In order to obtain an adequate supply of alpha-galactosidase for research and practical use, the fermentation, purification and identification of the recombinant coffee bean a-galactosidase were carried out. Baffled flasks containing 100mL BMGY were inoculated with the pPIC9K-Gal/GS115 strain and allowed to grow at 30 degrees C, 250- 300r/min until a maximum optical density at 600nm (OD600) between 2.0 to 6.0 was attained. Entire 400 mL seed culture was transferred aseptically to the 5-liter fermenter, which contained 4 liter sterilized basal salts medium and 4% glycerol. The batch culture grew at 30 degrees C, pH 5.0 until the glycerol was completely consumed, and a glycerol feed was initiated to increase the cell biomass prior to induction with methanol. The culture was centrifuged at 8000 x g and the supernatant was collected. Following ultrafiltration, the retentate was balanced in 20 mmol/L sodium formicate buffer, pH 3.8 and loaded onto a cation-exchange column, HiTrap SP. The column was washed with the same buffer and bound proteins were eluted with 1 mol/L NaCl. The fractions containing recombinant a-galactosidase were pooled and concentrated with PEG20 000. Subsequently, the biochemical properties of the enzyme were determined with typical methods. At last, the fresh human blood A and B erythrocytes were incubated with the purified alpha-galactosidase at 26 degrees C for 2 4 hours. Hemagglutinins were assayed by the standard method. After an elapsed fermentation times (EFT) of 18h, the fed-batch phase was initiated to increase the cell biomass. A cellular yield of nearly 200 g/liter wet cells was achieved when induction was initiated. 72h later, the alpha-galactosidase activity against artificial substrate PNPG (PNP-alpha-galactopyranoside) achieved 36 000u per liter culture. The crude fementation supernatant contained few impurities as detected by SDS-PAGE. The supernatant was purified by cation-exchange chromatography, the target alpha-galactosidase was eluted with 40% 1mol/L NaCl and showed a 41kD band on SDS-PAGE. After concentration, the final recovery was about 41%. The Michaelis constant of the recombinant alpha-galactosidase was determined as 0.275 mmol/L, which slightly lower than the nature enzyme and suggested a higher affinity with specific substrate. When human blood type B erythrocytes pretreated with 100u/mL recombinant alpha-galactosidase reacted with bood type B antiserum, no hemagglutination occurred. This suggested that the B antigens had been removed by the enzyme successfully. These results demonstrated that the recombinant alpha-galactosidase could be produced in largescale and made it possible to explore the application of alpha-galactosidase in more fields.
ABO Blood-Group System ; immunology ; Chromatography, Ion Exchange ; Electrophoresis, Polyacrylamide Gel ; Erythrocytes ; drug effects ; Fermentation ; physiology ; Hemagglutination ; drug effects ; Humans ; Pichia ; genetics ; metabolism ; alpha-Galactosidase ; genetics ; metabolism ; pharmacology

The aim of the study is to investigate the effect of salvianolic acid B (SalB) on blood-brain barrier (BBB) in rats after cerebral ischemia-reperfusion, and to illustrate its possible mechanisms. Cerebral ischemia-reperfusion was induced by middle cerebral artery occlusion in rats. The break-down of BBB was indicated by extravasations of immunoglobulin (IgG) monitored with immunohistochemistry. The expression of MMP-9 and NOS2 in the brain was determined by immunohistochemistry, and the expression of p-p38 and p-ERK1/2 was detected by Western blotting. It was shown that on day 2 after ischemia-reperfusion the IgG accumulated around the vascular boundary zone, suggesting the break-down of BBB, and the expression of MMP-9 and NOS2 up-regulated at the same time. The result of Western blotting suggested that the expression of p-p38 and p-ERK1/2 increased. On day 7 after ischemia-reperfusion the. expression of MMP-9 and NOS2 was about the same level as day 2, the expression of p-p38 was higher than that on day 2 and the expression of p-ERK1/2 was slightly lower than that on day 2. SalB (1 and 10 mg x kg(-1)) significantly alleviated the extravasations of immunoglobulin induced by cerebral ischemia-reperfusion (P < 0.05). On day 2 and day 7 SalB attenuated the expression of MMP-9 and NOS2 (P < 0.05). SalB (10 mg x kg(-1)) reduced the expression of p-p38 and p-ERK1/2 apparently on day 2 and 7 after ischemia-reperfusion (P < 0.05). SalB (1 mg x kg(-1)) inhibited the expression of p-p38 on day 7 after ischemia-reperfusion (P < 0.05). The results indicate that SalB protects blood-brain barrier in rats after cerebral ischemia-reperfusion by inhibiting the MAPK pathway.
Animals ; Benzofurans ; isolation & purification ; pharmacology ; Blood-Brain Barrier ; drug effects ; metabolism ; Brain Ischemia ; etiology ; metabolism ; pathology ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Infarction, Middle Cerebral Artery ; complications ; MAP Kinase Kinase Kinase 1 ; metabolism ; MAP Kinase Signaling System ; drug effects ; Male ; Matrix Metalloproteinase 9 ; metabolism ; Nitric Oxide Synthase Type II ; metabolism ; Phosphorylation ; Plants, Medicinal ; chemistry ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; metabolism ; pathology ; Salvia miltiorrhiza ; chemistry ; p38 Mitogen-Activated Protein Kinases ; metabolism

