ABSTRACT
L-asparaginase (L-ASN) is widely applied in the treatment of malignant tumor and low-acrylamide food production, however, the low expression level hampers its application. Heterologous expression is an effective strategy to increase the expression level of target enzymes, and Bacillus is generally used as the host for efficient production of enzymes. In this study, the expression level of L-asparaginase in Bacillus was enhanced through optimization of expression element and host. Firstly, five signal peptides (SPSacC, SPAmyL, SPAprE, SPYwbN and SPWapA) were screened, among which SPSacC showed the best performance, reaching an activity of 157.61 U/mL. Subsequently, four strong promoters (P43, PykzA-P43, PUbay and PbacA) from Bacillus were screened, and tandem promoter PykzA-P43 showed the highest yield of L-asparaginase, which was 52.94% higher than that of control strain. Finally, three Bacillus expression hosts (B. licheniformis Δ0F3 and BL10, B. subtilis WB800) were investigated, and the maximum L-asparaginase activity, 438.3 U/mL, was reached by B. licheniformis BL10, which was an 81.83% increase compared with that of the control. This is also the highest level of L-asparaginase in shake flask reported to date. Taken together, this study constructed a B. licheniformis strain BL10/PykzA-P43-SPSacC-ansZ capable of efficiently producing L-asparaginase, which laid the foundation for industrial production of L-asparaginase.
Subject(s)
Bacillus licheniformis/metabolism , Asparaginase/genetics , Bacillus/genetics , Protein Sorting Signals , Promoter Regions, Genetic/genetics , Bacillus subtilis/genetics , Bacterial ProteinsABSTRACT
<p><b>OBJECTIVE</b>To isolate, culture and identify of human endometrial stromal stem cells.</p><p><b>METHODS</b>Endometrial tissue was obtained from 10 women undergoing hysterectomy. Purified stromal stem cell suspensions were then obtained by selecting cells with magnetic bead sorting and colony-forming. The surface markers of stromal cell were identified by flow cytometry. Proliferation of stromal stem cell was observed by MTT methods. The osteogenic potential was evaluated by alizarin red staining, the adipogenic potential by oil red O staining. Then the adipogenic and osteogenic specific markers of differentiated cells assayed by RT-PCR method. Expression of cell surface antigen OCT-4 was detected with immunocytochemical staining.</p><p><b>RESULTS</b>Endometrial stem cells were successfully isolated from human endometrial tissue. They were stably proliferated and subcultured in vitro. Most of the passage cells expressed mesenchymal stem cell markers CD90, CD105 but not hemopoietic markers CD34, CD45. The analysis of cell cycle indicated that the percentage of G2-M phase and S phase cells increased. The growth curves of the third passage presented in "S" shape. After cultured in differentiation medium, the cells differentiated toward adipoblasts and osteoblasts as verified by positive staining with Oil Red O and alizarin red staining. Under induction,cells expressed osteogenic and adipogenic marker genes. The immunocytochemical staining of OCT-4 was positive.</p><p><b>CONCLUSION</b>Human endometrium contains endometrial stromal stem cells, which present characteristics of mesenchymal stem cells and may be used as seed cells for tissue engineering.</p>
Subject(s)
Adult , Female , Humans , Cell Culture Techniques , Cell Separation , Cells, Cultured , Endometrium , Cell Biology , Flow Cytometry , Mesenchymal Stem Cells , Cell Biology , Octamer Transcription Factor-3 , Metabolism , Stromal Cells , Cell BiologyABSTRACT
Objective To investigate the effects of remifentanil on NF-κB activity in a rabbit model of myocardial ischemia/reperfusion (I/R) injury. Methods Fifty New Zealand white rabbits were randomly divided into 5 groups (n = 10 each): sham operation group (group S); group I/R; low dose of remifentanil group (group L); median dose of remifentanil group (group M); high dose of remifentanil group (group H). In group I/R, L,M and H, myocardial I/R was produced by occlusion of left anterior descending artery for 30 min followed by 120 min reperfusion.In group S and I/R,10 min before ischemia,normal saline was infused at 5 ml·l·h-1 via the external jugular vein until the end of 120 min reperfusion. In group L, M and H, remifentanil was infused at 1, 5 and 10ug·kg·min-1 respectively 10 min before ischemia until the end of 120 min reperfusion,and the other procedures were the same as those in group I/R. The myocardial tissues were taken at the end of 120 min reperfusion for determination of NF-κB expression which was used to reflect the activity of NF-κB and microscopic examination. Results The activity of NF-κB was significantly higher in group I/R, L, M and H than in group S. The activity of NF-κB was gradually decreased with the increase in the dose of remifentanil in group L, M and H compared with group I/R. The microscopic examination showed that remifentanil significantly attenuated I/R-induced injury in a dose-dependent manner. Conclusion Infusion of remifentanil reduces myocardial I/R injury through decreasing the activity of NF-κB in a doee-dependent manner.
