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At least 955 records · Page 53Linked to original sources
PubMed2004

Individual expression of poliovirus 2Apro and 3Cpro induces activation of caspase-3 and PARP cleavage in HeLa cells.

The expression of individual viral genes enables the study of their effects on cellular functions. Our group previously generated stable HeLa cell lines that efficiently express poliovirus proteases 2A (clone 2A7d) and 3C (clone 3C7) under the control of tetracycline [Virology 266 (2000a) 352; J. Virol. 74 (2000b) 2383]. Upon induction of these proteases, the cells undergo drastic morphological alterations and eventually die. The present paper characterizes, in detail, the cellular and molecular events that lead to cell death in these lines. Several signs of apoptosis were observed in both 2A7d- and 3C7-induced cells, such as nuclear fragmentation, DNA breakdown (as determined by TUNEL), and phosphatidylserine translocation. Protease 2A induces the cleavage of poly-ADP-ribose-polymerase (PARP). This is blocked by the caspase-3 inhibitor DEVD in both 2A7d-On and 3C7-On cells suggesting that this enzyme might account for PARP cleavage in both cell lines. The results indicate that both poliovirus proteases induce apoptosis by mechanisms involving caspase activation, although the kinetics of apoptosis differs.

3C Viral Proteases↗
PubMed1997

Recombinant human extracellular superoxide dismutase produced in milk of transgenic rabbits.

Expression of human extracellular superoxide dismutase (EC-SOD), a glycosylated, tetrameric metalloprotein, was targeted to the lactating mammary gland of transgenic rabbits. Efficient expression of the recombinant whey acidic protein/ec-sod gene was achieved and up to 3 mg ml-1 of the enzyme was secreted into the milk. Rabbit milk-produced recombinant EC-SOD was primarily found in the whey and purified by a two-step chromatographic method. To evaluate the rabbit milk-produced human EC-SOD, comparisons with native and Chinese hamster ovary cell (CHO)-produced EC-SOD were performed. All proteins were tetrameric and N-glycosylated. The behaviour on SDS-PAGE and size-exclusion chromatography indicated that the masses, and thereby the extent of post-translational modification of the proteins was similar. The monosaccharide composition of both recombinant EC-SOD variants was analysed and indicated similarities in the attached N-glycans on the two proteins. Furthermore, the peptide maps of the three EC-SOD variants revealed that all proteins had similar polypeptide backbones.

Animals↗
PubMed1993

Insect resistant rice generated by introduction of a modified delta-endotoxin gene of Bacillus thuringiensis.

As a first step towards development of insect resistant rice we have introduced a truncated delta-endotoxin gene, cryIA(b) of Bacillus thuringiensis (B.t.) which has specific biological activity against lepidopteran insects into a japonica rice. To highly express the cryIA(b) gene in rice the coding sequence was extensively modified based on the codon usage of rice genes. Transgenic plants efficiently expressed the modified cryIA(b) gene at both mRNA and protein levels. Bioassays using R2 generation plants with two major rice insect pests, striped stemborer (Chilo suppressalis) and leaffolder (Cnaphalocrosis medinalis), indicated that transgenic rice plants expressing the CryIA(b) protein are more resistant to these pests than untransformed control plants. Our results suggest that the B.t. endotoxin genes will be useful for the rational development of new rice varieties resistant to major insect pests.

Amino Acid Sequence↗
PubMed1987

Baculovirus expression vectors: the requirements for high level expression of proteins, including glycoproteins.

The requirements for high level expression of three foreign proteins using the polyhedrin gene promoter of Autographa californica nuclear polyhedrosis virus (AcNPV, Baculoviridae) have been investigated. In Spodoptera frugiperda cells infected with the appropriate recombinant baculoviruses, the synthesis of the two S RNA coded genes of lymphocytic choriomeningitis virus (LCMV; i.e. the nucleoprotein, N, and glycoprotein precursor, GPC), or the haemagglutinin gene of influenza A virus, appears to be related to the degree of integrity of the 5' upstream sequence of the polyhedrin gene. No effect on the level of N protein expression was detected when all the polyhedrin gene coding sequences or some of the immediate 3' downstream sequences were deleted. Using the most efficient expression viruses derived from a new transfer vector, pAcYM1, it has been estimated that LCMV N protein represented approximately 50% of the total cellular protein, an observation consistent with the presence of numerous inclusion bodies in the cytoplasm of infected cells. For recombinant viruses derived from the pAcYM1 transfer vector containing the LCMV GPC gene, the level of synthesis of the arenavirus glycoprotein was equivalent to approximately 20% of the cellular protein. Thin sections of cells infected with the GPC recombinant revealed a highly vacuolated cytoplasm.

