To express feruloyl esterase A from Aspergillus oryzae in Pichia pastoris expression system and study its hydrolysis function, explore the conditions and effects of purification for ferulic acid extracts by macroporos resin. Using the total RNA from A. oryzae CICC 40186 as the template, we amplified coding sequence AorfaeA encoding a mature feruloyl esterase A (AorFaeA) by RT-PCR technique. Then, the coding sequence AorfaeA was successfully expressed in Pichia pastoris GS115 mediated by an expression plasmid pPIC9K. The purified recombinant AorFaeA (reAorFaeA) showed one single band on SDS-PAGE with an apparent molecular weight of 39.0 kDa. The maximum activity of reAorFaeA to methyl ferulate, measured by high-performance liquid chromatography (HPLC), was 58.35 U/mg. Then, reAorFaeA was used to release ferulic acid from de-starched wheat bran in the presence of xylanase. The purification tests for ferulic acid from the enzymatic hydrolysate were carried out with preselected macroporous resins. The results showed that macroporous resin HPD-300 had much higher adsorption and desorption capacities. Ferulic acid could be quantitatively recovered by 50% of the eluent concentration at a flow speed of 1 mL/min. Under the purification condition, the recovery ratio of ferulic acid was 92%, and the content of ferulic acid was increased from 0.13% in the raw material to 10.55%. This work exploits the breakdown of ferulic acid by recombinant enzymeand provids a good strategy to its "green production".
Aspergillus oryzae ; enzymology ; Carboxylic Ester Hydrolases ; biosynthesis ; genetics ; Cloning, Molecular ; Coumaric Acids ; chemistry ; Electrophoresis, Polyacrylamide Gel ; Hydrolysis ; Molecular Weight ; Pichia ; genetics ; metabolism

ObjectiveTo evaluate the clinical efficacy and safety of astragalus injection on the immune function in patients with senile sepsis.Methods Sixty patients with old age sepsis in Critical Care Medicine Department of Guangdong Provincial Traditional Chinese Medicine Hospital were enrolled and randomly assigned into control and treatment groups according to the table of random numbers, 30 cases in each group. According to 2012 sepsis guidelines for treatment, including antibacterial drug, mechanical ventilation, visceral function support, etc., the therapy was given to the control group; besides the treatment in the control group, intravenous drip of 60 mL astragalus injection(10 mL per ampoule) in 250 mL 0.9% normal saline was additionally given in the treatment group, once a day for 7 days. Before and after treatment, the immunological indexes, acute physiology and chronic health evaluationⅡ(APACHEⅡ) score, sequential organ failure assessment(SOFA) score, duration of mechanical ventilation and time of stay in intensive care unit(ICU), 28-day mortality and adverse drug reactions were compared between the two groups.Results Before treatment, there were no statistically significant differences in CD3+, CD3+CD4+, CD3+CD8+ and T helper cells /T suppressor cells(Th/Ts)levels between the two groups(allP>0.05), while CD3-NK+ of the control group was significantly higher than that in the treatment group〔(10.47±6.22)% vs. (6.26±4.13)%,P<0.05〕. After treatment in treatment group, CD3+, CD3+CD4+ and CD3-NK+ were increased, CD3+CD8+,Th/Ts were decreased compared with those before treatment; in the control group after treatment, CD3+,CD3+CD8+ and CD3-NK+ were decreased and CD3+CD4+ and Th/Ts increased compared with those before treatment. In the comparisons between the treatment group and control group after treatment, the differences in CD3+, CD3+CD4+ and CD3+CD8+ had statistical significance〔CD3+:(30.30±17.17)% vs.(41.91±22.29)%, CD3+CD4+:(31.54±13.24)% vs.(40.08±15.28)%, CD3+CD8+:(14.25±8.10)% vs.(9.52±9.33)%,allP<0.05〕; while the differences in Th/Ts and CD3-NK+ had no statistical significance(bothP>0.05). After treatment in the treatment group, IgG was increased compared with that in the control group〔IgG(g/L): 13.07±5.43 vs. 10.10±3.96,P<
0.05〕. The differences in IgA, IgM, complement(C3,C4) and total serum complement activity(CH50) in the comparisons between the two groups had no statistical significance after treatment(allP>0.05). The differences in APACHEⅡ score(13.83±6.18 vs. 15.90±7.48), SOFA score(7.38±4.66 vs. 6.89±4.19), time of stay in ICU(day: 11.63±5.13 vs. 13.62±8.08), invasive ventilation time(hour: 155.44±119.68 vs. 224.08±174.15) and noninvasive ventilation time(hour: 55.55±42.24 vs. 98.57±43.17) had no statistical significance in comparisons between the treatment group and control group after treatment(allP>0.05). The difference in 28-day mortality had no statistical significance in comparison between the treatment group and control group〔16.7%(5/30) vs. 20.0%(6/30),P>0.05〕. In 60 cases, there were 2 patients with adverse drug reaction, one diarrhea and another little rashes, the rest of the patients did not appear any drug side effect.ConclusionAstragalus injection combined with conventional western medicine therapy possibly has certain effect on adjustment of disturbance of immunologic functions in old patients with sepsis, and its therapeutic safety is well.

