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1.
Article in Chinese | WPRIM | ID: wpr-927895

ABSTRACT

Objective: To investigate the repair effect and JNK/NF-κB,SOX9 mechanisms of vibration exercise with different frequencies on articular cartilage in rats with early knee osteoarthritis. Methods: Forty-eight adult male SD rats were randomly divided into six groups(n=8):model control group(MC),high frequency vibration group 1 (GP1,60 Hz),high frequency vibration 2 group (GP2,40 Hz),medium frequency vibration group (ZP,20 Hz),minor frequency group(DP,10 Hz)and normal control group(NC). Except for NC group,the rats in each group were made into early knee osteoarthritis model after six weeks of knee joint cavity injection of papain solution and 2% mixture l-cysteine on the 1st,4 th and 7th day. Each exercise group was subjected vibration to 40 minutes a day with amplitude of 2~5 mm and 5 days a week. Four weeks later, the articular cartilage of the lateral femoral condyle of the both back leg knee joints were detected by HE staining,serine O staining and Mankin scores for morphological observation. The expression levels of JNK,NF-κB p65 and Sox9 mRNA in articular cartilage of the medial femoral condyle were detected by RT-qPCR,and the protein expressions of JNK,NF-κB p65 and Sox9 were detected by Western blot. Results: Compared with the NC group,the Mankin score in other groups was significantly higher (P<0.01). Compared with the MC group,the Mankin score of each vibration group was significantly lower(P<0.05),the mRNA and protein expressions of JNK and NF-κB p65 in each vibration training group were significantly lower (P<0.01),the expressions of Sox9 mRNA and protein in vibration training group were increased significantly (P<0.01). Compared with the higher frequency group,the Mankin score,the mRNA and protein expressions of JNK and NF-κB p65 of lower frequency group were significantly lower (P<0.05 or P<0.01). But the expressions of Sox9 mRNA and protein were significantly higher (P< 0.05 or P<0.01). Conclusion: Vibration exercise of different frequencies may present varying degrees of cartilage repair impact in rats with early knee osteoarthritis,and the cartilage repair by low-frequency vibration training is better than that by high-frequency vibration. This can be one of the mechanisms on controlling collagen synthesis by down-regulating JNK/NF-κB expression and increasing SOX9 activity of OA articular cartilage.


Subject(s)
Animals , Cartilage, Articular/metabolism , MAP Kinase Kinase 4 , Male , NF-kappa B/metabolism , Osteoarthritis, Knee/therapy , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , SOX9 Transcription Factor , Vibration
2.
Article in Chinese | WPRIM | ID: wpr-921526

ABSTRACT

Objective To investigate the differences of gut microbiota between patients with abdominal aortic aneurysm and atherosclerosis.Methods From December 2018 to June 2019,20 fresh stool samples were collected respectively from the patients with abdominal aortic aneurysm and atherosclerosis treated at the Department of Vascular Surgery,Peking Union Medical College Hospital.The 16S rDNA high-throughput sequencing was employed to compare the composition,abundance,and α and β diversities of gut microbiota between the two disease groups,and further determine the significantly differential genera.Results The two groups had great similarities in the composition of gut microbiota.There was no statistical difference in α diversity.Although β diversity did not have statistically significant difference,certain microbial taxa showed differences between the two groups.The LEfSe demonstrated that the abdominal aortic aneurysm group had higher relative abundance of


Subject(s)
Aortic Aneurysm, Abdominal , Atherosclerosis , Feces , Gastrointestinal Microbiome , Humans
3.
Journal of Experimental Hematology ; (6): 1868-1871, 2018.
Article in Chinese | WPRIM | ID: wpr-774370

ABSTRACT

BK virus infection is one of the common complications after hematopoietic stem cell transplantation(HSCT), which is also one of the reasons of the hemorrhagic cystitis.In recent years, although there are more studies of the risk factors related with human BK virus infection after hematopoietic stem cell transplantation, the risk factors related with BKV-associated hemorrhagio cystitis(BKV-HC) remain to be elucidated. Diagnosis of BK virus infection is mainly based on quantitative PCR of blood or urine. An effective strategy for treatment of these patients is the adoptive transfer of T lymphocytes specific to virus-associated antigens. In this review, the progressis of diagnosis and treatment of BK virus infection after hematopoietic stem cell transplantation are briefly summarized.


