Study on the purification technology of total triterpenoid extracts from Inonotus obliquus and their anti -tumor activity in vitro / 中国药房

OBJECTIVE To optimite the purification technology of total triterpenoid extracts from Inonotus obliquus ，and to investigate the anti -tumor activity of its purified products . METHODS Using inotodiol as control ，the method was established for the content determination of total triterpenoid in I. obliquus. The type of macroporous adsorption resin ，sample volume ，sample concentration，sample flow rate ，eluent volume ，eluent dosage and elution flow rate were selected by single factor experiments . The purification technology of the crude extract was determined and verified . The effects of total triterpenoid purified from I. obliquus on the proliferation ，migration and apoptosis of human cervical cancer HeLa cells were detected by cell proliferation test ， migration test ，flow cytometry and AO/EB kit . RESULTS The best purification technology of total triterpenoid crude extracts from I. obliquus was as follows ：AB-8 macroporous adsorption resin was used ；mass concentration of the sample solution was 2.0 mg/mL；sample volume was 140 mL，and the flow rate was 1.0 mL/min；the impurity was removed with 50% ethanol 40 mL， then eluted with 95% ethanol 160 mL，at the elution flow rate of 3.0 mL/min. After purification ，mass concentration of total triterpenoid from I. obliquus increased from 34.36% to 73.39%. The total triterpenoid of I. obliquus could inhibit the proliferation of HeLa cells ，and the 50% inhibitory concentration was 184.20 μg/mL. Compared with control group ，the purified products could significantly inhibit the migratio n and promote the apoptosis of HeLa cells （P＜0.05 or P＜0.01）. CONCLUSIONS The purification technology of total triterpenoids extracts from I. obliquus is successfully optimited . The purified product could inhibit the proliferation and migration of HeLa cells and induce their apoptosis.

Progress in the discovery of novel cytokines from human genome / 中华微生物学和免疫学杂志

Cytokines are small secreted proteins that play an important role in physiological and pathological processes. In the field of pharmacological development, cytokine drugs have achieved great suc-cess. In 2017, five of the world′s top ten best-selling drugs were inhibitors of cytokines or their receptors. The most innovative and challenging work in cytokine research is the discovery of novel cytokines with impor-tant functions and potential druggable prospects. In the post-genome era, a number of novel cytokines have been internationally discovered using reverse biology techniques, and from which, several cytokine drugs have been approved for marketing. This review highlights 10 novel cytokines found in the human genome by the author′s laboratory, including CTRP4, CCDC134, PSMP, VSTM1-v2, FAM3D, TMEM98, CSBF/C10orf99, FAM19A4, FAM19A5 and FAM19A1. Some of them have been shown to have important physio-logical and pathological functions. Identification of functional receptors, clinical specimen analysis or in vivo animal experiment has been carried out for them, which lays the foundation for further research in their clini-cal applications.

Cell-based Screening and Validation of Human Novel Gene TMEM9B Activated Nuclear Factor Signaling Pathway / 生物化学与生物物理进展

Nuclear factor κB (NF-κB) is an important cellular transcription factor. The important role of NF-κB-mediated cell signal transduction pathway in apoptosis is a hot topic at home and abroad. In order to discover new regulators in NF-κB signaling pathway, a high-throughput cell-based screening model based on dual luciferase reporters system was established, a number of genes that can activate NF-κB signal pathway were obtained by screening of 439 novel function genes. Among them, TMEM9B can obviously activate NF-κB signaling pathway. Further experiments showed that TMEM9B activated NF-κB signaling pathway in a dose-dependent pattern. Western blotting and EMSA experiments confirmed that TMEM9B can promote the degradation of IκBα (a cytoplasm inhibitor of NF-κB), and cause NF-κB shift from the cytoplasm to nucleus. At the same time, flow cytometry result demonstrated TMEM9B can induce apoptosis in HEK293T and HeLa cells. In short, a stable and effective screening system for NF-κB has been established, through which TMEM9B was identified to be able to significantly activate NF-κB signal transduction pathway and thus cause cells apoptosis.