Histone lysine methyltransferase EZH2 has been reported to be frequently overexpressed in hepatocellular carcinoma (HCC) tissues and associated with hepatocarcinogenesis. However, the exact mechanism of EZH2 up-regulation in HCC has not been determined. In this study, we used murine hepatocyte AML12 cells to investigate the role of hepatitis B virus X protein (HBx) in regulating the expression of mEZH2. Western blot analysis demonstrated that the expression level of mEZH2 protein in AML12 cells was up-regulated by HBx in a dose-dependent manner. To further investigate the mechanism of mEZH2 overexpression, the 2500 bp regulatory sequence upstream from the first exon of the mEZH2 gene was amplified from AML12 genomic DNA and constructed into a luciferase reporter plasmid. The luciferase activity of the mEZH2 promoter significantly increased in AML12 cells co-transfected with HBx plasmid, and deleting the -486/-214 promoter region decreased HBx-induced mEZH2 promoter activation by nearly 50%. The -486/-214 region was then analyzed in the TRANSFAC 6.0 database and a typical E2F1-binding site was found. Mutation of this E2F1-binding site or knockdown of E2F1 expression by RNAi led to a dramatic decrease in HBx-induced activation of the mEZH2 promoter and mEZH2 overexpression in AML12 cells. These results provide evidence that HBx up-regulates mEZH2 expression by transactivating the mEZH2 promoter through E2F1 transcription factor, thereby providing new epigenetic evidence for the carcinogenic effect of HBx.
Animals ; Binding Sites ; Cell Line ; E2F1 Transcription Factor ; genetics ; Enhancer of Zeste Homolog 2 Protein ; Hepatocytes ; cytology ; metabolism ; virology ; Histone-Lysine N-Methyltransferase ; genetics ; metabolism ; Mice ; Plasmids ; Polycomb Repressive Complex 2 ; Promoter Regions, Genetic ; genetics ; RNA, Small Interfering ; genetics ; Trans-Activators ; genetics ; metabolism ; Transfection ; Up-Regulation

Objective To determine the relationship between abnormal blood glucose and lipid levels and body mass index in patients with chronic respiratory diseases. Methods This research was conducted in Urumqi baseline survey population from "Xinjiang multi-ethnic cohort study" in which patients with chronic bronchitis, emphysema and chronic obstructive pulmonary disease (COPD) were screened.The patients were defined as angular, normal, overweight, obesity groups according to body mass index (BMI), and were compared with blood levels of lipid and glucose, and rate of abnormal metabolism. Results A total of 6 046 subjects were included in the 2018 Urumqi cohort study, including 545 patients with chronic respiratory diseases.The prevalence of chronic respiratory diseases in different age groups and at different physical activity levels was different, and the prevalence of emaciation group was significantly higher than that of the other three groups.However, there were no statistically significant differences among people with different genders, nationalities, educational levels and whether they smoked or not.There were statistically significant differences in fasting glucose, serum cholesterol (TC) and serum high-density lipoprotein (HDL) levels among respiratory patients with different BMI (P < 0.05).The detection rates of hyperglycemia, high TG and low HDL increased with the increase of BMI.The result of multi-factor analysis showed that blood glucose, TG and HDL were the influencing factors of BMI. Conclusion Increase of body mass index in patients with chronic respiratory diseases is associated with abnormal glucose and lipid metabolism, which is not significantly different from that in patients with non-respiratory diseases.However, the body mass index and nutritional status of patients with long-term diseases should be closely monitored, and timely intervention measures should be taken to delay the disease process.