Subject(s)
BK Virus , Cystitis , Hematopoietic Stem Cell Transplantation , Humans , Polyomavirus Infections , Tumor Virus Infections
4.
Chinese Journal of Immunology ; (12): 718-722, 2018.
Article in Chinese | WPRIM | ID: wpr-702804

ABSTRACT

Objective:To investigate the expression of PD-L1 in peripheral blood with gastric cancer patients and its clinical significance.Methods:Peripheral blood samples were collected from 44 gastric cancer patients (before and after the surgery) and 18 healthy subjects.The expression of PD-1 and PD-L1 on CD3+CD4+T,CD3+CD8+T cells and CD14+monocytes was detected by flow cy-tometry.Results:The expression of PD-L1 on CD14+monocytes of the gastric cancer patients was significantly higher than that of the healthy control[(29.2±16.7)% vs (17.5±9.7)%,P=0.007 3],while there was no statistical difference on CD4+T cells and CD8+T cells[(11.1±6.4)% vs(9.8±5.6)%,P=0.453 2;(13.9±12.0)% vs(12.0±7.1)%,P=0.558 9].The expression of PD-L1 on CD4+T cells and CD8+T cells in patients with gastric cancer was significantly higher than that before surgery[(15.8 ± 8.2)% vs (11.1±6.4)%,P=0.001 5;(22.5±13.3)% vs(13.9±12.0)%,P=0.000 2].However,no significant change was found on CD14+mononuclear cells[(33.8±17.3)% vs (29.2±16.7)%,P=0.082 8].There was no significant difference in the expression of PD-1 on CD4+T and CD8+T cells before and after surgery[(25.6 ±9.9)% vs (26.9 ±8.9)%,P=0.505 5;(26.5 ±14.6)% vs (29.9 ± 10.4)%,P=0.118 7].Conclusion:PD-L1 can be used as an effective marker to monitor the immune function and prognosis of patients with primary gastric cancer.

5.
Article in Chinese | WPRIM | ID: wpr-698280

ABSTRACT

BACKGROUND:Tetracycline hydrochloride not only promotes the proliferation of bone marrow mesenchymal stem cells (BMSCs),but also has a strong bone activity.Studies on the osteogenic differentiation of BMSCs induced by tetracycline hydrochloride will provide a new cell source for bone tissue engineering.OBJECTIVE:To observe the induction and differentiation activity of BMSCs into osteoblasts cultured by tetracycline hydrochloride in vitro.METHODS:BMSCs were separated and expanded by whole bone marrow adherent method and identified by cell morphology and BMSCs cell markers CD34,CD45,CD90,CD29.Passage 3 BMSCs were collected and cultured with tetracycline hydrochloride (experimental group) or cultured in L-DMEM containing 10% fetal bovine serum (control group).Osteogenic induction and differentiation activity of BMSCs cultured by tetracycline hydrochloride were detected by alkaline phosphatase activity detection,collagen I and osteocalcin immunohistochemistry staining,alizarin red staining of mineralized calcium nodules,and detection of collagen I and osteocalcin gene expression by RT-PCR.RESULTS AND CONCLUSION:Tetracycline hydrochloride continuously promoted alkaline phosphatase activity in the BMSCs,significantly different from that in the control group.After 21 days of culture,positive expression of collagen I and osteocalcin proteins,red calcium nodules shown by alizarin red staining,and mRNA expression of collagen I and osteocalcin were observed in the experimental group,while the negative expression was found in the control group.In conclusion,tetracycline hydrochloride has a significant role to promote the osteogenic differentiation of BMSCs.