15mm~2)of dural sac cross-sectional area to values smaller than 75 mm~2 was found during examination in axial loading,or if a suspected disc herniation,narrow lateral recess,narrow intervertebral foramen,or intraspinal synovial cyst changed to being obvious at the axial loading examination,they were regarded as additional information.Results After axial loading CT examination,AVI was found in 16 of 40 patients.A significant decrease of dural sac area was found in 13 patients.Intervertebral disc herniation was more severe in 7 patients,lateral recess or interverbral foramen narrowed in 4 patients,no intraspinal synovial cyst was found.After axial loading MRI examination,AVI was found in 19 of 60 patients.A significant decrease of dural sac area was found in 13 patients.Intervertebral disc herniation became severe in 10 patients,lateral recess or interverbral foramen narrowed in 8 patients,no intraspinal synovial cyst was found.AVI was found in 32 of 79(40.5%)patients with sciatica and 2 of 20(10.0%)patients with low back pain(?~2=7.45 P

Regarding the needling manipulation, Chifeng Yingyuan, there are different descriptions in ancient medical literature and the current teaching materials or clinical reports, especially for the understanding recorded in Jinzhenfu Poem of Golden Needle). In the current teaching materials and clinical reports, it is described that the needle is inserted deeply at first, and then shallowly; afterward, the needle tip is pushed to the middle layer and manipulated with lifting, thrusting and rotating technique, combined with twisting and flying method. After analyzing the original description in the book and the feasibility of function and operation, the authors introduced their recognition on its manipulation and techniques in this article. This manipulation should be: the needle is inserted to the middle layer, afterward, the needle body is pulled and tilted toward each of the four directions, on this basis, then twisting and flying method is applied.
Acupuncture ; education ; history ; Acupuncture Points ; Acupuncture Therapy ; history ; instrumentation ; methods ; China ; History, 20th Century ; History, 21st Century ; History, Ancient ; Humans ; Medicine in Literature ; Needles ; history

Objective To assess apoptosis mediated by the cell death receptor Fas in peripheral T lymphocytes of patients with abdominal aortic aneurysm. Methods The apoptotic pathway was triggered by anti-Fas monoclonal antibody in cultured and activated peripheral T-cells from 20 AAA patients. Control groups consisted of 15 patients with aortic atherosclerotic occlusive disease(AOD)and 25 healthy individuals. Cell survival and death rate were assessed. Results Cross-linkage of Fas receptor exerted a strong apoptotic response on T cells from AOD patients and healthy controls, while the effect on T cells was very limited from that of AAA patients. The evaluation of cell Survival rate showed a significantly higher percentage in AAA group(98.9%±10.3%)than in the AOD subjects(58.9%±15.2%)or the healthy group(59.4%±12.9%;P＜0.001＝.Apoptosis assessment by annexin V and propidium iodide staining and flow cytometry showed similar results. The defect in AAA group was not due to decreased fas expressed at normal levels. Moreover,it specifically involved the Fas system because cell death was induced in the normal way by methylprednisolone. Conclusions Fas-induced apoptosis in activated T cell from AAA patients is impaired. This may disturb the normal down-regulation of the immune response and thus provide a new insight into possible mechanisms and routes in the pathogenesis of AAA.