Base Sequence↗
PubMed1999

The Archaeoglobus fulgidus D-lactate dehydrogenase is a Zn(2+) flavoprotein.

Archaeoglobus fulgidus, a hyperthermophilic, archaeal sulfate reducer, is one of the few organisms that can utilize D-lactate as a sole source for both carbon and electrons. The A. fulgidus open reading frame, AF0394, which is predicted to encode a D-(-)-lactate dehydrogenase (Dld), was cloned, and its product was expressed in Escherichia coli as a fusion with the maltose binding protein (MBP). The 90-kDa MBP-Dld fusion protein was more efficiently expressed in E. coli when coexpressed with the E. coli dnaY gene, encoding the arginyl tRNA for the codons AGA and AGG. When cleaved from the fusion protein by treatment with factor Xa, the recombinant Dld (rDld) has an apparent molecular mass of 50 kDa, similar to that of the native A. fulgidus Dld enzyme. Both the purified MBP-Dld fusion protein and its rDld cleavage fragment have lactate dehydrogenase activities specific for D-lactate, are stable at 80 degrees C, and retain activity after exposure to oxygen. The flavin cofactor FAD, which binds rDld apoprotein with a 1:1 stoichiometry, is essential for activity.

ATP-Binding Cassette Transporters↗
PubMed1993

Rat renin promoter activity in cultured cells and transgenic mice.

Renin, a key enzyme controlling blood pressure, is mainly synthesized in kidney. To characterize the rat renin promoter, we have constructed a reporter gene containing the 238-bp putative regulatory region linked to a bacterial chloramphenicol acetyltransferase (CAT), and analyzed its promoter activity by in vitro transfection and introduction of the CAT fusion gene into germline of mice. CAT activity was detected in transfected embryonic kidney-derived 293 cells, but not in HeLa cells derived from cervical carcinoma, showing that the putative promoter region of the rat renin gene directs transcription in a cell type-dependent manner. To examine whether the sequence was sufficient to regulate the expression of the CAT chimeric gene in mice, we generated seven transgenic mice carrying the reporter construct. Unexpectedly, the transgene was not expressed in any of the independent transgenic mice examined. These results suggest one possibility that an additional control region may be required for efficient expression of the rat renin promoter in developing mice.

Animals↗
PubMed2004

[Construction and identification of yeast expression vectors containing human platelet-derived growth factor B-chain gene].

OBJECTIVE: To investigate the expression efficiency of human platelet-derived growth factor B-chain (PDGF-B) gene in yeast and assess the activity of the expressed product. METHODS: A full-length complementary DNA of human PDGF-B gene was amplified from the total RNA extracted from human vascular endothelial cells using reverse transcription (RT)-PCR and then cloned into pGEM-T vector. The PCR products with specific primers were recombined into yeast expression plasmids pMETB or pMETalphaA, followed by identification with restriction endonuclease. RESULTS: The 578 bp fragment encoding mature PDGF-BB peptide with signal peptide and the 340 bp fragment without signal peptide were identified and verified by restriction endonuclease mapping and sequencing, and two yeast expression vectors pMETB-PDGFB(1) and pMETalphaA-PDGFB(2) were reconstructed. CONCLUSION: The yeast expression vectors containing human PDGF-B gene have been successfully constructed for further studies of gene expression in yeast.

Becaplermin↗
PubMed2005

[Construction of DNA and RNA based on bifunctional replicon vector derived from Semliki Forest virus].