6.
Chinese Journal of Dermatology ; (12): 541-546, 2016.
Article in Chinese | WPRIM | ID: wpr-672344

ABSTRACT

Objective To evaluate the clinical efficacy and safety of compound polymyxin B ointment combined with desonide cream for the treatment of subacute or chronic eczema. Methods A multicenter, randomized, double?blind, parallel?group, controlled clinical study was conducted. Totally, 144 patients with subacute eczema and 144 patients with chronic eczema were enrolled into this study, and both randomly and equally divided into the test group and control group. The test group and control group firstly topically applied compound polymyxin B ointment and its vehicle respectively, then both topically applied desonide cream 3 hours later. The drugs or vehicle were applied twice a day in all the patients. Patients′ symptoms and signs (including degree of itching, inflammation, erosion/exudation and infiltration/thickening, as well as area of target lesions) were evaluated, and the time to onset and duration of itching?alleviating effect were recorded. The clinical efficacy and safety of treatments were analyzed and compared between the test group and control group. Results The total symptom and sign scores significantly decreased to different extents on days 7 and 14 in the test group(subacute eczema patients:6.09 ± 2.78 and 3.68 ± 3.18 vs. 13.44 ± 1.66; chronic eczema patients: 6.56 ± 2.68 and 4.38 ± 3.27 vs. 12.96 ± 1.16)and control group(subacute eczema patients:8.26 ± 3.17 and 5.28 ± 4.05 vs. 13.60 ± 1.75;chronic eczema patients: 8.84 ± 2.90 and 6.25 ± 3.78 and vs. 12.64 ± 1.18)compared with those at baseline. Moreover, the total symptom and sign score of patients with subacute or chronic eczema was significantly lower in the test group than in the control group on days 7 and 14(all P<0.05). A significant increment was observed in the degree of decrease in scores for itch, infiltration/thickening in patients with subacute eczema in the test group compared with that in the control group(all P<0.01), as well as in scores for itch, infiltration/thickening and area of target lesions in patients with chronic eczema in the test group compared with those in the control group (all P < 0.05). In addition, patients with subacute eczema in the test group showed significantly shorter onset and longer duration of itching?alleviating effect than those in the control group(both P<0.05). The time to onset of itching?alleviating effect was also significantly shorter in patients with chronic eczema in the test group than in those in the control group(P<0.000 1), but there was no significant difference in the duration of it between the two groups of patients with chronic eczema. Clinicians and patients were both more satisfied with therapeutic effects in the test group than in the control group(all P<0.05). Conclusions Topical compound polymyxin B ointment can increase the efficacy of topical desonide cream for the treatment of subacute or chronic eczema, especially subacute eczema. Compound polymyxin B ointment also shows a favorable therapeutic effect on itching and infiltration/thickening in patients with eczema.

7.
Article in Chinese | WPRIM | ID: wpr-256579

ABSTRACT

<p><b>OBJECTIVE</b>To evaluate the effect of the length of covered stents in the portal and hepatic veins on long-term clinical efficacy of transjugular intrahepatic portosystemic shunt (TIPS).</p><p><b>METHODS</b>We retrospectively reviewed 53 cases receiving TIPSs between January, 2008 and December, 2011. All the shunts were created with Fluency stents (8 mm in diameter). Bare metal grafts of the same diameter were implanted to extend the length in the portal and hepatic veins as deemed necessary according to angiographic images. The primary patency, hepatic encephalopathy and patient survival were evaluated during the follow up. The length of the covered stents within the hepatic vein (X1) and in the portal vein (X2), and the total length of stents placed in the hepatic vein (X3) and the portal vein (X4) were measured and their effects on primary patency and the patients'outcomes were evaluated.</p><p><b>RESULTS</b>The procedures were completed successfully in all the patients and the mean portosystemic pressure decreased from 29.80∓4.83 mmHg to 19.00∓3.92 mmHg (t=13.44, P<0.01) after the procedure. The patients were followed up for a median of 64 months (3 to 89 months, 39 months on average). Hepatic encephalopathy occurred in 23% (12/53) of the patients after TIPS. Shunt dysfunction occurred in 16 cases, and the cumulative primary patency rates at 1 to 5 years were 83%, 75%, 63%, 62%, and 54%, respectively. The cumulative survival rates of the patients at 1 to 5 years were 79%, 72%, 72%, 69%, and 69%, respectively. Cox proportional regression analysis showed a significant association between the length of covered-stent in the hepatic vein and the primary patency (OR=0.42, P<0.01), and there was a significant association between the length of stent in the portal vein and the patient survival. No significant correlation was found between these parameters and hepatic encephalopathy.</p><p><b>CONCLUSION</b>Increasing the length of the covered stent in the hepatic vein and decreasing the stent length in the portal vein can improve the primary patency and the patient survival receiving TIPS.</p>


Subject(s)
Angiography , Hepatic Encephalopathy , Hepatic Veins , General Surgery , Humans , Portal Vein , General Surgery , Portasystemic Shunt, Transjugular Intrahepatic , Retrospective Studies , Stents , Survival Rate , Treatment Outcome
8.
China Medical Equipment ; (12): 66-68, 2015.
Article in Chinese | WPRIM | ID: wpr-483780

