Different riboswitches on regulatory efficiency of downstream target genes: a comparative study / 军事医学

Objective To compare the regulation effects of different activated and inhibitory riboswitches , and to facilitate the precise regulation of gene circuits .Methods A green fluorescent protein amcyan expression vector regulated by different riboswitches (addA, M6, TPP and btuB) was constructed, and the expression level of amcyan under different ligand concentrations was analyzed by RT-qPCR and relative fluorescence intensity , and then compared with the expression level of a vector without any riboswitch .The dynamic control performance was analyzed .Results Under the control of addA and M6 activated riboswitches , the expression of green fluorescent protein increased with ligand concentrations , and addA riboswitch had more dynamic regulatory effect than M 6 riboswitch.However, under the control of TPP and btuB inhibitory riboswitches , the expression of green fluorescence decreased with the increase in ligand concentrations , and the dynamic regulation of btuB riboswitch was slightly greater than that of TPP riboswitch .Conclusion The regulation efficacy of different riboswitches which have the same mechanism varies .Activated riboswitch addA and inhibitory riboswitch btuB with dynamic regulation and control advantages are more suitable for precise metabolism regulation and target gene expression in Escherichia coli.

Preparation and preliminary evaluation of chitosan composite KGF-2 mutant thermal sensitive dressing / 军事医学

Objective To develop chitosan composite keratinocyte growth factor-2 mutant(KGF-2M)temperature-sen-sitive dressing and evaluate its physicochemical properties and dynamic release rule were used.Methods Chitosan, chi-tosan quaternary ammonium salt,β-glycerophosphate and other adjuvant materials to configure different formulations which were compounded with KGF-2M in order to develop temperature-sensitive dressing.Gelling time, temperature,the release rate of KGF-2M and other indicators were measured to analyze the physical and chemical properties of the temperature -sen-sitive dressing.Results Chitosan-KGF-2M composite dressing with temperature-sensitive properties was obtained by opti-mizing the formulation components of chitosan and related adjuvant materials.When the liquid dressing was above 35℃,it could be converted from liquid to solid gelatin within 10 minutes.The compound KGF-2M released from the gel was more than 98%at 4 h,and its bioactivity remained stable.Conclusion The thermo-sensitive gel has the characteristics of good conformability,moisturizing(moisture),isolation,wound healing,and a controlled release effect,which has great potential in wartime for wound repair.

Protective effect of interleukin-1 receptor antagonist on intestinal ischemia-reperfusion induced injury in rats / 中国药理学通报

Aim To explore the effects of interleukin-1 receptor antagonist (IL-1Ra) on intestinal ischemia-reperfusion (I/R) induced injury in rats. Methods Thirty-five male SD rats were randomly divided into sham operation group (S), model group (I/R), dif-ferent dosage drug groups(C1,C2,C3). Rat intesti-nal I/R model was established via clamping the superi-or mesenteric artery (SMA). After 1 h of ischemia, the arterial clamps were released for 1 h of reperfusion. 10,20,50 mg·kg-1of IL-Ra was injected via caudal vein 15min before reperfusion. Results After 2 h of I/R,compared with S group,I/R group rats exhibited severe damage on the intestinal mucosa, increase in MDA content, decrease in SOD activity, and signifi-cant release of TNF-α, IL-1β, IL-6. The results showed that, following the injection of IL-1Ra after clipping superior mesenteric artery, damage of the in-testinal mucosa was obviously relieved in different dos-age drug groups. Furthermore, there was different de-gree of relief on oxidative stress and inflammatory re-sponses. Conclusion IL-1Ra showed obvious protec-tive effect on intestinal I/R induced injury by relieving oxidative stress and inflammatory response,and it may potentially be used in the clinical treatment of intestinal I/R injury.