DNA-based replicon expression vector pSMCTA and helper vector pSHCTA were constructed by replacing the SP6 promoter used in the original system pSFV1 and pSFV-helper2 derived from Semliki Forest virus (SFV) with the RNA polymerase II -dependent cytomegalovirus immediate early (CMV IE) enhancer/promoter and T7 promoter, and inserting BGH transcription termination and polyadenylation signal downstream 3'-untranslated region (UTR). The RNA polymerase II -dependent cytomegalovirus immediate early (CMV IE) enhancer/promoter and T7 promoter in pSMCTA and pSHCTA could drive transcription to produce replicon RNA in vivo and ex vivo. High level expression of foreign genes (GFP and LacZ) could be demonstrated by transfecting BHK21 cells with the new replicon expression vectors based on both DNA and RNA, and recombinant virus particles (RVP) be prepared by cotranfecting the expression vectors with the helper vectors. Foreign genes were also highly expressed in cells (BHK21) which were infected with RVP activated by alpha-chymotrypsin. The bifunctional replicon vectors can be used in highly efficient expression of foreign genes and preparation of RVP ex vivo, also in development of replicon vaccines and gene therapy vectors in vivo.

3' Untranslated Regions↗
PubMed1995

Unaltered immunoglobulin expression in hybridoma cells modified by targeting of the heavy chain locus with an integration vector.

Chimeric antibodies against the murine T-cell antigen Thy-1.2 were generated in amounts sufficient for in vivo studies by substituting the constant gene segments via homologous recombination in the hybridoma cell. We show that an integration vector targets the heavy chain locus at high frequency even in a non-isogenic situation. Using this vector type, for the first time expression rates were obtained that were identical to the parental hybridoma. The use of the gpt selection marker seems to be crucial for efficient expression, and may overcome a recently claimed drawback of vector integration. A chimeric antibody produced by gene targeting was characterized in vitro and in vivo.

Animals↗
PubMed1996

Noninfectious gene transfer and expression systems for cancer gene therapy.

Gene therapy provides a significant opportunity to devise novel strategies for the control or cure of cancer. Success of this modality will ultimately depend on the ability to express a therapeutic gene of interest at high levels, and specific gene delivery to targeted tumor cells will minimize toxicities. Although current gene therapy trials typically use viral-based, infectious vectors to express suitable target genes in human cancer cells, these vectors have significant limitations in their expression characteristics, lack of specificity in targeting tumor cells for gene transfer, and safety concerns regarding induction of secondary malignancies and recombination to form replication-competent virus. These limitations have refocused efforts to develop noninfectious gene transfer technologies for in vivo gene delivery of plasmid-based expression vectors. This article reviews recent developments in non-infectious gene transfer techniques, including liposome and receptor-mediated methods, which can efficiently deliver plasmid vectors into tumor cells in vivo. Additionally, strategies are reviewed for efficiently expressing target genes in tumor cells, including use of tissue-specific promoters, inducible promoters, and replication-control sequences to regulate extrachromosomal amplification of vector DNA in human tumor cells. Optimal coupling of these noninfectious gene transfer and expression technologies have the potential to yield safe and effective gene therapies for patients with cancer.

Gene Transfer Techniques↗
PubMed1997

[A promoter responsible for over-expression of cholera toxin B subunit in cholera toxin A subunit structure gene].

A promoter sequence, which promotes the transcription of cholera toxin B subunit gene, was found in cholera toxin A subunit structure gene. The transcription starts at the adenine Located at +833, that is 456bp upstream to the A of the initiation codon ATG of cholera toxin B gene. Under the control of the promoter, cholera toxin B subunit was over-expressed as high as 200 mg/L at an optimized culture condition. The chloramphenicol acetyl transferase gene and beta-galactosidase could also be efficiently expressed under the direction of the promoter. This promoter may be responsible for the 6 fold and 7 fold higher expression level of cholera toxin B subunit than cholera toxin A subunit in V. cholerae and Escheria coli respectively. The over-expression of CTB may be useful in preparing vaccine against cholera and facilitating the construction of peptide-bearing immunogenic hybrid proteins.

Base Sequence↗
PubMed1996

Functional and immunological properties of the baculovirus-expressed hemagglutinin of African swine fever virus.

A recombinant baculovirus harboring the hemagglutinin (HA) gene of African swine fever virus, with homology to the T-lymphocyte surface antigen CD2, was constructed. The efficient expression of the HA gene was determined by immunofluorescence and Western blot studies on insect cells infected with the recombinant baculovirus. The baculovirus-expressed HA showed hemadsorption and erythrocyte-agglutinating activities characteristic of the CD2 homolog protein induced by the virus in infected macrophages. Pigs immunized with the recombinant HA developed hemagglutination-inhibition and temporary infection-inhibition antibodies that recognize a 75-kDa structural protein and were protected against lethal infection.