ObjectiveTo explore the clinical diagnosis and surgical treatment of primary retroperitonealneurilemoma(schwannoma). MethodsClinicaldataof 47patientsof primary retroperitoneal schwannoma admitted and surgically treated from January 1995 to December 2009 were retrospectivelly reviewed.ResultsAs diagnosed by pathology there were 36 cases of Benign schwannoma,with a median age at onset of 41years, among those 11 patients were symptomatic, and 25 were asymptomatic. There were 11 malignant 11 cases, the median age was 38 years, among those 6 patients were symptomatic, and 5 were asymptomatic. The positive diagnostic rate of preoperative CT and MRI were 36. 2％ ( 17/47 ) and 58. 3％ ( 7/12 ) respectively. Immunohistochemically positive rates of S-100 were 100％ and 81.8％(9/11) in benign and malignant group respectively.All cases underwent surgical treatment. Surgical resection rates for benign and malignant groups were 100％ and 90. 9％(10/11)respectively. There was no perioperative death, Overall 5-year survival rates were 100％ and 45.5％ for benign and malignant tumors groups respectively. In benign group 2 cases recurred, in malignant group 4 cases recurred, and 3 had distant metastasis.ConclusionsPrimary retroperitoneal schwannomas are less common. It is difficult to make an accurate preoperative diagnosis. Surgery is the most effective therapy.Prognosis is good for benign and poor for malignant retroperitoneal neurilemomas.

OBJECTIVETo determine the expression of Skp2 in different prostate cancer (PCa) cell lines and tissues, and explore its influence on the androgen receptor (AR) signaling pathway and development of castration-resistant prostate cancer (CRPC).

METHODSThe expression levels of Skp2 and AR in different PCa cell lines were detected by Western blot. After knockdown of Skp2 in the C4-2 and 22RV1 cells transfected with shRNA, the expressions of AR and P27 were determined and the activity of ARR3-Luc measured by dual-luciferase reporter gene assay following treatment with dihydrotestosterone (DHT). The expressions of AR and Skp2 in human naïve PCa or CRPC specimens were detected by immunohistochemical staining followed by analysis of their differences and correlation.

RESULTSThe Skp2 protein expression level was significantly higher in the C4-2 or 22RV1 cells than in the LNCaP cells. DHT treatment increased the expression of Skp2 in the C4-2 cells, but knock-down of Skp2 significantly up-regulated the expression of the well-known downstream protein P27 and down-regulated that of AR. Consistently, DHT treatment increased the activity of ARR3-Luc, while knockdown of Skp2 remarkably decreased it in the C4-2 and 22RV1 cells (P < 0.05). In addition, significantly higher expressions of Skp2 and AR were observed in the CRPC than in the naïve specimens (P < 0.05), with a positive correlation between the two proteins (r = 0.658 1, P < 0.05).

CONCLUSIONSkp2 can enhance the expression and transcription activity of the AR protein in CRPC cells or tissues and is promising to be a critical molecular therapeutic target.

Androgens ; pharmacology ; Cell Line, Tumor ; Dihydrotestosterone ; pharmacology ; Disease Progression ; Gene Knockdown Techniques ; Humans ; Male ; Neoplasm Proteins ; genetics ; metabolism ; Prostatic Neoplasms, Castration-Resistant ; metabolism ; Receptors, Androgen ; genetics ; metabolism ; S-Phase Kinase-Associated Proteins ; physiology ; Transcriptional Activation ; Up-Regulation

