcium intervention for 24 h and 48 h. Conclusion In this study, calcium has no significant effect on the proliferation and apoptosis of ALC cells. Calcium, however, has an effect on the ALC cell cycle. Results of this study show that calcium ions has no obvious toxic or side effects on the ameloblasts, which could be used to explore the possible mechanism and effect of calcium on dental fluorosis.

Objective To explore the effectiveness and safety of vaginal paravaginal repair(VPVR) plus vaginal bridge repair in the treatment of female pelvic organ prolapse (POP). Methods Sixty-five patients with different defects of pelvic floor underwent VPVR or plus vaginal bridge repair for posterior vaginal wall. Patients were followed up after operation. The cure rate was estimated subjectively and objectively. The patients' quality of life was evaluated by the pelvic floor distress inventory short form 20 (PFDI-20). Results All 65 cases were treated by vaginal hysterectomy and anterior vaginal repair, in which there were 33 cases underwent VPVR while 32 cases underwent VPVR plus middle area repair. Forty concomitant procedures for vaginal bridge repair were also performed. The average operative time was (110.00±20.12) min and blood loss was (119.52±45.33) ml. The symptom of stress urinary incontinence of 25 cases significantly released after operation. Four incision recovery delayed and there were no other complicatious occurred. Patients were followed up for 6-29 months,the objective cure rate was 100.00% (65/65) and subjective cure rate was 92.31%(60/65), and 58 cases (89.23%)improved significantly with the quality of life comparing with that of pre-operation by completing PFDI-20 (P<0.01). Conclusions It is an effective and safe procedure for VPVR plus vaginal bridge repair to correct median to severe anterior vaginal prolapse and posterior vaginal wall prolapse. More clinical trials are needed to evaluate their long-term outcome.

This study was aimed to construct a pEGFP-N1 vector of von Willebrand factor cleaving protease (ADAMTS13, a disintegrin and metalloprotease with a thrombospondin type 1 motifs 13) so as to pave the way for further studying its synthesis and secretion. Human full-length cDNA sequence of ADAMTS13 was acquired by polymerase chain reaction (PCR) with Phusion(®) High-Fidelity (NEB), then the PCR product was double digested with EcoRI and XhoI. After digestion, the ADAMTS13 cDNA sequence was purified and recombined with the pEGFP-N1 vector. The DNA sequence analysis showed that ADAMTS13 was ligated to the pEGFP-N1 vector correctly. After transient expression in HeLa cells, the expression of EGFP could be detected by fluorescent microscopy, and the expression of ADAMTS13 protein could be detected by SDS-PAGE and Western blot. It is concluded that the ADAMTS13-pEGFP-N1 vector is successfully constructed, and it can be widely used in further research on the mechanism of the synthesis and secretion of ADAMTS13.
ADAM Proteins ; genetics ; ADAMTS13 Protein ; Gene Expression ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; HeLa Cells ; Humans ; Transfection

OBJECTIVETo establish an HPLC method for determination of chlorogenic acid and hydrochlorothiazide in Zhenjujiangyapian.

METHODThe HPLC method was carried on C18 column using methanol-0.1% phosphoric acid(20: 80) as mobile phase, and the detection wavelength was 327 nm, the flow rate was 1.0 mL x min(-1) and the temperature of column was 40 degrees C.

RESULTIn the HPLC method, the calibration curve for chlorogenic acid, hydrochlorothiazide were linear in the range of 0.049 6-0.496 (r = 0.999 5) and 1.002-10.02 microg (r = 0.999 8). The average recovery for chlorogenic acid, hydrochlorothiazide were 101.0% and 100. 1%. RSD were 2.0% and 1.4% (n = 9), respectively.

CONCLUSIONThe method is convenient, precise and reliable for determining the content of chlorogenic acid and hydrochlorothiazide in Zhenjujiangyapian.

