A fusion protein of glucagons-like peptide-1 and human serum albumin (GLP-1/HSA) was expressed and secreted into the fermentation broth with recombinant Pichia pastoris. The productivity of expressed GLP-1/HSA could reach 63.6mg/L in 10L fermentor. After concentrated with hollow-fiber ultrafiltration membrane, GLP-1/HSA was purified from fermentation broth by hydrophobic chromatography, negative ion exchange chromatography and gel filtration chromatography in turn. The HPLC analysis showed that the purified GLP-1/HSA had an overall purity of 95.8%. Furthermore, the analysis of in vivo activity indicated that GLP-1/HSA had the bioactivity of native GLP-1, and could significantly reduce blood glucose level 4h after intraperitoneal administration. It was concluded that a great deal of GLP-1/HSA with higher purity could be harvested by Pichia pastoris expression system and the established purification methods. Preliminary studies show a new potential for developing the long-acting GLP-1 analogs for clinical applications.

Streptomyces sp. A048 was cultured in a complete medium to the last stage of log phase,the hyphae were washed and collected by centrifugation. Then the hyphae were inoculated in liquid medium for chitinase production using two-step fermentation. Activity of chitinase produced by two-step fermentation was 1.1 times higher than that from one-step fermentation,and ferment cycle was for 54 hours,which was 66 hours shorter than that of one-step fermentation. The hyphae and the powder of chitin were co-immobilizated and cultured in liquid medium for 36 hours,activity of chitinase was 1.8 times higher than that from one-step fermentation,and ferment cycle was 54h shorter than that of one-step fermentation. By adding 0.4% cellulose to two-step fermentation,activity of chitinase was 18.52 U/mL that was 4 times higher than that from the control and 10 times higher than that from one-step fermentation. Two step fermentation with chitin and cellulose may be the optimal fermentation process to produce Chitinase from Streptomyces sp. A048.

Objective To investigate dental caries in 3-year-old children in Jing′an District of Shanghai and analyze the influence of oral cleaning behavior on caries in children. Methods Six hundread 3-year-old children from 12 kindergartens in Jing′an District, Shanghai were randomly selected by cluster sampling method for oral examination, and a self-administered questionnaire survey on children′s oral cleaning behavior was completed by parents.SPSS 22.0 software package was used for statistical analysis. Results Among the 600 children, the prevalence of dental caries was 42.50%, and the average dmft index was 1.76±2.92.Children who did not brush their teeth had a statistically significant increase in rate of dental caries(χ²=29.995, P < 0.001).Children who brushed their teeth for more than three minutes each time had a statistically significant lower rate of caries(χ²=3.918, P=0.048).Children whose parents helped their children brush their teeth every day had a lower rate of caries than those who brushed teeth by themselves, and the difference was statistically significant (χ²=8.718, P=0.013).Logistic regression analysis showed that age of beginning brushing teeth, times of brushing teeth per day and length of brushing teeth per time were all influence factors for caries in children (P < 0.05). Conclusion For the prevention and control of dental caries in 3-year-old, proper oral cleaning behavior is very important.We should make parents as a priority group for doing oral hygiene in children.Schools should strengthen oral health education for parents.Medical institutions should guide and monitor the effectiveness of oral hygiene in children.It is important to reduce the rate of dental caries in preschool children from three aspects:family, school and medical institutions.

Objective To explore the alteration of brain pain functional areas in patients with functional dyspepsia (FD) irritable bowel syndrome (IBS) overlap by rectal balloon volume stimulation and magnetic resonance imaging (MRI) and the differences with IBS alone patients and healthy individuals were compared.Methods A total of 11 IBS alone patients,16 IBS overlapped with FD patients (IBS-FD) and 10 healthy controls were recruited.Sensory thresholds and visual analogue scale (VAS) were recorded during the rectal balloon air injection process. The changes of brain activated areas were analyzed by functional MRI (fMRI) when the rectum was stimulated at the volume of 50,100 and 150 ml.The data were analyzed by least significant difference (LSD) test.Results Under rectal volumetric stimulation,the sensory thresholds of IBS-FD group and IBS alone group were (53.14 ± 16.05) ml and (59.20 ± 20.55) ml and the difference was not statistically significant (LSD test,P＞0.05).There was no significant difference in VAS score between IBS alone group and IBS-FD group (LSD test,P＞0.05).Rectal stimulated under different volume,the results of fMRI indicated the activation of anterior cingulated cortex, dorsolateral prefrontal cortex,postporietal cortex,thalamus and insular cortex in both IBS alone group and IBS-FD group.And there was no significant difference in activated areas and intensity between IBS alone group and IBS-FD group (LSD test,P＞0.05).Conclusions There was no significant difference in activations of brain areas between IBS alone and IBS-FD patients under rectal volumetric stimulation. Under rectal volumetric stimulation,although symptoms overlapped,there was no evidence of the overlap of braingut axis and visceral hypersensitivity between IBS alone and IBS-FD.