Objective To find out the cerebral blood flow velocity and its relationship with attention and execu?tion function in patients with major depressive episode. Methods A total of 70 patients with major depressive episode and 65 healthy controls were included. The 24 items Hamilton depression scale (HAMD-24) was adopted for patients' depressive symptoms assessment. The Cancellation Test (CT) and Wisconsin Card Sorting Test (WCST) were adopted for cognitive function assessment. And the blood flow velocity of cerebral arteries were detected by transcranial Doppler ul?trasonography (TCD). Results Compared with the controls, the patients showed significantly slower average blood flow ve?locity in basilar artery, left middle cerebral artery, right middle cerebral artery, left anterior cerebral artery and right ante?rior cerebral artery (P<0.05). Compared with those in the controls, the net score of each stage and the total net score of CT were significantly lower in the patients, while the total number of responses, response errors, perseverative errors and the necessary response number to complete the first category of WCST were significantly higher (P<0.01). The blood flow velocity in the cerebral basilar artery (r=0.25), left middle cerebral artery (r=0.46), right middle cerebral artery (r=0.25) and right posterior cerebral artery (r=0.26) showed positive correlations with total net score of CT (P<0.05), and the cere?bral artery average blood flow velocity showed negative correlations with the total number of responses, response errors and perseverative errors of WCST in the patients (P<0.05). Conclusions Compared with healthy controls, patients with major depressive episode suffer from lower average cerebral artery blood flow velocity, bad attention and executive func?tion. The change of average cerebral artery blood flow velocity may be responsible for impaired cognitive function in epi?sode major depression patients.

The aim of this paper is to compare the cytotoxicity and cellular uptake efficiency of three kinds of poly(b-benzyl-L-amino) block-poly(ethylene glycol) nanoparticles (PXA-PEG-NPs) using Calu-3 cells, and select one as a nasal drug delivery vector for curcumin (Cur). Poly(gamma-benzyl-L-glutamate) block-poly(ethylene glycol) nanoparticles (PBLG-PEG-NPs), poly(gamma-benzyl-L-lysine) block-poly(ethyleneglycol) nanoparticles (PZLL-PEG-NPs) and poly(gamma-benzyl-L-aspartate) block-poly(ethylene glycol) nanoparticles (PBLA-PEG-NPs) were prepared by emulsion-solvent evaporation method. MTT assays were used to evaluate the cytotoxicity of PXA-PEG-NPs against Calu-3 cells. The cellular uptake of nanoparticles was visualized by an inverted fluorescence microscope and quantified by a flow cytometer. The results indicated that even at high concentration of 2 mg x mL(-1) the three nanoparticles had no cytotoxicity on Calu-3 cells. Compared to the curcumin solution, the three curcumin-loaded PXA-PEG-NPs showed significantly higher cellular uptake efficiency on Calu-3 cells (at equal concentration of curcumin with 5 microg x mL(-1) Cur solution), PBLG-PEG-NPs group was the highest. The cellular uptake increased with incubation time, and has positive correlation with nanoparticle concentration. In brief, PXA-PEG-NPs are conducive to delivery Cur into cells, and PBLG-PEG-NPs might be provided as a good nasal drug delivery carrier.

Objective: To observe the clinical efficacy of combining auricular point sticking and a healthy diet to treat simple obesity in children aged 6-9 years old.Methods: A total of 190 eligible obese kids were divided into an observation group and a control group using the random number table method, with 95 cases in each group. The observation group was intervened by auricular point sticking plus guide on a healthy diet, while the control group was only provided with the guide on a healthy diet. The therapeutic efficacy was observed after intervention for three consecutive months, as well as the changes in body mass (BM), body mass index (BMI), waist circumference (WC), hip circumference (HC), and subcutaneous fat thickness. Results: After the 3-month intervention, the total effective rate was 91.6％ in the observation group, versus 74.7％ in the control group, and the between-group difference was statistically significant (P<0.01); in both groups, the BM, BMI, WC, HC, and subcutaneous fat thickness all decreased significantly (P<0.05), and were lower in the observation group than in the control group, showing statistical significance (P<0.05). Conclusion: Auricular point sticking plus a healthy diet is safe and effective in treating simple obesity in children, producing more significant efficacy than healthy diet intervention alone.