Mechanisms of autophagic eukaryotic cells in Acinetobacter microvilli removal and protein histological study on apoptosis induced by macrophages / 国际检验医学杂志

Objective To observe the mechanisms of autophagic eukaryotic cells in Acinetobacter microvilli removal and protein histological study on apoptosis induced by macrophages .Methods A model of Acinetobacter baumannii infection was established in 24 female OCR mice .The mice were randomly divided into control group (n= 12) and observation group (n= 12) .The control group was injected with normal saline ,and the observation group was injected with autophagy eukaryotic cells ,the histopathological changes of Acinetobacter and the induction of macrophage apoptosis were observed .Results There was no significant difference in the bacterial counts between the two groups of mice immediately after implantation (P>0 .05) ,the bacterial counts in the 24 and 48 h in the observation group was significantly lower than that in the control group (P<0 .05) .The lung tissue of mice in the ob-servation group injected after autophagy was normal ,the alveolar cavity was open ,no abnormal substances were found ,the alveolar wall was not obviously thickened ,and no inflammatory cell infiltration was found in the wall .The mice in the control group were in-jected with normal saline and lacked the ability to remove Acinetobacter ,resulting in a large number of inflammatory cell infiltra-tion ,vasodilatation ,and congestion in some mice .Conclusion Autophagic eukaryotic cells injected with Acinetobacter baumannii can increase the clearance rate ,induce apoptosis of macrophages and improve the quality of Acinetobacter baumannii .

Mechanisms of autophagic eukaryotic cells in Acinetobacter microvilli removal and protein histological study on apoptosis induced by macrophages / 国际检验医学杂志

Objective To observe the mechanisms of autophagic eukaryotic cells in Acinetobacter microvilli removal and protein histological study on apoptosis induced by macrophages .Methods A model of Acinetobacter baumannii infection was established in 24 female OCR mice .The mice were randomly divided into control group (n= 12) and observation group (n= 12) .The control group was injected with normal saline ,and the observation group was injected with autophagy eukaryotic cells ,the histopathological changes of Acinetobacter and the induction of macrophage apoptosis were observed .Results There was no significant difference in the bacterial counts between the two groups of mice immediately after implantation (P>0 .05) ,the bacterial counts in the 24 and 48 h in the observation group was significantly lower than that in the control group (P<0 .05) .The lung tissue of mice in the ob-servation group injected after autophagy was normal ,the alveolar cavity was open ,no abnormal substances were found ,the alveolar wall was not obviously thickened ,and no inflammatory cell infiltration was found in the wall .The mice in the control group were in-jected with normal saline and lacked the ability to remove Acinetobacter ,resulting in a large number of inflammatory cell infiltra-tion ,vasodilatation ,and congestion in some mice .Conclusion Autophagic eukaryotic cells injected with Acinetobacter baumannii can increase the clearance rate ,induce apoptosis of macrophages and improve the quality of Acinetobacter baumannii .

stg fimbrial operon from S. Typhi STH2370 contributes to association and cell disruption of epithelial and macrophage-like cells

BACKGROUND: Salmonella enterica serovar Typhi (S. Typhi) stg operon, encoding a chaperone/usher fimbria (CU), contributes to an increased adherence to human epithelial cells. However, one report suggests that the presence of the Stg fimbria impairs the monocyte-bacteria association, as deduced by the lower level of invasion to macrophage-like cells observed when the stg fimbrial cluster was overexpressed. Nevertheless, since other CU fimbrial structures increase the entry of S. Typhi into macrophages, and considering that transcriptomic analyses revealed that stg operon is indeed expressed in macrophages, we reassessed the role of the stg operon in the interaction between S. Typhi strain STH2370 and human cells, including macrophage-like cells and mononuclear cells directly taken from human peripheral blood. RESULTS: We compared S. Typhi STH2370 WT, a Chilean clinical strain, and the S. Typhi STH2370 Astg mutant with respect to association and invasion using epithelial and macrophage-like cells. We observed that deletion of stg operon reduced the association and invasion of S. Typhi, in both cellular types. The presence of the cloned stg operon restored the WT phenotype in all the cases. Moreover, we compared Salmonella enterica sv. Typhimurium 14028s (S. Typhimurium, a serovar lacking stg operon) and S. Typhimurium heterologously expressing S. Typhi stg. We found that the latter presents an increased cell disruption of polarized epithelial cells and an increased association in both epithelial and macrophage-like cells. CONCLUSIONS: S. Typhi stg operon encodes a functional adhesin that participates in the interaction bacteria-eukary-otic cells, including epithelial cells and macrophages-like cells. The phenotypes associated to stg operon include increased association and consequent invasion in bacteria-eukaryotic cells, and cell disruption.