Differential expression of the RNA-binding motif protein 3 in human astrocytoma / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The RNA-binding motif protein 3 (RBM3), which is transcriptionally induced by low temperature and hypoxia, has recently been found to be upregulated in human tumors. However, its expression status in human astrocytoma is not well defned. This article focuses on the differential expression of RBM3 in human astrocytomas of different grades and normal brain tissues.</p><p><b>METHODS</b>RBM3 was detected in astrocytomas and normal brain tissues by quantitative real-time PCR, immunohistochemistry, and Western blotting. Analysis of variance was performed on the data from quantitative real-time PCR. The Fisher's exact test was used to analyze the immunohistochemistry results. A P-value of less than 0.05 indicates a statistically significant difference.</p><p><b>RESULTS</b>On one hand, the mRNA expression levels of three X-chromosome-related RBM genes (RBMX, RBM3, and RBM10) were detected by quantitative real-time PCR. The results showed that there were no significant differences in RBMX and RBM10 mRNA expression levels in human astrocytomas of different grades and normal brain tissues. However, RBM3 mRNA expression levels were elevated in high-grade (World Health Organization (WHO) Grade III-IV) astrocytomas versus low-grade (WHO Grade I-II) astrocytomas (5.06 ± 0.66 vs. 1.60 ± 0.58; P < 0.05) or normal controls (5.06 ± 0.66 vs. 1.03 ± 0.22; P < 0.05) as determined by quantitative real-time PCR analysis. On the other hand, immunohistochemistry showed an increased RBM3 labeling index in astrocytomas of different grades and normal brain tissues (positive staining rate: astrocytoma Grade IV, 92.9%; astrocytoma Grade III, 81.8%; astrocytoma Grade I-II, 50%; normal brain tissues, 37.5%; high-grade astrocytoma versus normal brain tissues, P < 0.05; high-grade astrocytoma versus low-grade astrocytoma, P < 0.05). The higher protein levels of RBM3 were also validated in high-grade astrocytomas and low-grade astrocytomas compared with normal brain tissues by Western blotting.</p><p><b>CONCLUSIONS</b>These data suggest that the overexpression of RBM3 may serve as an important molecular mechanism underlying astrocytic carcinogenesis. Moreover, RBM3 may have proliferative and/or proto-oncogenic functions in human astrocytomas.</p>

Quantitative method for detection of plasma lumbrokinase content and its assessment / 中国实验血液学杂志

This study was aimed to set up and evaluate a quantitative method for detecting lumbrokinase level in plasma. The lumbrokinase was used to immunize rabbit and BALB/c mouse for preparation of rabbit or mouse-derived polyclonal antibodies, and then the standard curves were drawn up by detecting the lumbrokinase diluted in PBS using the double antibody sandwich ELISA. This method further was analyzed for its specificity, precision and recovery rate. This established double antibody sandwich ELISA was used to assay the lumbrokinase in human plasma, and the assayed results were assessed. The results showed that a double antibody sandwich ELISA for the detection of lumbrokinase has been established. And the standard curve fitting R value > 0.99, the precision assessment showed that the measured values of coefficient of variation (CV) in 3 batches were all < 15%; recovery assessment in 3 batches showed that all the measured recovery rates were > 80%; the quantitative low limit was assessed as 5 ng/ml (precision CV < 15%, recovery rate > 85%). It is concluded that this method is consistent with the criteria stipulated by the Pharmacopeia, which provides a reliable measurement method for quantitative detection of plasma lumbrokinase in clinical trials.

Influence of long-used staphylokinase derivative on hemoagglutinative and fibrinolytic systems / 中国实验血液学杂志