ABSTRACT

Objective:To investigate the effects of static ultrasound elastography on the elastic modulus of normal breast tissue.Methods: (1)Fifteen cases of normal breasts was tested by static ultrasound elastography at the same locations. Intercepted two pictures each time with one stress-free and the other compressed the maximum amount. For these two cases we measured the breast thickness and calculated the minimum stretch ratios(λ). (2)Fifty breasts with no breast nodules were prospectively enrolled in the study. Intercepted two pictures each time with one stress-free and the other compressed to the minimum stretch ratios(λ). For these two cases we measured the Elastic modulus (E0, Em, kPa). Calculate the elastic modulus change ratio (Rm/0).Results: (1)The minimum stretch ratio caused by static ultrasound elastography was 0.74±0.07. (2)The elastic modulus change ratio of static ultrasound elastography was 4.38±1.72. Conclusion: The normal operation of static ultrasound elastography caused 74 percent deformation in normal breast tissue. There was 4.38 times elastic modular increasing caused by 74 percent deformation in normal breast tissue.

9.
Article in Chinese | WPRIM | ID: wpr-448012

ABSTRACT

Objective To assess the relationship between pulse wave velocity(PWV) and carotid atherosclerosis by UltraFast imaging.Methods 476 cases from Medical Examination Center of Beijing Friendship Hospital were enrolled in this study.According to intima-media thickness(IMT) of carotid artery,all subjects were divided into two groups:IMT without thickening group (G0) of 283 cases and IMT thickening group (G1) of 193 cases.Through UltraFast imaging,carotid artery PWV was measured,including the PWV at the beginning of the systole(BS) and the PWV at the ending of the systole(ES).Carotid IMT and plaque situation were recorded under gray-scale ultrasound.BS and ES were compared between the two groups.Spearman rank correlation was used to analyse the correlation between IMT of carotid artery and BS,between IMT of carotid artery and ES.Two independent samples t test to compare BS,ES between the two groups.Results BS of G1 and G0 were (6.03 ± 1.33)m/s and (5.51 ± 1.13)m/s,t =-4.571,P =0.000;ES of G1 and G0 were (8.42 ± 2.13)m/s and (7.34 ± 2.02) m/s,t =5.619,P =0.000.BS and ES of G1 were larger those of G0 respectively.BS (r =0.192,P =0.000) and ES (r =0.249,P =0.000) were correlated with the IMT of carotid artery.Conclusions Ultrafast imaging technique can quickly measure the carotid artery PWV.BS and ES of carotid artery can be quick,convenient,safe,noninvasive parameters for evaluating carotid atherosclerosis.

10.
Chinese Journal of Dermatology ; (12): 196-198, 2010.
Article in Chinese | WPRIM | ID: wpr-390734

ABSTRACT

Objective To measure the expressions of IL-10, IL-23 and CD86 in lesions of epidermodysplasia verruciformis (EV), and to explore the relationship between cellular immune abnormality and EV pathogenesis. Methods Immunohistochemistry was used to measure the expressions of IL-10, IL-23 and CD86 in tissue samples from 10 patients with EV and 10 normal human controls. Results Three cytokines were observed in all the samples of EV, with the expression score ranging from 3 to 6 and expression intensity from moderate to high. However, of the control specimens, only 1 was positive for IL-10 with the expression score being 3, and expression intensity being moderate. Conclusion The pathogenesis of epidermodysplasia verruciformis may be correlated with the expression abnormality of some cytokines secreted by keratinocytes.

11.
Braz. j. microbiol ; 40(4): 734-746, Oct.-Dec. 2009. ilus, graf, tab
Article in English | LILACS | ID: lil-528155

ABSTRACT

Bitespiramycin, a group of 4"-O-acylated spiramycins with 4"-O-isovalerylspiramycins as the major components, was produced by recombinantspiramycin-producing strain Streptomyces spiramyceticus harboring a 4"-O-acyltransferase gene. The experiment was initially performed in synthetic medium with 0.5 g l-1 Valine, Isoleucine or Leucine feeding at 36 h cultivation. When valine was fed, the biological titer of bitespiramycin was 45.3 percent higher than that of the control group, but the relative content of total isovalerylspiramycin components decreased by 22.5 percent. In the case of ilecine, the biological titer of bitespiramycin and the total isovalerylspiramycins alone were 85 percent and 72.1 percent of the control group, respectively. In contrast, the relative content of other acylated spiramycins increased by 54.41 percent. However, leucine feeding increased the relative content of total isovalerylspiramycins by 41.9 percent while the biological titer of bitespiramycin was nearly equal to that of the control group. The improvement effect of leucine on the biosynthesis of isovalerylspiramycins was further confirmed by feeding of 2.0 g l-1 leucine to the culture with complex medium. After batch feeding with a total amount of 2.0 g l-1 leucine to the culture from 70 h to 90 h, the biological titer of bitespiramycin was almost unreduced, and the final relative content of total isovalerylspiramycins increased from 31.1 percent to 46.9 percent.