Prokaryotic cells: structural organisation of the cytoskeleton and organelles

For many years, prokaryotic cells were distinguished from eukaryotic cells based on the simplicity of their cytoplasm, in which the presence of organelles and cytoskeletal structures had not been discovered. Based on current knowledge, this review describes the complex components of the prokaryotic cell cytoskeleton, including (i) tubulin homologues composed of FtsZ, BtuA, BtuB and several associated proteins, which play a fundamental role in cell division, (ii) actin-like homologues, such as MreB and Mb1, which are involved in controlling cell width and cell length, and (iii) intermediate filament homologues, including crescentin and CfpA, which localise on the concave side of a bacterium and along its inner curvature and associate with its membrane. Some prokaryotes exhibit specialised membrane-bound organelles in the cytoplasm, such as magnetosomes and acidocalcisomes, as well as protein complexes, such as carboxysomes. This review also examines recent data on the presence of nanotubes, which are structures that are well characterised in mammalian cells that allow direct contact and communication between cells.

Transient expression and biological activity identification of human pigment epithelium-derived factor in mammary cell line SP2/0 / 中华围产医学杂志

Objective To construct eukaryotic expression plasmid pIRESneo3-pigment epithelium-derived factor (PEDF) and detect its transient expression in SP2/0 cells. Methods Specific primers were designed based on the mature peptide sequence of human PEDF cDNA in the GenBank. Human PEDF gene was cloned into the eukaryotic expression vector pIRESneo3. The PEDF DNA was transfected into SP2/0 with LipofectamineTM 2000. The recombinant human PEDF protein expressed in SP2/0 cell culture supernatant was identified by Western blot and enzyme-linked immunosorbent assay. The biological activity of the recombinant human PEDF was measured by 3-(4,5-dimethylthiazol-z-y1)-2,5-diphenytetrazolium bromide(MTT) method. Results PCR amplification, restriction enzyme digestion and DNA sequencing confirmed that the mature peptide sequence of human PEDF cDNA was successfully cloned into the eukaryotic expression vector pIRESneo3. And the plasmid was transfected into SP2/0 cells, which could secret PEDF. Western blot analysis showed that there was only one obvious band at the position of relative molecular weight of 50 000, and it is equivalent to the expected value. Enzyme-linked immunosorbent assay suggested that the content of PEDF began to rise after transfection, and peaked at 36 h [(0.92±0.04) μg/ml]. The proliferation of human umbilical vein endothelial cell line was significantly inhibited by supernatant after transfection of 36 h (P<0.05). Conclusions The eukaryotic expression plasmid pIRESneo3-PEDF had been successfully constructed and active human PEDF was transiently secreted, which made a foundation for further study of stable expression and purification of PEDF. This protein could be a potential medication for preventing and managing retinopathy of prematurity.

Cloning and expression of genotype B and C hepatitis B virus in eukaryotic cells / 中华传染病杂志

Objective To construct recombinant full length genotype B and C hepatitis B virus (HBV)and to examine HBV DNA replication and hepatitis B surface antigen(HBsAg),hepatitis B e antigen(HBeAg)expressions in Huh7 cells. Methods The full length genotype B and C HBV DNA were extracted and amplified from two HBV infected patients. The recombinant plasmids were constructed by inserting the amplified HBV fragments into the eukaryotic expression vector,pHY106,which were then transfected into Huh7 cells. The cells transfected with blank pHY106 vector were used as control. HBV DNA replication at 72 hours of transfection was detected by Southern blot. The HBV DNA levels in Huh7 cells at 24,48,72,96 and 120 hours of transfection were determined by real-time polymerase chain reaction(PCR).Meanwhile, the HBsAg and HBeAg expression levels in the supernatants at 24,48,72,96 and 120 hours were determined by enzyme linked immunosorbent assay(ELISA).Results The recombinant plasmids expressing genotype B or C HBV DNA were successfully constructed.The HBV replicative intermediates in HBV core particles,including rcDNA dsDNA and ssDNA,were detected by Southern blot.HBV DNA level could reach 8 lg copy/mL which was by real-time PCR. HBsAg and HBeAg levels determined by ELISA peaked at 72 hours after transfection and then declined gradually. Conclusions The recombinant plasmids inserted with genotype B or C HBV DNA are constructed successfully, which can express high levels of HBsAg and HBeAg in Huh7 cells. This system provides a platform for studying the pathogenesis of B and C genotype HBV, the interaction between HBV and host, as well as exploiting new drugs against HBV.