ABSTRACT
Objective To study the mechanism of Chinese Medicine in treating adenomyosis. Methods Thirty-seven patients in the Chinese Medicine treated group (17 cases of adenomyosis) and the control group (20 cases of adenomyosis, non-drug treated) underwent hysterectomy. Endometrium was looked as eutopic endometrium and adenomyosis as ectopic endometrium. Expression of MMP-9 and TIMP-1 in eutopic and ectopic endometrium were measured by immunohistochemical techniques. Meanwhile the levels of CA125 in serum were measured. Results The ectopic endometrium of the Chinese Medicine treated group expressed lower level of MMP-9, higher level of TIMP-1 and lower ratio of MMP-9/TIMP-1 than the ectopic endometrium of the control group (P<0.05). The levels of CA125 in serum were significantly decreased in Chinese Medicine group after treated as contrast with those in control group. Conclusion Chinese Medicine could reduce the level of CA125 and the ratio of MMP-9 and TIMP-1 to prevent the progression of adenomyosis.
ABSTRACT
Objective To study the mechanism of Chinese Medicine in treating adenomyosis.Methods Thirty-seven patients in the Chinese Medicine treated group(17 cases of adenomyosis) and the control group(20 cases of adenomyosis,non-drug treated) underwent hysterectomy.Endometrium was looked as eutopic endometrium and adenomyosis as ectopic endometrium.Expression of MMP-9 and TIMP-1 in eutopic and ectopic endometrium were measured by immunohistochemical techniques.Meanwhile the levels of CA125 in serum were measured.Results The ectopic endometrium of the Chinese Medicine treated group expressed lower level of MMP-9,higher level of TIMP-1 and lower ratio of MMP-9/TIMP-1 than the ectopic endometrium of the control group(P
ABSTRACT
Objective To investigate the expression of H19 imprinted genes in the villus tissues of women with spontaneous abortion.Methods Reverse transcription-polymerse chain reaction method(RT-PCR) was employed to detect H19 imprinted genes allele-specific expression levels of the villus tissues extracted from 45 cases of spontaneous abortion and 30 cases of normal pregnancy.Results Nineteen cases of 21 heterozygous cases in spontaneous abortion samples showed billelic expression of H19(19/21),whereas no billelic expression was found in the normal pregnancy samples(0/13)(P
ABSTRACT
Objective To study the loss of imprinting(LOI) of insulin-like growth factor Ⅱ(IGF2) and H19 genes in cervical carcinomas.Methods Polymerase chain reaction(PCR) method was used on DNA samples extracted from 40 cases of cervical carcinoma and 20 cases of normal control tissues to detect the heterozygosity of IGF2 and H19 genes.The LOI of IGF2 and H19 genes was detected by RT-PCR.Results The heterozygous frequency of IGF2 was 52% in cervical cancer samples.Comparatively,in 20 matched normal samples,13 cases showed the heterozygosity(65%).The biallelic expression of IGF2 was detected in 10 cases among the 21 informative cervical cancer samples(47.6%),however,1 case in 13 heterozygous samples of control group(7.7%)((P0.05).Conclusion LOI and LOH of IGF-2 and H19 genes are involved in cervical carcinomas.LOI of IGF2 and H19 may be involved in the initiation stage of carcinogenesis of cervical carcinoma.
ABSTRACT
Objective To study the relationship between fetal growth retardation(FGR) and expression of insulin-like growth factor-2 and H19 imprinted genes.Methods Immunoradioassay was used to detect the insulin-like growth factor-2(IGF2)level in cord blood from 42 pregnant women with FGR(FGR group) and 30 normal pregnant women(normal group).Reverse transcription-polymerase chain reaction method(RT-PCR) was used to detect H19 imprinted genes allele-specific expression level in placenta.Results The level of IGF2 in cord blood from FGR group was(1.52?0.20)?g/L.It was significantly decreased as compared with that from the normal group(1.97?0.21)?g/L(P