Subject(s)
Amino Acids/analysis , Amino Acids/biosynthesis , Spiramycin/analysis , Spiramycin/biosynthesis , Leucine/analysis , Leucine/biosynthesis , Protein Biosynthesis , Methods , Methods
12.
Article in Chinese | WPRIM | ID: wpr-259085

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the expressions of CD1a and CD83 of Langerhans cells (LC) in the lesions of epidermodysplasia verruciformis (EV) patients.</p><p><b>METHODS</b>We used immunohistochemical method to detect the expressions of CD1a and CD83 in the lesions of 10 patients with EV lesions and in the skins of 10 normal subjects.</p><p><b>RESULTS</b>No CD83 + LCs was detected in all EV patients and normal controls, but CD1a + LC was found in all cases. The quantity of CD1a + LCs in the lesions of EV patients was significantly lower than that in the normal skin (P < 0.01); furthermore, the distribution of LCs in EV lesions was uneven.</p><p><b>CONCLUSION</b>The functions of LCs may be inhibited in EV patients.</p>


Subject(s)
Antigens, CD , Genetics , Antigens, CD1 , Genetics , Epidermodysplasia Verruciformis , Allergy and Immunology , Pathology , Humans , Langerhans Cells , Allergy and Immunology , Leukocyte Immunoglobulin-like Receptor B1 , Receptors, Immunologic , Genetics , Skin , Allergy and Immunology , Pathology
13.
Article in Chinese | WPRIM | ID: wpr-259065

ABSTRACT

Epidermodysplasia verruciformis (EV), a rare inherited disease, is believed to be associated with human papillomavirus (HPV) infection. EVER1/2 genes, dendritic cells, T lymphocytes, and the biological characteristics of HPV itself may play roles in the pathogenesis of HPV infection.


Subject(s)
Dendritic Cells , Allergy and Immunology , Epidermodysplasia Verruciformis , Genetics , Allergy and Immunology , Virology , Humans , Membrane Proteins , Genetics , Mutation , Papillomaviridae , Virulence , Papillomavirus Infections , T-Lymphocytes , Allergy and Immunology
14.
Article in Chinese | WPRIM | ID: wpr-293377

ABSTRACT

Immobilized penicillin acylase was used for bioconversion of penicillin PG into 6-APA in aqueous two-phase systems consisting of a light-sensitive polymer PNBC and a pH-sensitive polymer PADB. Partition coefficients of 6-APA was found to be about 5.78 in the presence of 1% NaCl. Enzyme kinetics showed that the reaction reached equilibrium at roughly 7 h. The 6-APA mole yields were 85.3% (pH 7.8, 20 degrees C), with about 20% increment as compared with the reaction of single aqueous phase buffer. The partition coefficient of PG (Na) varied scarcely, while that of the product, 6-APA and phenylacetic acid (PA) significantly varied due to Donnan effect of the phase systems and hydrophobicity of the products. The variation of the partition coefficients of the products also affected the bioconversion yield of the products. In the aqueous two-phase systems, the substrate, PG, the products of 6-APA and PA were biased in the top phase, while immobilized penicillin acylase at completely partitioned at the bottom. The substrate and PG entered the bottom phase, where it was catalyzed into 6-APA and PA and entered the top phase. Inhibition of the substrate and products was removed to result in improvement of the product yield, and the immobilized enzyme showed higher efficiency than the immobilized cells and occupied smaller volume. Compared with the free enzyme, immobilized enzyme had greater stability, longer life-time, and was completely partitioned in the bottom phase and recycle. Bioconversion in two-phase systems using immobilized penicillin acylase showed outstanding advantage. The light-sensitive copolymer forming aqueous two-phase systems could be recovered by laser radiation at 488 nm or filtered 450 nm light, while pH-sensitive polymer PADB could be recovered at the isoelectric point (pH 4.1). The recovery of the two copolymers was between 95% and 99%.