Influence of ATP-binding cassette transporter A1 protein in eukaryocyte and its expression on arsenic resistance / 中国地方病学杂志

Objective To examine the expression of ATP-binding cassette transporter A1(ABCA1)in eukaryotie cells and the effect of arsenic resistance after the transfection of eukaryotic expression vector containing ABCA1 gene.Methods HeLa cells were transfected with the recombinant plasmid by lipofectaonmine 2000 (recombinant plasmid group),empty plasmid and untransfected HeLa cell as the control group.The level of the mRNA was examined by real-time PCR,and the expression of ABCA1 protein wag examined by Western blot,the change of cell survival rate was examined by methyl thiazolyl tetrazolium(MTT)after exposure in a series of arsenic [0(contro1),4,8,16,32,64,128 μmol/L]for 48 hours.Results Expression level of ABCA1 mRNA in recombinant plasmid,empty plasmid and untransfeeted groups was(2.09±0.08)×10-4,(0.09±0.02)×10-4,(0.08±0.02)×10-4,there was a significant difference between the groups(F=1499.23,P<0.01).The level of ABCA1 mRNA in recombinant plasmid group was higher than empty plasmid and untransfected group(all P<0.01).Western blot showed that specific protein straps existed at 254×103 in all the three groups,with a similar size to the ABCA1 protein.The amount of the recombinant plasmid group was higher than the other two groups.MTT shows that arsenic concentration at 4,8,16,32,64,128 μmol/L,the survival rates of recombinant plasmid group was(94.8±0.9)%,(86.5 ± 2.6)%, (77.8 ± 2.0)%, (56.0 ± 2.0)%, (23.8 ± 1.7)%, (18.6 ± 0.6)%, higher than that of empty plasmid group[ (85.3 ± 1.1)%, (78.7 ± 0.6)%, (67.8 ± 2.4)%, (43.2 ± 1.5)%, (14.5 ± 1.3)%, (8.0 ± 0.4)%], the difference of survival rate had a statistical signifieance(t = 18.985,6.689,5.922,9.504,9.481,32.634, all P < 0.01). Conclusions ABCA1 protein is over expressed in HeLa cells after transfect ABCA1 gene. ABCA1 protein increases resistance of arsenic in HeLa cells.

Construction and further identification of eukaryotlc expression plasmid containing rat hepatocyte growth factor gone and augmenter of liver regeneration gene / 中华传染病杂志

Objective To construct and identify an eukaryotic expression plasmid containing rat hepatocyte growth factor(rHGF)gene and rat augmenter of liver regeneration(rALR)gene,so that to provide experimental basis for developing new treatments of hepatic fibrosis.Methods The gene fragments of rHGF and rALR were amplified from recombinant prokaryotic plasmid pUC18-rHGF and pUC18-rALR by polymerase chain reaction(PCR),respectively,then were spliced by overlap extension PCR with a linker,and the fusion gene rHGF-linker-rALR was constructed.The fusion gene was directionally inserted into the eukaryotic expression plasmid pcDNA3.1 between restriction sites of Kpn Ⅰ and Xba Ⅰ to construct the recombinant eukaryotic expression plasmid pcDNA3.1-rHGF-linker-rALR,and the new constructed recombinant plasmid was identified by double restriction digestion and DNA sequencing.Results DNA fragments of 2200 bp and 400 bp were observed after the electrophoresis of products amplified from recombinant prokaryotic plasmid pUC18-rHGF and pUC18-rALR,respectively,which was consistent with the theoretical value.The electrophoresis of fusion gene rHGF-linker-rALR obtained by overlap extension PCR technique showed only a 2 600 bp DNA fragment,which was in accordance with the expected value.Electrophoresis of products of pcDNA3.1-rHGF-linker-rALR digested with Kpn Ⅰ and Xba Ⅰ showed two DNA fragments with 2600 bp and 5400 bp,which were both consistent with the expected value.The sequences were confirmed correctly by DNA sequencing.Conclusion The recombinant eukaryotic expression plasmid pcDNA3.1-rHGF-linker-rALR is successfully constructed,which provides experimental basis for developing gene therapy of hepatic fibrosis.