Antihypertensive Agents ; analysis ; Chlorogenic Acid ; analysis ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; analysis ; Hydrochlorothiazide ; analysis

The theoretical basis and needling techniques of RUAN's needling method in treatment of insomnia are introduced in this paper. Ruan's needling method follows the theory of traditional Chinese medicine and acupuncture, stresses the theory of taking brain as the marrow sea in treatment of insomnia acupuncture. The characteristics of his needling method are that emphasis on acupoints, including positioning accuracy and proper compatibility; think highly of needling method that combines with perpendicular needling, oblique needling, parallel needling, deep needling and shallow needling; emphasis on manipulation and identify qi under the needle to decide reinforcing or reducing method by arrival of qi, excess or deficiency. And the clinical observation of RUAN' s needling method on 30 cases of insomnia is attached.
Acupuncture Points ; Acupuncture Therapy ; instrumentation ; Adult ; Aged ; Female ; Humans ; Male ; Middle Aged ; Sleep Initiation and Maintenance Disorders ; therapy

To evaluate the clinical efficacy and safety of Qinghouyan lozenge in the treatment of acute pharyngitis due to Lung-heat and Yin-deficiency, and compare with Qinghouyan oral Liquid. Totally 144 subjects were enrolled and randomly divided into two groups (72 in the test group and 72 in the control group). The participants in the test group were given Qinghouyan lozenge for 5 days, and those in the control group were given Qinghouyan oral Liquid for 5 days. The effectiveness evaluation indexes were pharyngalgia/odynophagia disappearance rate, overall efficacy of TCM syndromes, TCM syndrome scores, and single syndrome and sign disappearance rate. During the test, the safety was evaluated by vital sign, lab examination indexes and adverse events. The results for the full analysis set showed that the couth disappearance rate, the incidence rate of TCM syndromes, and the throat/uvula congestion disappearance rate of the test group were higher than that of the control group (P < 0.05), with significant differences in the changes in syndrome scores between the two groups (P < 0.05). Altogether 3 adverse events were observed in the test group while 6 adverse events in the control group, without significant differences in the adverse event rate between the two groups (P < 0.05), serious abnormal laboratory examinations and vital signs. In conclusion, Qinghouyan lozenge has better efficacy in treatment of acute pharyngitis due to Lung-heat and Yin-deficiency than Qinghouyan oral liquid, with good safety.
Acute Disease ; Double-Blind Method ; Humans ; Medicine, Chinese Traditional ; Pharyngitis ; drug therapy

Objective To explore the relationship between insulin resistance and other metabolic disorders in patients with essential hypertension (EH).Methods Glucose metabolism rate (GMR) was measured by euglycemic insulin clamp technique,and salt sensitivity was tested by increase in blood pressure after salt load and its decreases alter depletion of sodium in 26 healthy subjects and 84 patients with EH.Results GMR lowered significantly in patients with EH than that in healthy subjects,P

Objective To assess the value of preoperative MRI in predicting the incidence of cement leakage into adjacent discs during percutaneous vertebral augmentation (PVA) for osteoporotic vertebral compression fracture (OVCF).Methods Clinical and radiological characteristics of 127 patients who were treated with PVA for OVCF were analyzed retrospec tively.The following clinical data of these patients were analyzed,including gender,age,location of treated vertebral body and surgical approach.The image features of endplate injury,fracture line extended to the endplate,adjacent intervertebral dick injury and intravertebral cleft were evaluated on the preoperative MRI.The incidence of cement leakage into the adjacent disc were compared for the above factors with statistical methods.Results Totally 127 patients were enrolled in our study,including 179 treated vertebral bodies,358 endplates and 341 adjacent intervertebral discs.The incidence of intradiscal cement leakage was 57.73％ (56/97) in endplate injury sign,60.98％ (25/41) in fracture line extended to endplate sign,35.91％(51/142) in adjacent discs injury sign and 55.56％(20/36) inintravertebral cleft sign.The differences were statistically significant on preoperative MRI in patients with the above signs compared to those who had not (P＜0.05).The incidence of intradiscal cement leakage in percutaneous kyphoplasty (PKP) and percutaneous vertebroplasty (PVP) were 26.67％(16/60) and 42.86％(51/119),respectively,which was significant different (P=0.035).For bone cement volume ≤ 5 ml vertebral bodies,the incidence of intradiscal cement leakage was 31.19 ％ (34/109),lower than bone cement volume ＞ 5 ml vertebral bodies (47.14％ [33/70],P=0.031).There was no statistically significant association between intradiscal cement leakage and age,gender and location of treated vertebral body (P＞0.05).Conclusion Preoperative MRI can predict the leakage of bone cement into adjacent discs effectively during PVA.The PKP and the lower volume of bone cement injection can reduce the risk of intradiscal cement leakage.