African Swine Fever↗
PubMed1992

Expression of poliovirus nonstructural proteins in Escherichia coli cells. Modification of membrane permeability induced by 2B and 3A.

The poliovirus nonstructural proteins 2B, 2C, 2C3A, 2C3AB, 3A, and 3AB have been cloned and efficiently expressed in Escherichia coli cells. Each individual protein, or combinations of some of them, were cloned using polymerase chain reaction techniques and correspond to the genuine poliovirus protein plus an additional methionine. The system used to express them uses pET vectors containing the promoter of gene 10 of phage T7. Expression of protein 2C in BL21 (DE3) pLysS cells, which express the T7 lysozyme, is not toxic, and the bacteria synthesize this protein for several hours after induction. In contrast, the expression of proteins 2B, 3A, or 3AB is not tolerated by BL21 (DE3) pLysS cells which could make them only for a limited period of time. Protein 3AB was particularly toxic and induced a rapid lysis of the recombinant clone after its induction with isopropyl-1-thio-beta-D-galactopyranoside alone or with both isopropyl-1-thio-beta-D-galactopyranoside and rifampicin. Further analyses showed that 3AB induced profound modifications in membrane permeability to o-nitrophenyl-beta-D-galactopyranoside, labeled uridine, and nonpermeant translation inhibitors. Cloning and expression of proteins 2B, 3A, and 3AB in BL21 (DE3) cells that do not contain the T7 lysozyme lead to a more sustained expression of these proteins without detectable cell lysis. Changes in permeability to low molecular weight compounds such as radioactive uridine, o-nitrophenyl-beta-D-galactopyranoside, and hygromycin B readily appeared upon induction of 2B, 3A, and 3AB. Our results indicate that the poliovirus nonstructural polypeptides 2B and 3A (or 3AB) are lytic for the bacteria. In fact, both proteins 2B and 3A contain hydrophobic domains in a potential amphipathic helix; this is one characteristic shared with a number of membrane-active peptides.

Autoradiography↗
PubMed1984

Immunoselection of B cell hybridomas with anti-idiotypic antisera to study the role of heavy and light chains for idiotype expression and antibody activity.

Conventional reconstitution experiments with heavy (H) and light (L) offins of hybridoma anti-poly-(Glu60Ala30Tyr10) (anti-GAT) antibodies demonstrated that H and L chains are required for efficient expression of anti-GAT activity and the previously defined common CGAT idiotype. On the basis of this observation, we treated a CGAT+ hybridoma cell line (F9.195.6), which was produced by fusion with an MOPC 21 (gamma 1, K) secretor line, with anti-CGAT idiotypic antibodies plus complement to select CGAT-variants. Such treatment greatly enhanced the frequency of isolating hybridoma variants. Molecules secreted from these variants bear anti-GAT mu-chain and MOPC 21 L chain (as judged by serology and Southern blot studies), lack GAT binding activity and CGAT idiotype, and exhibit a "new" private idiotype (E8). Anti-private idiotypic sera were successfully used to select a series of CGAT+, E8- clones that secrete molecules bearing only the appropriate anti-GAT mu- and K chains. From E8- hybridoma supernatants, CGAT- molecules bearing MOPC 21 H chain and anti-GAT L chain could be obtained. The data indicate that immunoselection of hybridoma clones with anti-idiotypic antibodies is a convenient means of preparing reconstituted antibody molecules. Furthermore, the data demonstrate that CGAT idiotype determinants are expressed on antibody molecules composed of appropriate H and L chains.

Animals↗
PubMed2004

[Cloning, expression and antitumor effect of mouse costimulatory molecule 4-1BBL].