Expression.purification and antigenicity of a recombinant respiratory syncyttal virus fusion gene encoded protein(amino acids 168-289)in the insect cells / 中华检验医学杂志

Objective To explore the antigenicity of the recombinant respiration syncytial virus (RSV)fusion protein (amino acids 168-289) encoded by 546-881 bases of the fusion gene expressed in insect baculoviruses expression system.Methods The fragment F' of fusion gene 546-881 bases was amplified from viral RNA( Long strain ) by the reverse transcription-polymerase chain reaction(RT-PCR).F' was inserted into transfer vector pBacPAK9 and the recombinant plasmid pBacRSV F' was constructed.Sf9 insect cells were then co-transfeeted with a mixture of recombinant plasmids pBacRSV F' and linearized BacPAK6 viral DNA( Bsu36 Ⅰ -digested).The recombinant baculoviruses BacPAK F' was constructed and was able to express the recombinant protein in Sf9 insect cells.The recombinant protein was purified by Ni2+ NTA chromatography and its antigenicity was identified by Western blot(WB) analysis.Specimens including the nasopharyngeal aspirates(NPAs) and sera were collected from 33 infants and young children with acute lower respiration tract infection. Indirect immunofluorecenee assay (IFA) and WB were used to detect the RSV antigen in NPAs and the anti-RSV antibody in sera respectively.Results The recombinant protein (molecular weight 13 000) was expressed in Sf9 insect cells.WB analysis demonstrated that the purified recombinant protein had a specific RSV antibody-binding activity. The recombinant protein could be recognized by positive serum infected by RSV.The positive rate was 27.3% and 33.3% respectively.There was no significant difference between them(X2 = 0.29 ,P ＞ 0.05).Conclusion The recombinant respiratory syncytial virus fusion gene (546-881 bp) encoding protein is expressed in Sf9 insect cells and it has strong antigenicity and could have clinical application value for detection of RSV infection.

Construction and identification of eukaryotic expressed plasmid vector pLenti6/V5- DEST-Cx43 in human / 中华胸心血管外科杂志

Objective To construct eukaryotic expressed plasmid vector pLenti6/V5- DEST-Cx43 in human for further study on bioinformative communication between myocardial cell, embryonic development of heart and differentiation of myocardial cell. Methods Cx43 cDNA fragments were obtained from plasmid p18-T by restriction enzyme BamH1、Xho1, after the agarose gel electrophoresis was performed, the Cx43 cDNA was retrieved. pENTR11 were extracted by enzyme BamH1、Xho1, then connected with Cx43 cDNA by T4 DNA ligase. LR reaction was performed and Cx43 cDNA were cloned into Lentiviral vector pLenti6/V5-DEST. Results Agarose electrophoresis and sequent examination, identified that Cx43 cDNA was cloned into Lentiviral vector pLenti6/V5-DEST. Conclusion The present experiment demonstrate a successful cloning of human Cx43 cDNA into the Lentiviral vector pLenti6/V5-DEST.

Construction of pcDNA3.1(+) glial cell line derived neurotrophic factor(GDNF) vector and its expression in eukaryotic cells / 中华神经科杂志

Objective To construct pcDNA3 1(+)GDNF recombinant eukaryotic expression plasmid and to investigate its expression in eukaryotic cells. Methods The coding sequence of GDNF was amplified from rat astrocytes by reverse transcription PCR (RT PCR) and cloned into pcDNA3 1(+) eukaryotic expression vector The recombinant pcDNA3 1(+)GDNF plasmid was then transfected into eukaryotic cells mediated by using Fu Gene 6 method Analysis by restricting enzyme digestion and DNA sequencing were carried out to demonstrate the sequence of the plasmid GDNF protein and its activity were then determined using pcDNA3 1(+)GDNF plasmid transfected eukaryotic cells Results RT PCR product is 640 bp specific segment Analysis by restricting enzyme digestion and DNA sequencing of pcDNA3 1(+)GDNF recombinant showed results from restricting enzyme were 640 bp and 300 bp segments respectively DNA sequencing revealed that GDNF cloning was successful The recombinant plasmid can express active GDNF protein in eukaryotic cells Conclusion The study on the role of both GDNF and gene therapy is significant in the treatment of Parkinson disease