Construction and identification of plasmid with luciferase reporter gene for detection of T-bet expression activity / 中华微生物学和免疫学杂志

Objective To construct a T-bet response reporter gene, for the detection of T-bet tran-scriptional activity and application in high-throughput screening for the functional genomies. Methods The cis-acting DNA element, ThRE, based on CNS-22 T box site of IFN-γ gene, was recombined into a reporter vector pLUC-MCS. The reporter gene was transfected into HEK 293T cells to detect its response to T-bet. And the binding of T-bot to TbRE was identified with electrophoretic mobility shift assay(EMSA). Results ThBE was successfully cloned into pLUC-MCS, named as TbRE-LUC. Using a luciferase assay, expression of the reporter gene is found to be induced by T-bet in a dose dependent manner and correlate with T-bet ex-pression positively with activation up to 20 folds. Moreover, the binding specificity of T-bet to TbRE is vali-dated by EMSA. Conclusion We successfully constructed a T-bet response reporter gene, ThRE-LUC, which responds to T-bet keenly and specifically. TbRE-LUC will be a useful tool in high-throughput screen-ing for human genes associated with transcription activity of T-bet.

Expression and functional study of human recombinant chemokine-like factor I in Drosophila S2 cells / 中华微生物学和免疫学杂志

Objective To express human chemokine-like factor 1 (CKLF1) in Drosophila S2 cells and study its function. Methods The pMT/V5-His-CKLF1 expression plasmid was constructed and transfected into Drosophila S2 cells. The positive clones were selected through PCR and RT-PCR. The culture medium was analyzed by Western blot with anti-CKLF1 polyclonal antibody. Chemotaxis and MTT assays on human peripheral blood and C2C12 cells, respectively, were then carried out with the medium. Results CKLF1 was transcribed efficiently in S2 cells. The expressed CKLF1 protein could be detected in the culture supernatant by Western blot, which showed weak chemotactic activity on both human peripheral blood neutrophils and lymphocytes as well as enhancing effect on the proliferation of C2C12 cells. Conclusion CKLF1 was expressed successfully in Drosophila S2 cells and secreted into the culture medium. The recombinant CKLF1 expressed in Drosophila cells can chemoattract leucocytes and promote the proliferation of C2C12 cells.

Programmed Cell Death 10,Beyond an Apoptosis-related Molecule / 生物化学与生物物理进展

Homo sapiens PDCD10(programmed cell death 10,alias,"TF-1 cell apoptosis related gene 15,TFAR15"),cloned by means of cDNA-representational differences analysis,had been initially identified associated with cell apoptosis.Recent research suggested mutations within the PDCD10 gene or deletion were responsible for cerebral cavernous malformations,and PDCD10 was the third CCM gene.On the other hand,other research demonstrated that PDCD10 was strictly modulated and up regulated in many kinds of tumors,which implicated that PDCD10 participated in tumorous signal transduction.The recent research confirmed that PDCD10 interacts with MST4,a member of Ste20-related kinases,and the interaction promoted cell proliferation and transformation via modulation of the ERK-MARK pathway.In conclusion,all these demonstrate that PDCD10 has many biological effects,which suggests that it is a novel player in vascular morphogenesis and/or remodeling,as well as tumorigenesis and cancer progression.

Preparation and identification of monoclonal antibodies against SARS-CoV putative protein X4 / 北京大学学报(医学版)

Objective: To obtain monoclonal antibodies against putative protein X4 of SARS-CoV for further study of the structure and function of protein X4. Methods: Balb/c mice were immunized with recombinant Protein X4, hybridoma cell lines secreting monoclonal antibodies against protein X4 were screened by regular cell fusion and subcloning approach. The specificities of these monoclonal antibodies were determined by Western blotting and Immunofluorescecence assay. Results: Three hybridoma cell lines (8A5, 8H6 and 4A2) stable in secreting specific monoclonal antibodies were successfully obtained. They could bind specifically to protein X4 proved by Western blotting. All of them belonged to the IgG2a isotype proved by antigen mediated ELISA. Indirect Immunofluorescence assay indicated that they could specifically bind to protein X4 expressed in SARS-CoV infected Vero E6 cells. Conclusion: Monoclonal antibodies of high specificity against protein X4 have been successfully prepared, which laid the foundation for the further study of protein X4.

Characterization of programmed cell death 5 (PDCD5) gene in human cartilage and its possible significance / 北京大学学报(医学版)

Objective: To characterize the expression of programmed cell death 5 (PDCD5) in normal and osteoarthritic human cartilage. Methods: Articular cartilage specimens were obtained from 20 patients with osteoarthritis and 10 with femoral neck (normal cartilage) at the time of arthroplasty. Expression of PDCD5 was detected by flow cytometry, immunofluorescence, RT-PCR and immunohistochemical analysis. Results: PDCD5 expression in osteoarthritis cartilage was significantly higher than in normal cartilage especially in the nucleus. PDCD5 positive chondrocytes were mainly observed in the superficial and deep zone of osteoarthritis tissue sections,and in contrast, in the superficial and middle regions of normal controls. Conclusion: Since apoptotic chondrocyte death occurs more frequently in osteoarthritis compared to normal cartilage and PDCD5 is an apoptosis related protein, the different expression patterns of PDCD5 in osteoarthritis and normal cartilage suggest that it might be involved in the pathogenesis of osteoarthritis.