This study was aimed to explore the influence of staphylokinase derivative (SAKD) on the hemoagglutinative and fibrinolytic systems, and to determine the safety of the staphylokinase derivative in application. The normal and model rats each 30 were divided into normal saline, SAKD and rSAK groups. The hemorrhage, bleeding time (BT), blood platelet count (BPC), activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen (Fg), D-dimer (D-D), plasminogen (PLG) and plasmin inhibitor activity (PI) were detected before and after the administration with staphylokinase derivative 0.5 mg/kg body weight, once three days for consecutive 15 days. The results indicated that one case of normal rats with SAKD and two cases of high fat diet model group had mild hemorrhage, all of which showed automatic hemostasis; and 3 cases in rSAK group had mild hemorrhage. And the platelet counting, D-D, PLG and PI in all groups did not significantly change. The rats of high fat diet group treated with SAKD showed the significant extension of APTT, PT and TT times, and the decrease of Fg time (p < 0.05). All the experimental results demonstrated that the influence of SAKD on the hemagglutination of the normal animals was lower, however, which can improve the high-hemagglutination status of the rats with high fat diet. It is concluded that the SAKD at the dosage of this study has the higher safety, which can alleviate the high hemagglutination symptoms of the rats with high fat diet.

Construction and characterization of soluble HLA-A*0201-PR1 complex / 中国实验血液学杂志

This study was aimed to construct the soluble HLA-A*0201-PR1 complex for preparation of HLA-A*0201-PR1 tetramer. The recombinant HLA-A*0201-BSP (BirA substrate peptide) fusion protein as heavy chain and beta(2)-microglobulin (beta(2) m) as light chain were expressed highly as insoluble aggregates in Escherichia coli and then purified with gel filtration, and the final purity reached above 90%. The two subunits were refolded to form an HLA-A*0201-peptide complex by dilution method in the presence of an antigenic peptide PR1, a HLA-A2-restricted peptide from proteinase 3 (aa 169 - 177, VLQELNVTV). Refolded HLA-A*0201-PR1 complex was biotinylated using a BirA enzyme and purified by anion exchange chromatography on a Q-Sepharose (fast flow) column. The extent of reconstitution of the HLA-A*0201-PR1 complex was analyzed by HPLC gel filtration. The refolded and biotinylated products were detected by Western blot and ELISA with monoclonal antibody BB7.2 that recognized the natural conformations of HLA-A2 and streptavidin. The results showed that the refolded complex was composed of HLA-A*0201-BSP aggregate, HLA-A*0201-PR1 complex and beta(2) m, and reconstitution yields of 18% with PR1 was obtained. Refolded HLA-A*0201-PR1 complex could be confirmed by practical immunological method and biotinylated efficiently. It is concluded that the refolding and biotinylation of HLA-A*0201-PR1 complex is successfully obtained. This work provides the basis for the preparation of HLA-A*0201-PR1 tetramer.

Identification of heplipin inducing apoptosis-related genes in KG-1 human leukemia cells by differential display RT-PCR / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate whether Heplipin can induce KG-1 cell apoptosis and explore apoptosis related differentially expressed genes in KG-1 leukemia cell before and after Heplipin induction.</p><p><b>METHODS</b>DNA distribution and DNA electrophoresis were used to prove that Heplipin can induce KG-1 cell apoptosis. The differential display reverse transcription-polymerase chain reaction (DDRT-PCR) was adopted to screen differentially expressed genes before and after Heplipin induction of KG-1 cells for 16 hours and 20 hours. The differentially expressed genes were cloned and analyzed.</p><p><b>RESULTS</b>Heplipin could induce KG-1 cell apoptosis. There were differentially expressed genes in KG-1 cells before and after induction. Wnt13 and ATPase 3 were apoptosis related differentially downregulated genes after Heplipin induction. Conclusion Heplipin can induce KG-1 cell apoptosis. Heplipin induced KG-1 cell apoptosis is related with Wntl3 and ATPase3 (PSMC3). It is the first report that Wnt13 was detected in leukemia cell line.</p>

Soluble high-expression, purification and bioassay of IGFBP-3 / 生物工程学报

cDNA for Insulin-like growth factor binding protein 3 was cloned and constructed a prokaryotic expression vector--pET-DsBA-IGFBP3. The construct was transformed into E. coli BL21 (DE3)plysS. The induced fusion protein (D-IGFBP3) was expressed successfully in soluble form. We obtained D-IGFBP3 the purify of which is over 95% after purification by His affinity chromatography. The product was identified by Western-blot. The cell assay showed that the obtained fusion protein can inhibit the growth of MCF-7 and bind with IGF-I in vitro.