To prepare ginseng saponin Compound K with ultrasound-assisted total zymolytic ginseng saponins. The conversion rate was taken as the index to detect the pre-treatment factors such as ultrasonic power and ultrasonic time, as well as the impact of enzymatic factors, such as pH value, temperature, concentration of substrate, dosage of enzyme and reaction time, on the conversion rate. The response surface method was used to optimize the preparation conditions. The enzymolytic products were identified with MS, 1H-NMR and 13C-NMR. The results showed that the optimum conditions of the ultrasound-assisted enzymolysis were 250 W for ultrasonic power, 15 min for ultrasonic time, 5.5 for enzymolytic pH, 50 degrees C for enzymolytic temperature, 36 h for enzymolytic time, 4:5 for enzymolytic dosage: substrate and 1.0 g x L(-1) for concentration of substrate. The relative molecular mass of reaction products was 622.4. Therefore, the nuclear magnetic map verified that the reaction product was rare ginseng saponin Compound K. Under the above conditions, based on the total zymolytic ginseng saponins, the conversion rate of rare ginseng saponin Compound K was 6.91% in proportion to the total of ginsenosides. The process features gentle reaction conditions, high conversion rate and simple and reliable process, which is suitable for industrial production.
Chemistry, Pharmaceutical ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Enzymes ; chemistry ; Panax ; chemistry ; Plant Roots ; chemistry ; Saponins ; chemistry ; isolation & purification ; Ultrasonics ; methods

Objective To investigate whether expression of XAF1 mediated by edenovirus vector AdS/F35 could induce apoptosis in pancreatic cancer cell line BxPC-3 and its possible mechanisms.Methods Preconstructed recombinant Ad5/F35-XAF1 virus and negative control Ad5/F35-Null was tranfected into BxPC3:the expression of XAF1 mRNA and protein before and after tranfection was,analyzed by semi-quantitative RT-PCR and Western blot.The expressions of proteins including Caspase-3,PARP,Caspase-8 and bcl-2 were detected by Western blots.Cell apoptosis was assessed by Annexin-v/PI and TUNEL staining.Results After Ad5/F35-XAF1 tranfection,XAF1 mRNA and protein expression significantly increased,and the difference was statistically significant when compared with control group and Ad5/F35-Null group(P＜0.05).The apoptosis rate was(19.90±3.09)%and(9.29±2.13)%,which was significantly different(P＜0.01)from those in the Ad5/F35-Null group[(6.72±0.7)6%,(2.73±0.51)%]or in the control group[(7.22±1.53)%,(1.56±0.47)%].The expression of Caspase-3,PARP and Caspase-8 significantly increased,but the expression of bcl-2 decreased.Conclusions XAF1 plays a major role in the apoptosis of pancreatic cancer that acts thriugh the activation of death receptor pathway and mitochondrial pathway of apoptosis.

BACKGROUND:There are reports concerning differentiation of adipose-derived mesenchymal stem cells(ADSCs)into chondrocytes using gene transfection technique.However,the transfection of adenovirus and adeno-associated virus into ADSCs is various.It is controversial whether adeno-associated virus(AAV)can transfect ADSCs.OBJECTIVE:To observe the enhanced green fluorescent protein(EGFP)expression following Ad5-EGFP and rAAV2-EGFP transfection into ADSCs,and investigate the cell proliferation ability following transfection.METHODS:ADSCs were isolated from the adipose tissue,which was from 6-month-old New Zealand albino rabbit back and neck by mechanical digestion and enzyme digestion,then ADSCs were cultured and amplified in vitro.ADSCs were infected with Ad5-EGFP and rAAV2-EGFP,and the EGFP expression was observed.A total of 2 μL sodium butyrate(1 mol/L)was added into the medium after rAAV2-EGFP transfection.MTT assay was used to detect the gene transfection effects on reproductive activity of ADSCs.RESULTS AND CONCLUSION:ADSCs isolated and cultured in vitro were flat,long-spindle and amplified stabry.The cell morphology was uniform.Many green fluorescent cells were observed in Ad5-EGFP and rAAV2-EGFP groups.Transfection efficiencies were about 88% and 83%.Adenovirus and adeno-associated virus vector can be transfected with ADSCs,and transfection efficiency is high.Adeno-associated virus needs sodium butyrate to increase its level of gene expression.