Construction of eukaryotic expression plasmid of APM1 gene and its expression In HEK 293 cells / 中华内分泌代谢杂志

Objective To construct an eukaryotic expression vector with human adipose most abundant gene transcript 1 (APM1) gene,and to investigate the transfection and expression of pCDEF-APM1 eukaryotic expression plasmid in HEK293 cells.Methods pCDEF-APM1 eukaryotic expression plasmid was constructed by DNA recombinant method.Expression vector pCDEF-APM1 was transfected into HEK293 cells with Effectene reagent.The level of human adiponectin protein in the supernatant of cell culture media was detected with double antibody sandwich ELISA.Results The sequence of DNA fragment from constructed pCDEF-APM1 plasmid was identical to that published in GenBank.There was raised human adiponectin protein level in culture supernatant of HEK293 cells tnmsfected with pCDEF-APM1.Conclusion The pCDEF-APM1,an eukaryotic expression plasmid for APM1 gene is successfully constructed.High protein expression of adiponectin can be obtained in HEK293 cells transfected with pCDEF-APM1 eukaryotic expression plasmid.

Construction of eukaryotic expression plasmid pcDNA3.1(-)/PSMA7 and its expression in A549 cell line / 解放军医学杂志

0.05). The data proved that PSMA7 was overexpressed in pcDNA3.1(-)/PSMA7-transfected A549 cell line, both in mRNA level and in protein level. Conclusion Eukaryotic expression plasmid pcDNA3.1(-)/PSMA7 has been successfully constructed, and the PSMA7 is stably expressed in A549 cell line. This would pave the way for further study of PSMA7.

Cloning and eukaryotic expression of human CD38 / 第二军医大学学报

Objective:To clone and express the full length cDNA of human CD38. Methods:The full length cDNA of the human CD38 antigen was amplified from total RNA of Daudi cell by RT-PCR, and it was inserted into pGEM-T. The validity on the sequences was confirmed by automatic DNA sequencing. Inserting the valid CD38 gene into pcDNA3.1(+) plasmid to obtain recombinant mammalian expression vector pcDNA3.1(+)/CD38Z; Using lipofectin gene transfer technique system, recombinant expression vector containing CD38 gene was transfected into COS7 cells. The expression of CD38 molecules on the surface of COS7 cells was detected by FACS and immunohistochemical technique. Results:DNA sequencing showed that the cloned full length cDNA sequence was identical with reported. The result of FACS and immunohistochemical technique indicated that CD38 molecules were expressed on the surface of COS7 cells. Conclusion:The full length cDNA of human CD38 is obtained, recombinant mammalian expression vector pcDNA3.1(+)/CD38Z is successfully constructed, and the CD38 molecules is expressed on the surface of COS7 cells,this may facilitate studies on the biochemistry and function of CD38 antigen.

Construction of hantavirus Z37 envelope glycoprotein genes G1 and G2 recombinant and its expression in eukaryocyte / 中华传染病杂志

Objective To construct hantavirus Z37 envelope glycoprotein genes G1 and G2 recombinant and express in eukaryocyte. Methods Using three pairs of primers based on hantavirus Z37 M gene sequences. PCR products F1, F2 and G2 were obtained by PCR from the Plasmid pGEMZ37(containing partial cDNA of G1) and Plasmid pCUMZ37(containing partial cDNA of G1 and whole cDNA of G2). G1 PCR products were also obtained by fusing F1 and F2 PCR products. The G1 and G2 PCR products were then digested by BamHⅠ and XhoⅠ and cloned into the corresponding sites of expression vector pcDNA3.1 respectively. The recombinants pcDNA3.1 G1 and pcDNA3.1 G2 were identified by digestion with endonuclease BamHⅠ and XhoⅠ and confirmed by sequencing. After transfecting COS 7 cells by Calcium phosphate/DNA precipitating method, indirect immunofluorescence assay (IFA) was used to verify the transient expression of HV Z37 G1 or G2 protein in COS 7 cells. Results The recombinant expression plasmids pcDNA3.1 G1 and pcDNA3.1 G2 were constructed. After transfection with the above recombinant expression plasmids, specific antigens were detected within cells by IFA. Conclusions The reombinant expression plasmids pcDNA3.1 G1、pcDNA3.1 G2 were constructed successfully and expressed transiently in eukaryocyte.