External snapping hip(ESH) is a vague term used to describe palpable or auditory snapping with hip movements with or without pain. The pathogenesis of ESH is related to the specific anatomical structure and friction factor. The clinical symptom is auditory snapping during activities, physical examination, X-ray, magnetic resonance imaging(MRI), dynamic ultrasound and other imaging techniques can be used to diagnose. Conservative medical management includes rest, avoidance of aggravating activities, and antiinflammatory medications. Treatment Patients with mild symptoms can achieve good results by medication, rest and physiotherapy. Surgical treatment for patients with ineffective conservative treatment was performed. All kinds of open surgery method can achieve good clinical curative effect, arthroscopic surgery is gradually been promoted due to small trauma, less complications. Besides, there are some reports that traditional treatments such as massage, acupuncture and acupotomology have achieved good clinical results, which deserve further study and promotion.

Objective To explore the effects of valsartan and MEK1/2 inhibitor U0126 on atrial fibrosis and connexin40 (Cx40) remodeling in rats treated with isopreterenol (ISO).Methods 32 male SD rats were randomly divided into control group (A),ISO (5 mg · kg-1 · d-1 for7 days) + DMSO group (B),ISO + U0126 (0.5 mg · kg-1 · d-1 for 28 days) group (C,U0126 was dissolved in DMSO),ISO +valsartan (30 mg · kg-1 · d-1 for 28 days) + DMSO group (D).Rats were sacrificed after 28 days.The AngⅡ content in myocardial tissue was measured by radioimmunoassay,P-MEK1/2,P-ERK1/2 and Cx40 was detected by immunohistochemistry,atrial fibrosis was determined on HE and Masson stained heart sections.Results The content of Ang Ⅱ was significantly higher in group B,C and D compared with group A [ (368.243 ±6.283 ) ng/L,( 357.175 ± 5.944 ) ng/L,( 359.908 ± 2.496 ) ng/L vs ( 250.380 ± 4.261 )ng/L,P ＜0.01 ] ; the degree of atrial fibrosis was significantly lower in group C and D compared with group B [CVF(10.260 ±0.525)％,(10.238 ±0.524)％ vs (78.710 ± 1.587)％,P＜0.01 ] while there was no atrial fibrosis in group A [ CVF(9.025 ± 0.456)％ ] ; the expression of P-MEK1/2 and P-ERK1/2 was significantly upregulated in group B compared with group A ( P-MEK1/2:0.311 ± 0.007 vs 0.203 ± 0.009,P ＜0.01 ; P-ERK1/2:0.259 ±0.003 vs 0.173 ±0.006,P ＜0.01 ) and significantly lower in group C and D compared with group B (P-MEK1/2:0.212 ± 0.004,0.213 ± 0.005 vs 0.311 ± 0.007,P ＜0.01,P-ERK1/2:0.178 ±0.004,0.175 ±0.007 vs 0.259 ±0.003,P ＜0.01 ).The content of Cx40 was obviously reduced and the distribution of Cx40 was disordered in group B compared with A (0.199 ±0.007 vs 0.241 ±0.004,P＜0.01) which could be partly reversed in group C and D ( 0.239 ±0.037,0.235 ±0.006 vs 0.199 ± 0.007,P ＜ 0.01 ).All parameters in group C and D were similar ( P ＞ 0.05 ).Conclusion The chronically elevated Ang Ⅱ content in myocardium may be related to atrial fibrosis and Cx40 remodeling in this model,valsartan and U0126 were equivalent on attenuating atrial fibrosis and Cx40 remodeling by inhibiting ERK pathways at different levels.

Objective To investigate a scientific and effective way of nursing quality feedback to achieve the purpose of continuous improvement of nursing quality.Methods Nursing quality,nursing defect correction number and satisfaction rate by written and oral feedback in 2010 were the control group,while the same nursing evaluation items by multi-media with written feedback were the experimental group,and the results of different nursing quality feedback pathways were compared between two groups.Results The score of ward management,disinfection and isolation,intensive care,first aid items,nursing documents,basic nursing quality was respectively (97.60 ± 1.34),(98.35 ± 0.80),(98.21 ± 1.17),(98.19 ± 1.51),(94.75 ±1.56),(97.13 ± 1.34)in the experimental group,and (89.81 ±2.08),(89.86 ± 1.01),(91.48 ±2.10),(90.47 ±0.86),(88.79 ± 1.83),(85.62 ± 2.26) in the control group.The differences were statistically significant (t =10.90,22.79,9.69,15.34,8.67,15.75,respectively;P ＜ 0.05).The number of nursing defect correction was 38 in the experimental group and 18 in the control group,and the difference was statistically significant (x2 =-3.35,P ＜ 0.05).And there was also statistically significant difference of patients' satisfaction rate between two groups (x2 =7.585,P ＜ 0.05).Conclusions Multi-media with written feedback is a scientific and effective way of continuous improvement of nursing quality,which can improve nurses' quality and service consciousness as well as shorten the cycle of nursing defects correction,thus truly achieving the purpose of continuous improvement of nursing quality.