Subject(s)
Catalysis , Enzymes, Immobilized , Metabolism , Hydrogen-Ion Concentration , Kinetics , Penicillanic Acid , Chemistry , Metabolism , Penicillin Amidase , Metabolism , Penicillin G , Chemistry , Metabolism , Phase Transition , Polymers , Chemistry , Substrate Specificity
15.
Chinese Journal of Biotechnology ; (12): 878-884, 2007.
Article in Chinese | WPRIM | ID: wpr-327930

ABSTRACT

Redox electrode was used to control redox potential at four different levels (-50 mV, -100 mV, -150 mV, - 230mV) for the study of ethanol fermentation. The result showed that there was notably influence on the yield of ethanol, the formation of glycerol, the secretion of organic acid, biomass and the death of cell by controlling redox potential at different levels. For example: the biomass of ORP at -50 mV was 1.26, 1.86, 2.59 times higher than ORP at -100 mV, -150 mV, -230 mV respectively, the final glycerol concentration was 1.2, 1.1, 1.7 times higher but final ethanol concentration was 0.87, 0.49, 0.51 times lower compared to the latest three ORP control level. And take biomass, ethanol yield, glycerol concentration, and unconsumed glucose into consider, we get the conclusion that it is very favorable for ethanol fermentation by control ORP at 150 mV. So it give us a apocalypse that we can use redox electrode to control the ethanol fermentation exactly on bioreactor scale.


Subject(s)
Electrodes , Ethanol , Metabolism , Fermentation , Industrial Microbiology , Methods , Oxidation-Reduction , Saccharomyces cerevisiae , Metabolism
16.
Article in Chinese | WPRIM | ID: wpr-686300

ABSTRACT

13C metabolic flux analysis(13CMFA)have been the research hotspots of metabolic engineering internationally due to its accuracy and applicability.It is vital that the measurement of 13C labelling pattern of proteinogenic amino acids for 13C metabolic flux analysis.To acquire 13C-labelling proteinogenic amino acids,Pseudomonas denitrifican which products Vitamin B12 was firstly fed with mimimal culture medium contained 20% U-13C and 80% natural glucose,after the culture reached a steady state,then about 20 mg biomass was hydrolyzed by 1 ml of 6 mol/L hydrochloric acid for 24h at 110℃.Then amino acids was separated,concentrated,evaporated in a vacuum,and derivatized with MBDSTFA,TBDMS-derivatized amino acids can be observed by GC-MS last we get 13C labelling pattern of fifteen aminio acids through mass spectrum.The experimental methods and sample preparation offers referential value for the development of 13C metabolic flux analysis in our courtry.

17.
Article in Chinese | WPRIM | ID: wpr-685594

ABSTRACT

Immobilized penicillin acylase was used for bioconversion of penicillin G into 6-APA in aqueous two-phase systems consisted of a light-sensitive polymer PNBC and a pH-sensitive polymer PADB.Partition coefficients of 6-APA was found to be:about 5.78,in the presence of 1% NaCl.Enzyme kinetic showed that reaction reached equilibrium at 7h or so.The 6-APA mole yields were 85.3%(pH 7.8,and 20 ℃) and this value was about 20%higher than control in reaction of single aqueous phase buffer.Partition coefficient of penicillin G(Na) washardly changeable,while partition coefficient of product,6-APA and phenylacetate acid was significantly changeable.Reason is due to Donnan effect of phase systems andhydrophobicity of products.The change of partition coefficients of products also affects bioconversion yield of products.In the aqueous two-phase systems,substrate,penicillin G,products 6-APA and phenylacetate acid are biased in top phase,while immobilized penicillin acylase is completely partitioned in bottom.Substrate,penicillin G enters into bottom phase,and it is catalyzed into 6-APA and phenylacetate acid,then the products enter into top phase.Finally,inhibition of substrate and products is removed to result in improvement of products yield.Moreover,immobilized enzymehashigher efficiency than immobilized cells and occupy smaller volume.Comparing with free enzyme,immobilized enzymehashigher stability,longer use life,completely partitioned in bottom phase and recycle.Bioconversion in two-phase systems using immobilized penicillin acylase showed outstanding advantage.The light-sensitive copolymer forming aqueous two-phase systems could be recovered by laser radiation at 488 nm or filtrated 450 nm light,while pH-sensitive polymer PADB could be recovered by isoelectric point(pH 4.1).The recovery of the two copolymers was 95%~99%.

