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

Objective To understand the related factors of rabies epidemic and provide the basic data for rabies control and prevention in China by statistic and retrospective analysis of rabies surveillance data in 2010.Methods We used descriptive epidemiology method and statistic analysis to analyze the epidemiological characteristics of rabies in 2010 of China.Results 2048 rabies cases were rabies cases were reported in 817 counties (districts) in 2010,which dropped 7.46％ compares to 2009.The incidences in children and elder people were high; farmers are main occupation of the cases,the male to female ratio of the cases was 2.44:1.Children and older people are higher acquired rabies than other age population.640 cases reported through national rabies sentinel surveillance system,87.50％ cases were caused by exposed to dogs,bite was the main exposure reason.The situation of deposing wounds was poor,and the use of vaccine was still low in individual cases,but in the rabies clinic cases under surveillance,the vaccine usage can reach 98％,the usage of immunoglobulin (RIG) or anti-serum for category Ⅲ exposure in either group cases was not high.Conclusion The epidemic of the rabies in 2010 was eased,Out-patient post-exposure prophylaxis was in good station,but there are still lots of problem existed: post-exposure prophylaxis of individual case was not desirable yet.

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 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 prepare the immunoconjugate composed of Adriamycin nanoparticles and VEGFR 2 monoclonal antibody(conjugate of ADM-NP and VEGFR 2-MAb) and study its pharmacokinetics property. Methods Adriamycin nanoparticles were prepared by using double emulsion method, with PLA and O-CMC as materials. Conjugate of ADM-NP and VEGFR 2-MAb was prepared by using molecule conjugate technology. Immunoreactivity of the conjugate with type IV collagenase and H 22 cell were analyzed by using ELISA. Pharmacokinetics parameters of the immunoconjugate were obtained by using SD rats as study objects. Results The prepared ADM-NP was sphere particles under SEM, which diameters were (160±34) nm. The drug loading rate and entrapment rate were (30.15±3.5)% and (80.56±4.24)% respectively. Conjugate of ADM-NP and VEGFR 2-MAb was successfully prepared, which had immunoreactivity with type IV collagenase and H 22 cell. The immunoconjugate showed good ADM control-release ability and could prolong the retention time of ADM in vivo. Conclusion Conjugate of ADM-NP and VEGFR 2-MAb keeps the immunoreactivity of VEGFR 2-MAb and shows good ADM control-release ability.

ObjectiveTo investigate the clinical and pathological features of Hirschprung disease (HD), intestinal neuro-nal dysplasia (IND) and hypoganglionosis (IH) in children.MethodsThe clinical data and pathologic slices from 238 children with intestinal dysganglionosis were retrospectively analyzed. The age, sex, involved intestinal length of children and prognosis were compared.ResultsIn 238 patients, 138 (58.0%) were diagnosed by rectal mucosal biopsies. There were 122 HD patients whose median age at diagnosis was 9 months and the ratio of male to female was 4.3:1, without involvement of whole colon. There were 45 IND patients whose median age at diagnosis was 14 months and the ratio of male to female was 1.05:1, and the whole colon of 33.3% patients was involved. There were two male IH patients whose ages at diagnosis were 12 years and 18 years respectively, and their whole colon was involved. There were 59 patients with HD complicated by IND whose median age at diagnosis was 13 months and the ratio of male to female was 5.56:1 and the whole colon of 16.9% patients was involved. There were 10 male patients with HD complicated by IH whose median age at diagnosis was 11.5 months and the whole colon of 80.0% patients was involved. The ages at diagnosis, the sex ratio, the rates of whole colon involved, and the cure rates among 5 groups were signiifcantly different (allP<0.01).ConclusionsThe rectal mucosal biopsy was the main method in diagnosis of intestinal dysganglionsis in children. Patients with HD had higher incidence and mild condition and favorable prognosis. Patients with IH or patients with HD complicated by IH had lower incidence rates and severe condition and poor prognosis, followed by patients with IND or patients with HD complicated by IND.

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.