The objective of this study was to investigate the method and effect of blocking the specific reaction between lymphocyte HLA-I antigen and its antibody. The lymphocytes were disposed with 12 mmol/L methoxypolyethelene glycol benzotriazol carbonate (mPEG-BTC) in concentration gradient in PBS (pH 7.4) at 22 degrees C. The effect of the modified lymphocytes was detected by microlymphocytotoxicity assay. The results showed that lymphocytes modified by mPEG-BTC did not react with related HLA-I antibodies in microcytotoxicity test. It is suggested that the specific reaction between HLA-I antigen of lymphocyte and HLA-I antibodies can be completely camouflaged by mPEG-BTC in PBS (pH 7.4) under 22 degrees C room temperature.
Antigen-Antibody Reactions ; Cytotoxicity, Immunologic ; Histocompatibility Antigens Class I ; immunology ; Humans ; Lymphocytes ; immunology ; Polyethylene Glycols ; pharmacology ; Triazoles ; pharmacology

OBJECTIVETo analyze the clinical features and prognosis of the primary myelodysplastic syndrome with myelofibrosis (MDS-MF) patients and to improve the cognition of MDS-MF.

METHODSFour hundred and sixty-six primary MDS patients with bone marrow (BM) biopsy were divided into two groups according to whether BM associated with fibrosis, the clinical features and prognosis of the two groups were analyzed retrospectively.

RESULTS167 (35.8%) MDS cases revealed myelofibrosis, of which MF-1 123 cases (26.4%), MF-2 40 cases (8.6%), MF-3 4 cases (0.9%). The proportion of hepatosplenomegaly in MDS-MF group was significantly higher than in MDS without MF group, the difference had statistical significance (P = 0.031). The proliferation of BM biopsy in MDS-MF group was significantly more active than in MDS without MF group. The number of blasts, megakaryocytes and abnormal megakaryocytes in MDS-MF group were significantly higher than in MDS without MF group, the differences had statistical significance (P < 0.05). Among the 345 patients who had available results of cytogenetic analysis, 121 cases were MDS-MF patients, the proportion of middle and high-risk prognostic group according to IPSS karyotype prognosis groups in MDS-MF group were significantly higher than in MDS without MF group, the differences had statistical significance (P = 0.047). The median survival was 17 (1 - 60) months in MDS-MF group, and was 32 (1 - 62) months in MDS without MF group. The difference had statistical significance (P = 0.001). Myelofibrosis had independent prognostic significance by multi-variable analysis (P = 0.019).

CONCLUSIONThe myelofibrosis in MDS is main the proliferation of reticular fiber. The proliferation of reticular fiber is closely related with the number of blast cells, the proliferation and developmental abnormalities of megakaryocytes and the karyotype. The prognosis of MDS-MF patients is poor.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Child ; Female ; Humans ; Karyotyping ; Male ; Middle Aged ; Myelodysplastic Syndromes ; complications ; diagnosis ; pathology ; Primary Myelofibrosis ; complications ; diagnosis ; pathology ; Prognosis ; Retrospective Studies ; Young Adult

OBJECTIVETo investigate the changes in the percentages and balance of CD4T cell subsets including T helper cells (Thl, Th2, and Thl7) and T regulatory cells (Treg) in patients with ovarian cancer.

METHODSPeripheral blood samples were collected from 30 patients with ovarian cancer and 20 healthy subjects for analysis of the percentages of Thl, Th2, Thl7 and Treg using flow cytometry.

RESULTSCompared with the control subjects, the patients with ovarian cancer showed significantly increased percentages of Th2, Thl7 and Treg (P<0.05) but significantly decreased percentage of Th1 in the peripheral blood of patients with ovarian cancer (P<0.05). The changes in CD4T cell subsets were significantly correlated with the clinical stage of the tumor (P<0.05) but not with the histological type or cell differentiation (P>0.05). The Th1/Th2 ratio was significantly decreased in ovarian cancer patients (P<0.05) with obvious Th2 polarization compared with control group. The Treg/Th17 ratio was significantly increased in ovarian cancer patients (P<0.05).

CONCLUSIONPatients with in ovarian cancer have abnormal expressions of CD4T cell subsets in the peripheral blood with Th1/Th2 and Treg/Th17 imbalance, and these findings provide evidence for clinical immunotherapy of ovarian cancer.