In silico data mining of the human programmed cell death 5 (PDCD5) sequences / 北京大学学报(医学版)

Objective:To lay foundation for the functional studies of programmed cell death 5 ( PDCD5 ) and develop new technical pathway for bioinformatics analysis of human functional genes. Methods:Using PDCD5 as the target molecule, intensive bioinformatics analysis of the nucleic acid and protein sequences were conducted. Data mining and comprehensive analysis by sequence against database similarity searching, ortholog structure comparison, expression profile analysis and gene “neighbor” listing were performed. Results: Two human putative pseudogenes on chromosomes 12 and 5, and one mouse putative pseudogene on chromosome 1 were identified. The methanobacterium thermoautotrophicum ortholog was classified as the same fold as ubiquitin and ribosomal protein S13. The C. elegans ortholog, ubiquitin and IAP (inhibitor of apoptosis proteins) belonged to the same expression profile cluster. This cluster was related to biosynthesis and protein synthesis. PDCD5 orthologs in various genomes were adjacent to various ribosomal proteins on the chromosome. Conclusion: The human genome contains at least two processed pseudogenes of PDCD5 . Besides the relationship with cell apoptosis, PDCD5 is predicted to have functional relationship with ubiquitin and participate in the translation regulation.

Effects of chemokine-like faotor 1 (CKLF1) on proliferation and metabolism of chondrocytes / 北京大学学报(医学版)

Objective: To investigate the effects of chemokine like factor 1 (CKLF1) on proliferation and metabolism of chondrocytes. Methods: Cell culture, 3H TdR and 3H Proline incorporation methods were used. Chondrocytes were harvested from rabbit articular cartilage. First passage cells were seeded on 96 well plates. After synchronization,the medium was replaced by DMEM containing 5% FCS with various concentration of CKLF1 conditioned medium. The synthesis of mucopolysaccharide was detected by Saffraan O staining. The transcription of inducible nitricoxide synthase (iNOS) was detected by semi quantitative RT-PCR. Results: CKLF1 inhibited the DNA, collagen and mucopolysaccharide synthesis significantly, meanwhile, stimulated the transcription of iNOS. Conclusion: CKLF1 inhibits the proliferation and matrix synthesis of chondrocytes, which might be an important factor resulting in cartilage destructive lesions. CKLF1 may exert its effects on chondrocytes through iNOS pathway.

Expression of human VEGF(121) cDNA in mouse bone marrow stromal cells / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To construct a retroviral vector carrying human vascular endothelial growth factor (hVEGF (121)) cDNA for evaluation of the possibility of VEGF gene therapy in ischemic bone disease.</p><p><b>METHODS</b>hVEGF(121) cDNA was obtained from the plasmid pCDI/VEGF(121) and cloned into retroviral plasmid pLXSN. Recombinant plasmid was transferred to the retro virus packaging cell, PT-67, by lipofectamine mediated gene transfer. Mouse bone marrow stromal cells (MSCs) were transfected by the retrovirus. The integration of the hVEGF(121) cDNA into MSC genomic DNA and expression of the VEGF gene was detected. Proliferation assays of human umbilical vein endothelial cells (HUVECs) by VEGF(121) in culture medium were performed.</p><p><b>RESULTS</b>Recombinant pLXSN/VEGF(121) was correctly constructed and confirmed by restriction endonuclease analysis and DNA sequencing analysis. hVEGF(121) gene was integrated into MSC genomic DNA after transfection, and the VEGF(121) protein was expressed. Proliferation assays showed VEGF(121) in culture medium was a biologically active protein and had a mitogenic effect on HUVEC.</p><p><b>CONCLUSIONS</b>Recombinant retroviral vector carrying hVEGF(121) cDNA was successfully constructed. VEGF (121) protein expressed by MSCs had mitogenic effect biologically. This provides a further foundation for VEGF gene therapy for bone ischemic disease and bone tissue engineering.</p>

Construction of adenoviral vector encoding human VEGF(121) cDNA and its expression in vitro / 中华外科杂志