Cloning of Human beta2-microglobulin gene and efficient expression in Escherichia coli / 中国实验血液学杂志

Human beta(2)-microglobulin (beta(2)m) is the light chain of major histocompatibility complex (MHC) class I molecule. High-yield production of this protein is a prerequisite to the preparation of MHC class I tetramer. The present study was aimed to obtain recombinant human beta(2)m expressed in Escherichia coli (E. coli) for preparing MHC class I tetramers. For cloning of human beta(2)m gene, a pair of specific primers was designed based on the published sequence of this gene. A 300 bp specific DNA fragment corresponding to the encoding region of beta(2)m lack of the signal peptide sequence was obtained by RT-PCR from the total RNA of human leukocytes. The amplified cDNA was inserted into the IPTG-inducible expression plasmid pET-17b by Nde I and Bam H I sites and its sequence was confirmed by DNA sequence analysis. The recombinant plasmid pET-beta(2)m was transformed to the competent cells of E. coli BL21 (DE3). The results showed that beta(2)m was expressed in the form of inclusion body and amounted to over 32% of total cell proteins after IPTG induction. After washing with triton X-100 and urea, the inclusion body was dissolved with 4 mol/L urea and then purified with Sephacryl S-200 HR, and the final purity reached above 95%. The denatured protein was renatured by dilution method. Western blot assay indicated that the monoclonal antibody against human native beta(2)m could react specifically with the recombinant protein. In conclusion, the human beta(2)m gene was cloned successfully and expressed efficiently in E. coli BL21 (DE3). This work establishes a convenient approach for renaturation and purification of large quantity of recombinant beta(2)m. This provides the basis for the preparation of MHC tetramers.

Role of B-cell-activating-factor in immune regulation--review / 中国实验血液学杂志

B cell activating factor (BAFF) is one of the TNF family member, regulates the survival and maturation of B lymphocyte. BAFF binds to three receptors: BCMA, TACI and BAFF-R. In recent years, studies have revealed important roles of BAFF and its receptors in immune regulation of antibody isotype switching, germinal center maintenance, and T cell co-stimulation, that may provide new drugs in the future for the treatment of autoimmune disorders, lymphoma and B cell immunodeficiencies. Therefore, the structure, expression, receptors, biological function and clinical application of BAFF are briefly summarized in this review.

Cloning and expression of HLA-A*0201-BSP / 中国实验血液学杂志

High-yield production of HLA-A*0201 heavy chain is a prerequisite to the preparation of HLA-A2 tetramer. The present study was aimed to construct the expression vector of recombinant HLA-A*0201-BSP fusion gene for preparing HLA-A2 tetramers. The extracellular region HLA*0201 was cloned by RT-PCR from HLA-A2(+) donor, and a 15-amino acid biotin-protein ligase (BirA) substrate peptide (BSP) for BirA-dependent biotinylation was added to the COOH-terminus of HLA-A*0201 heavy chain. Then the fusion gene was cloned into pBV220 vector at EcoRI and Bam HI sites and its sequence was confirmed by DNA sequence analysis. The recombinant plasmid pBV220-HLA-A*0201-BSP was transformed to the competent cells of E.coli DH5alpha. The results showed that the HLA-A*0201-BSP fusion protein was successfully expressed in the form of inclusion body and amounted to over 28% of total cell proteins via induction at 42 degrees C. After washed with triton X-100 and urea, the inclusion body was dissolved with 8 mol/L urea and then purified with Sepharcyl S-300 HR, and the final purity reached above 90%. It is concluded that the HLA-A*0201-BSP fusion gene was cloned successfully and expressed efficiently in E.coli DH5alpha. This work establishes a convenient approach for purification of large quantity of recombinant HLA-A*0201-BSP. This provides the basis for the preparation of HLA-A2 tetramers.