AIM: To clone mouse 4-1BBL gene, construct its eukaryotic expression vector, and evaluate antitumor activity of the expression product. METHODS: RT-PCR was used to amplify mouse 4-1BBL gene from total RNA of C57BL/6 splenocytes stimulated by PHA. Then m4-1BBL cDNA was subcloned into eukaryotic expression vector pcDNA3.1(+) and transfected into mouse hepatocellular carcinoma cell line Hepa1-6. The expression of m4-1BBL in transfected cells was detected by RT-PCR, indirect immunofluorescence staining, and flow cytometry. Non-adherent splenocytes from non-immunized C57BL/6 mice were incubated with mitomycin-treated non-transfected Hepa1-6(Hepa1-6-wt) or transfected Hepa1-6 cells (Hepal-6-m4-1BBL), respectively. Then the lymphocytes were tested for cytotoxic activity to Hepa1-6-wt cells. RESULTS: The Hepa1-6 cells transfected by pcDNA3.1(+)-m4-1BBL could efficiently express m4-1BBL. As compared with Hepa1-6-wt cells,Hepa1-6-m4-1BBL cells could induce more efficiently cytotoxic activity of lymphocytes to Hepa1-6-wt cells (P<0.01). CONCLUSION: The expression of m4-1BBL by tumor cells is effective in inducing antitumor immune response.

4-1BB Ligand↗
PubMed1997

The bacteriophage T7 binary system activates transient transgene expression in zebrafish (Danio rerio) embryos.

The bacteriophage T7 binary expression system is widely used in vitro for high level selective expression of cloned genes but its application to in vivo models has not yet been investigated. In the present work, we show that coinjection into fertilized zebrafish eggs of pE1T7R, an expression plasmid bearing the T7 RNA polymerase gene driven by the cytomegalovirus (CMV) promoter, together with reporter vectors containing the Escherichia coli lacZ gene driven by the T7 promoter, resulted in the efficient expression of the reporter gene in 24-h mosaic transgenic embryos. Conversely, embryos receiving an unrelated CMV-expression plasmid, instead of pE1T7R, lacked significant reporter gene activity, indicating the strict requirement of T7 polymerase to activate the T7 promoter in these embryos. The present study demonstrates the possibility of applying efficiently the bacteriophage T7 binary system in vivo to a vertebrate model.

Animals↗
PubMed2005

Expression of both Chlamydia pneumoniae RNase HIIs in Escherichia coli.

Both genes encoding the RNase HIIs from Chlamydia pneumoniae AR 39 (discriminated as CpRNase HIIa and CpRNase HIIb in this report) were cloned and efficiently expressed in Escherichia coli. These genes amplified from Chlamydial genomes with PCR were digested with restriction endonucleases and then cloned into plasmid pET-28a predigested with the same enzymes. DNA sequencing confirmed that the constructs were correct in translation frame and coding sequence. Recombinant RNase HIIs were over-expressed by 0.5 mM IPTG induction. CpRNase HIIa existed mainly as inclusion bodies while CpRNase HIIb mainly as soluble fractions in E. coli. The soluble proteins were 20% of total expressed CpRNase HIIa and 65% of total expressed CpRNase HIIb, respectively. Native purification and denaturing Ni-NTA purification were performed to recover the recombinant CpRNase HIIs from induced bacteria. 3.36 mg CpRNase HIIa and 18 mg CpRNase HIIb were, respectively, obtained from 1 g wet bacteria with native Ni-NTA purification. Denaturing Ni-NTA purification recovered 14.48 mg CpRNase HIIa and 10.4 mg CpRNase HIIb from 1 g wet bacteria, respectively. Although the proteins recovered by denaturing Ni-NTA purification were inactive, re-folding by dialysis against decreased concentrations of urea could generate CpRNase HIIa and CpRNase HIIb as active as those recovered by native Ni-NTA purification. These efforts offered basis for further study on the structure-function relationships and their biological importance of Chlamydial RNase HIIs.

Base Sequence↗
PubMed2002

Expression, characterization, and reaction of recombinant monkey metallothionein-1 and its C33M mutant.

After we modified the protocol of purification, monkey metallothionein-1 (mkMT-1) and its mutant at position 33 (C33M mutant) were efficiently expressed and purified by using the glutathione-S-transferase fusion protein system. The protein yield has been considerably improved (8 mg/L culture for mkMT-1 and 10 mg/L culture for C33M mutant). The recombinant MT-1 and C33M mutant were characterized by ESI-MS, UV, and CD spectra. The reactions of MI-1 and C33M mutant with 5,5'-dithiobis(2-nitrobenzoic acid) and EDTA also have been carefully studied. The pH titration of MT-1 and C33M mutant has been studied by UV and CD spectra. The mutation of cysteine-to-methionine at position 33 mostly maintains the alpha-domain structure similar to that in wild-type mkMT-1, but the C33M mutant has significant loss of stability and cooperative properties of the domain.

Amino Acid Sequence↗