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.

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

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.

Aim To study the protective effects of sphingosine 1-phosphate (S1P) postconditioning on rat myocardial cells injured by hypoxia/reoxygenation in reperfusion injury salvage kinase ( RISK ) signal path-way. Methods The cultured rat H9c2 cells were ran-domly divided into seven groups: ( 1 ) control group;(2) hypoxia/reoxygenation (H/R) group; (3) S1P group;(4) S1P+LY294002 group(S1P+LY); (5) LY group; ( 6 ) S1 P +PD98059 group ( S1 P +PD );(7) PD group. The viability of H9c2 cells was detec-ted using MTT method. The content of MDA in the cultured medium and the activity of T-SOD and Mn-SOD were measured with colorimetry. The concentra-tion of intracellular free calcium ion was detected by confocal microscopy. The rate of cell apoptosis was de-termined by flow cytometric analysis. Western blot was used to assess phosphorylation of Akt and ERK1/2 in H9c2 cells. Results Compared with the H/R group, S1P significantly increased vaibility of cells, lowered the rate of apoptosis, decreased the content of MDA in the culture medium, increased the activity of T-SOD and Mn-SOD, reduced concentration of intracellular calcium and increased the phosphorylation of Akt and ERK1/2 . When added LY294002 or PD98059 , the effects of S1P above were inhibited. Conclusion S1P protects H9 c2 cells against hypoxia/reoxygenation inju-ry. The protection of S1P was inhibited by LY294002, the inhibitor of PI3 K/Akt and PD98059 , the inhibitor of ERK1/2 . S1 P protects H9 c2 cells against hypoxia/reoxygenation injury via RISK pathway.

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.

U1 trastuctrural changes of myocardium and lungs from 6 cases died of cranioce-rebral penerating gunshot wound 2 hours after injury is reportcd.In all cases theelectron microscopy of the myocardial and lung tissue samples showed the similar ultrastructural morphological changes of the cells and interstitial tissues.The mostpr-ominent ultrastructural changes of myocardium were disorderly arrangement of the Zband.focal dissociation of the myofibrills,mitochondrial swelling with decreasing ofmatrix density and disruption of cristae,and interstial edema.The changes of theung tissue were increasing of width of alveolar septa with decreasing of the electron density.Aggregation of neutrophils in the capillaries of alveolar septa and some alveolar space was observed.The significance and the pathogenesis of the mainpathological changes were discussed.It is suggested that the pulmonary interstitialedema was neurogenic.The pulmonary edema may be manifested as interstitial edemaor intra-lveolar edema depending upon the time elapsed after the gunshot injury.