<p><b>OBJECTIVE</b>To construct the adenoviral vector bringing hVEGF(121) cDNA for evaluation of the possibility of VEGF gene therapy in ischemic bone disease.</p><p><b>METHODS</b>Human vascular endothelial growth factor (hVEGF(121)) cDNA obtained from the plasmid pCDI/VEGF(121) was cloned into plasmid pshuttle and further cloned to Adeno-X Viral DNA. The recombinant adenoviral plasmid was identified and then transferred to the adenoviral packaging cell HEK293 by lipofectamine mediated gene transfer method to pack the virus. After titilating the virus, the mouse bone marrow stromal cells (MSC) were transfected by the adenovirus and the expression of VEGF gene was detected.</p><p><b>RESULTS</b>The recombinant Adeno-VEGF(121) was correctly constructed and confirmed by restriction endonuclease analysis and DNA sequencing analysis. After MSCs were tranfected by the virus, RT-PCR showed that hVEGF(121) mRNA was transcripted from the hVEGF(121) gene. Western blot and immune histochemistry showed VEGF(121) protein was expressed in transgene MSCs.</p><p><b>CONCLUSION</b>The recombinant adenoviral vector bringing hVEGF(121) cDNA was successfully constructed and the transgene MSC expressed hVEGF gene in vitro, it provided the further foundation of VEGF gene therapy for bone ischemic diseases.</p>

Expression of IL-6 in recombinant non-replicating vaccinia virus and studying its effects on immunogenicity / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To investigate expression of IL-6 in non?replicating vaccinia virus and its immune effects on recombinant virus.</p><p><b>METHODS</b>The recombinant non replicating vaccinia virus RVJ123 delta CK11 beta 75IL6 was constructed with non?replicating vaccinia virus vector pNEOCK11beta75IL6 and replicating vaccinia virus RVJ123. In animal model, immunization with the recombinant virus was carried out and its immune response was studied.</p><p><b>RESULTS</b>The recombinant virus could express IL-nd HBsAg simultaneously. Southern blot analysis demonstrated that the genes between vaccinia virus Hind? C and K fragments were deleted and IL-6 gene was integrated stably. Given intranasal inocula of the virus to immunize BALB/c mouse and New Zealand Rabbit, the elevated anti-HBsAg IgA and IgG antibody secreting cells in mouse lung lymphoid to vectors expressing IL-6 was at about two?fold higher level than those elicited by control virus at day 14 after immunization. Authors also could detect elevated anti-HBsAg IgA and IgG antibody conversion in mouse serum and lung fluid, rabbits serum, lung fluid, saliva, vagina and nasal washing samples.</p><p><b>CONCLUSIONS</b>IL-6 expressed by non-replicating recombinant vaccinia virus could enhance the induced?immune effects, it could serve as the effective adjuvant for recombinant vector vaccine.</p>

Nuclear translocation of apoptosis-related protein TFAR19 in TF-1 cells undergoing apoptosis / 北京大学学报(医学版)

Objective: To study the expression and localization of apoptosis-related protein TFAR19 in TF-1 cells undergoing apoptosis. Methods: Using monoclonal antibody against TFAR19, the expression level and cell localization of TFAR19 were examined by fluorescence microscope, confocal laser scan microscope(CLSM) and flow cytometry. Simultaneously, we also analyzed the relationship of TFAR19 protein with phosphatidylserine (PS) externalization and cell nuclear DNA fragmentation. Results: The level of TFAR19 proteins expressed in TF-1 cells treated with GM-CSF withdrawal was significantly increased compared with normal TF-1 cells, then translocated rapidly from cytoplasm to the nucleus of cells. Appearance of TFAR19 in the nucleus of apoptotic cells preceded the detection of PS externalization and DNA fragmentation. Conclusion: Nuclear translocation of TFAR19 protein is one of the earliest events of cell apoptotic process. These data provided a new clue to further approach to the biological function of TFAR19 and study of cell apoptosis.

The expression and purificiation of human Annexin Ⅴ overexpressed in E. coli and the detection of apoptosis / 北京大学学报(医学版)

Objective: in order to provide rapid and reliable method. Methods: Encoded Annexin Ⅴ cDNA was amplifyed from U937 cDNA libary by PCR and then subcloned into E coli expression vector. MS2-Annexin Ⅴ fusion protein could be overexpressed in E　coli. The MS2 bacteria protein could be removed by thrombin digestion.The mature Annexin Ⅴ was obtained by ion exchange chromatography and the FITC labled Annexin Ⅴ could be used in the detection of apoptosis. Results:Up to 37% of the total bacterial proteins was rhAnnexin Ⅴ as showed by SDS-PAGE. The purification of Annexin Ⅴ is over 99%. The FITC labled Annexin Ⅴ could efficiently detect apoptosis. Conclusion: We successfully established the technique procedure of obtaining a large quantity of Annexin Ⅴ and provided the basic routine for popularizing the detection of apoptosis' with high effciency.