With low molecular weight chitosan and poloxamer 188 as the joint carriers, ginsenoside Rg3 solid dispersions were prepared by using the solvent evaporation method for an in vitro dissolution test. Subsequently, differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and X-ray diffraction (X-RD) were adopted for a phase analysis. The results showed that the 60 min in vitro cumulative dissolution rate of ginsenoside Rg3 solid dispersions prepared with low molecular weight chitosan and poloxamer 188 at the ratio of 2:1 exceeded 90%, and the drug was dispersed in carriers in an amorphous state. Therefore, ginsenoside Rg3 solid dispersions prepared with low molecular weight chitosan and poloxamer 188 could help significantly improve the drug dissolution, with a practical application value.
Chitosan ; chemistry ; Drug Compounding ; methods ; Ginsenosides ; chemistry ; Molecular Weight ; Poloxamer ; chemistry ; Solvents ; chemistry

Objective To preliminarily optimize the acupuncture protocol in treating migraine in acute stage. Method Ninety patients with migraine in acute stage were observed, with Visual Analogue Scale (VAS) as the evaluation index and an orthogonal design. Acupoints groups [Taiyang (EX-HN5), Fengchi (GB20), Taiyang (EX-HN5) and Fengchi (GB20)], insertion directions (perpendicular, downward penetration, and backward penetration), stimulation dosage (1 needle, 3 needles, and 5 needles) and acupuncture duration (30 min, 1 h, and 2 h), altogether 4 factors and 3 levels, formed up different acupuncture protocols to observe the analgesic efficacy in treating migraine in acute stage, so as to determine the role of the four factors (Chi-square test), advantage of the 3 levels (multiple comparisons) and the optimal grouping of the 4 factors and 3 levels. Result Acupoints group, insertion direction and stimulation dosage were the major factors in acupuncture analgesia, and the acupuncture duration was the secondary factor (P<0.05). The analgesic effect of Taiyang (EX-HN5) and Fengchi (GB20) was more significant than either Taiyang (EX-HN5) or Fengchi (GB20) (P<0.05). Penetration puncture from Naokong (GB19) towards Fengchi (GB20) (downward penetration) produced a more significant analgesic effect than from Fengchi (GB20) towards Wangu (GB12) and from Tianyou (TE16) towards Fengchi (GB20) (both backward penetration) (P<0.05). Acupuncture with Five needles and 3 needles (parallel horizontal insertion) were superior to that with 1 needle (P<0.05); acupuncture with 5 needles was better than that with 3 needles but without a statistical significance (P>0.05). Needle retaining for 2 h produced a better analgesic effect than retaining for 30 min and 1 h, but without statistical significances (P>0.05). Conclusion Taiyang (EX-HN5) plus Fengchi (GB20), downward penetration acupuncture, 5-needle parallel horizontal acupuncture, and 2-h needle retaining combine an optimal acupuncture protocol in treating migraine in acute stage. Nevertheless, 3-needle parallel acupuncture with 30-60-min needle retaining can also be chosen according to the condition of the patients.

Objective To investigate the morphological and functional alterations of peritoneum in uremic rats undergoing peritoneal dialysis(PD). Methods Thirty-five male Wistar rats were randomly divided into control group(sham operated group,n=6),uremia group(5/6 nephrectomy,n=6) and uremia with PD group(n=18).Uremia with PD group was subdivided into three subgroups according to different dialysis period(10 d,4 weeks and 8 weeks,n=6).Omenta were obtained for morphological examination,and peritoneal equilibration tests(PET) were performed to assess the transport function of peritoneal membrane. Results The number of blood vessels per high-power field in the uremia group,uremia with PD group and uremia with PD subgroups(5?3,10?5,17?5 and 19?4) were significantly increased compared with the control group(1?1),and that was much bigger in the uremia with PD group than the uremia group(P