18.
Chinese Journal of Biotechnology ; (12): 311-315, 2006.
Article in Chinese | WPRIM | ID: wpr-286290

ABSTRACT

The effect of induction temperature on aggregation of consensus interferon-alpha expressed by Pichia pastoris was investigated. The cell growth and cIFN level were analyzed and compared when Pichia pastoris was grown at 30,25,20 degrees C during induction phase, using 5.0L fermentor. The result suggested that the cell growth was not affected much under the different induction temperature, but the protein level declined markedly with the decrease of the induction temperature. The total protein ammount induced at 20 degrees C was 67.8 percent of that at 30 degrees C. SDS-PAGE and native-PAGE as well as Western blotting analysis were further conducted. The electrophoresis results revealed that cIFN formed aggregates after secreted into media when protein was induced at 30 degrees C but this problem can be restored by decreasing the induction temperature to 20 degrees C. cIFN monomer in supernatant arrived at 570mg/L and bioactivity of fermentation broth reached 1.05 x 10(9) IU/mL at 20 degrees C of induction temperature. The amount of cIFN monomer and bioactivity in supernatant elevated 7.2 and 38.7 times, respectively, when the induction temperature was controlled at 20 degrees C instead of conventional 30 degrees C.


Subject(s)
Fermentation , Humans , Interferon Type I , Genetics , Interferon-alpha , Pichia , Genetics , Metabolism , Recombinant Proteins , Genetics , Temperature
19.
Chinese Journal of Biotechnology ; (12): 198-203, 2005.
Article in Chinese | WPRIM | ID: wpr-249925

ABSTRACT

Apolipoprotein AI (apo AI), the major protein component of human high-density lipoprotein (HDL), is a single-chain polypeptide of 243 amino acids. Several epidemiological studies have shown that the plasma concentrations of HDL has the role of reverse cholesterol transport (RCT) and inversely correlated with the incidence of coronary artery disease. Because apo AI lacks post-translational modifications, it is convenient to express human apo AI in Escherichia coli expression system. However, there is a poor stability of the mRNA and the apo AI protein in E. coli, it is difficult to express mature apo AI in recombinant bacteria, moreover, even as a fusion protein, apo AI is still sensitive to degradation and can not be cleaved efficiently from the fusion tags. In contrast, proapolipoprotein AI (proapo AI, having an additional polypeptide containing the amino acids Arg-His-Phe-Trp-Gln-Gln at the amino-teminal of the mature protein) proved stable and undegraded in Escherichia coli, and therefore, in this research, an expression system of E. coli including a plasmid of P(R)P(L) tandem promoter was adapted to produce proapo AI. Furthermore, site-directed mutagenesis of the proapo AI cDNA was performed to generate a Clu8Asp mutation in the amino-terminal sequence of proapo AI which created an acid labile Asp-Pro peptide bond between amino acid 8 and 9, and permitted specific chemical cleavage to remove pro-peptide. After inducing with a shift of temperature, yields of recombinant proapo AI achieved about 40% of total cell protein and the recombinant proapo AI expressed proved as a form of inclusion body in cells, so protein need to renature. First of all, the protein was dissolved in buffer with denaturant, and renaturation was carried out on a hydrophobic interaction column (Phenyl Sepharose), ion-exchange chromatography and gel-filtration chromatography were then used to further purify the protein. The purified recombinant apo AI was detected by a set of tests including Western-blotting, Circular dichroism spectra and lipid-binding test, the results shown that recombinant apo AI has similar structural and lipid-binding properties identical to those of native plasma apo AI, which facilitates further research and application.


Subject(s)
Apolipoprotein A-I , Genetics , Chromatography, Ion Exchange , Methods , Escherichia coli , Genetics , Metabolism , Humans , Mutagenesis, Site-Directed , Mutation , Protein Precursors , Genetics , Recombinant Proteins , Genetics
20.
Article in Chinese | WPRIM | ID: wpr-622619

ABSTRACT

Medicine is intended for human beings and the crowd.The constant variation of the record of diseases and the continuous emergence of the technical means of diagnosis and intervening measures to promote health make it urgent to have the idea of life-long study and cultivating the creative talents of medicine.Therefore the medical higher learning must renew the idea of running schools,change the model and goals of cultivation,push the teaching reform forward including paying attention to pushing the curriculum system,teaching methods and the reform of teaching management system,emphasize creative education to create the condition for cultivation of the creative talents.

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