Effect of human chemokine-like factor-1 expression in vivo on the development of systemic lupus erythematosus in BXSB mice / 中华风湿病学杂志

Objective　To probe into roles of hCKLF-1 in the development of systemic lupus erythematosus in BXSB mice in vivo.Methods　The recombinant eukaryotic expression plasmid for hCKLF-1 (pCDI-hCKLF-1) was transferred and expressed in lupus-prone BXSB mice with the technique of muscle-mediated transgene by electric pulse;the levels of serum IgM and IgG anti-DNA antibodies as well as serum BUN and urine protein were detected.Results　hCKLF-1 expression in vivo did not make any effects on the levels of IgM and IgG anti-DNA antibodies in female and male BXSB mice,but markedly enabled abnormal elevation of urine protein in male BXSB mice in short time,suggesting its ability for accelerating glomerulonephritis.Conclusion　hCKLF-1 may be one of inflammation mediators,playing an important role in the lupus glomerulonephritis of male BXSB mice,and its target cells may be monocyte and macrophage.

Expression and purification of truncated human recombinant nuclear apoptosis-inducing factor 1 in E.coli / 中国免疫学杂志

Objective:Nuclear apoptosis-inducing factor 1(NAIF1) is a novel apoptosis gene cloned in laboratory. To analyze the binding proteins of NAIF1 by pulldown method, the fusion expression vector of truncated human nuclear apoptosis-inducing factor 1〔NAIF1(73-327)〕 was constructed, were expressed and purified the recombinant GST-NAIF1(73-327) fusion protein in E.coli.Methods:The cDNA encoding human NAIF1(73-327) was amplified by PCR and cloned into pGEX-KG vector. The GST-NAIF1(73-327) fusion protein was expressed in E.coli and purified by affinity chromatography. The purified protein was detected by SDS-PAGE, Western blot and ESI-Q-TOF-MS/MS.Results:A prokaryotic expression vector of GST-NAIF1(73-327) was constructed and the GST-NAIF1(73-327) fusion protein was expressed in E.coli at high level. SDS-PAGE analyses indicated that the purified protein was about 53 kD. Western blot and MS/MS analyses verified the recombinant fusion protein.Conclusion:An efficient method for obtaining recombinant GST-NAIF1(73-327) fusion protein had been established and it could be used for further studies on the structure and function of NAIF1.

Expression of TFAR19 in Apoptotic Processes of Jurkat Cells Induced with Various Methods / 中国实验血液学杂志

A new apoptosis-related gene TFAR19 was recently cloned. A primary functional analysis indicates its role in the apoptotic process of TF-1 cells. To clarify TFAR19 was involved in which apoptotic pathway, the changes in TFAR19 expression were observed during the apoptotic processes of Jurkat cells induced with various methods: serum deprivation, VP-16 treatment and Fas McAb activation. Then TFAR19 expression in Jurkat cells was detected with flow cytometry and Western blot, and TFAR19 mRNA with RT-PCR. Our results showed that expression of TFAR19 in Jurkat cells was increased at 12 hours after serum deprivation, 2 hours after VP-16 treatment and 2 hours after Fas McAb activation, respectively. These observations suggested that TFAR19 was involved in the apoptotic process of Jurkat cells induced with serum deprivation, DNA destruction and death receptor activation. It might take effect in the early apoptotic process. TFAR19 is a participant of the "final common pathway" in the apoptotic pathway.

Homology Modeling for the Structure of the Soluble Segment of TRAIL / 中国肿瘤生物治疗杂志

Objective: In order to further investigate the structural/functional relationship of TRALL. Methods: We did homology modeling for the extracellular segment of TRAIL, which is from R117 to G281, totally 165 aa residues long. The modeling software is Insight II from MSI/Biosym and the modeling work is based on the three dimensional structure of TNF-?. Results: From the modeling result, it can be seen that the modeled structure of TRAIL contains 10 ?-sheets and homologs for all these sheets could be found in TNF-?. This just confirms with the principle that the structurally con-seived regions within molecules of the same structure family should experience relatively small sequence mutations. In addition, the credibility of the modeled structure is checked by the way of inverse folding from Profile-3D. Conclusion: The result shows modeled structure is generally correct.