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At least 343 records · Page 19Linked to original sources
PubMed2000

[The selection of fluoroacetate-resistant mutant from E. coli MMR204 and its influence on the expression of heterologous GL-7ACA acylase].

In the cultivation of gene engineered strain of Escherichia coli on glucose medium, excretion and accumulation of acetic acid inhibit not only cell growth but also the the expression of heterologous protein. It is obvious that the desirable host strain maintaining acetate at a low level is one of the approaches to increase the production of recombinant protein. The present article deals with the selection of mutants of E. coli DP19, DP8, which grow on the medium containing pyruvate as the sole carbon source in the presence of 50 mmol/L fluoroacetic acid. It is shown that mutant DP19 is defective in its phosphotransacetylase(PTA) activity and accumulates less acetate in the medium, while DP8 is defective in acetate kinase (ACK) and accumulates similar level of acetate comparing with its parent. Using pta- mutant E. coli DP19 as host, the expression of GL-7ACA acylase gene on the recombinant plasmid pMR24 is improved, and the yield of enzyme activity in flask fermentation is about twice as much as its parent.

Acetate Kinase↗
PubMed1995

Genetic engineering in plants.

Until now most research, and its funding, has been focused on animal and human health care as well as simple microbiological model systems such as Escherichia coli and yeast. Molecular plant studies have generally lagged behind, often simply adapting discoveries from the animal field to plants. Clearly, good health and the efficient tackling of diseases is crucial for the well-being of humans, and good remedies have a high economic value for the pharmaceutical industry. However, one should not forget that plants are an essential component of the large ecosystem that is our planet. They are not only the basic food producers but they are also necessary for a balanced atmosphere (oxygen production) and stable and viable climates. Especially in this period of demographic explosion and growing environmental deterioration, there is a need to rebuild our agricultural systems. Plants also have a wide variety of 'non-food' uses, for instance as energy sources, construction materials, or cosmetics. Last, but not least, they produce a lot of chemicals that can be used as pharmaceuticals. The growing awareness of the importance of plants has coincided with the development of plant molecular biology. Specific features make them ideally suited for gene engineering and genetic studies in general.

Biotechnology↗
PubMed1991

[The use of the indirect hemagglutination reaction for the serodiagnosis of HIV infection].

An assay system, based on the passive hemagglutination test and permitting the serodiagnosis of HIV infection with correct results in more than 99% of cases, has been developed. Three kinds of freeze-dried erythrocyte diagnostica (with shelf life exceeding 6 months), possessing high serological activity and sensitized with recombinant gene-engineering polypeptides, have been obtained. The proposed assay system is highly promising for mass examination of sera for the presence of antibodies to HIV due to the simplicity of assay techniques, the possibility of storing the diagnostica within a wide range of temperatures (4 degrees-30 degrees C) and obtaining results in a short time (3 hours).

AIDS Serodiagnosis↗
PubMed2005

Toward a gene therapy for dominant disease: validation of an RNA interference-based mutation-independent approach.

The intragenic heterogeneity encountered in many dominant disease-causing genes represents a significant challenge with respect to development of economically viable therapeutics. For example, 25% of autosomal dominant retinitis pigmentosa is caused by over 100 different mutations within the gene encoding rhodopsin, each of which could require a unique gene therapy. We describe here an RNA interference (RNAi)-based mutation-independent approach, targeting as an example murine rhodopsin. Native transcripts are suppressed by a single RNAi molecular species, whereas transcripts from replacement genes engineered at degenerate third-codon wobble positions are resistant to suppression. We demonstrate suppression of murine rhodopsin transcript by up to 90% with full concomitant expression of replacement transcript and establish the validity of this approach in cell culture, retinal explants, and mouse liver in vivo.

Animals↗
PubMed2002

Effect of gene location, mRNA secondary structures, and RNase sites on expression of two genes in an engineered operon.

The effects of endoribonuclease sites, secondary structures in mRNA, and gene placement on protein production and mRNA stability and steady-state levels were tested in a dual-gene operon containing the genes encoding beta-galactosidase (lacZ) from Escherichia coli and green fluorescent protein (gfp) from Aequorea victoria. Two previously identified RNase E sites were placed separately between the coding regions to direct cleavage in this area and produce two secondary transcripts, each containing a single-gene coding region. Novel secondary structures were engineered into the 3' and 5' ends of each of the coding regions to protect the transcript from inactivation by endoribonucleases (5' hairpins) and degradation by exoribonucleases (3' hairpins). In addition, the effects of relative gene placement were examined by switching the locations of the two coding regions. Depending on the particular secondary structures and RNase E sites placed between the genes the relative steady-state transcript and protein levels encoded by the two reporter genes could be changed up to 2.5-fold and 4-fold, respectively. By changing gene location and incorporating secondary structures and RNase E sites the relative steady-state transcript and protein levels encoded by the two reporter genes could be changed up to 100-fold and 750-fold, respectively.

Arabinose↗
PubMed1992

Construction of a diphtheria toxin A fragment-C180 peptide fusion protein which elicits a neutralizing antibody response against diphtheria toxin and pertussis toxin.

A genetically engineered gene fusion was constructed which encoded a nontoxic derivative of the A fragment of diphtheria toxin joined to the C180 peptide of the S1 subunit of pertussis toxin. The product of this gene fusion, termed the DTA-C180 protein, was purified from the periplasm of Escherichia coli to approximately 80% purity. The DTA-C180 protein possessed an apparent molecular weight of 43,000 by reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The DTA-C180 protein was cleaved into two tryptic peptides, which migrated with apparent molecular weights of approximately 22,000. One tryptic peptide reacted with diphtheria antitoxin, while the other tryptic peptide reacted with anti-C180 peptide immunoglobulin G. The DTA-C180 protein did not inhibit protein synthesis or stimulate clustering morphology in Chinese hamster ovary cells. The DTA-C180 protein elicited an immune response, in guinea pigs, against both the DTA and C180 peptide components of the fusion protein, with alum being a more efficient adjuvant than Freund's adjuvant for eliciting neutralization titers. Neutralization titers elicited by DTA-C180 protein were weaker than those elicited by diphtheria toxoid and pertussis toxin 9K/129G, a genetically engineered double mutant of pertussis toxin. Three doses of DTA-C180 protein yielded a neutralization titer of 1/750 against pertussis toxin in Chinese hamster ovary cells and a neutralization titer of 1/50 against diphtheria toxin in Vero cells. This is the first report of a protein derived from a recombinant S1 subunit that elicits a neutralizing titer against pertussis toxin.

Adenosine Diphosphate Ribose↗
PubMed2001

Regulated expression of myosin II heavy chain and RacB using an inducible tRNA suppressor gene.

An inducible expression system that indirectly regulates gene expression through the use of an inducible suppressor tRNA has been used to express both endogenous and exogenous genes in Dictyostelium. The tetracycline repressor and tRNA suppressor (Glu) are expressed from a single G418 selectable vector, while a gene engineered to contain a stop codon is expressed from a separate hygromycin selectable vector. beta-Galactosidase could be induced over 300 fold with this system, and the extent of induction could be varied depending upon the amount of tetracycline added. It took 3 days to fully induce expression, and about 3 days for expression to decrease to baseline after removal of the tetracycline. Dictyostelium myosin II heavy chain could also be expressed in an inducible manner, although the induction ratio was not as high as beta-galactosidase and the maximum expression level was not as high as wild-type levels. A significant accumulation of the truncated peptide indicates that complete suppression of the stop codon was not achieved. Partial phenotypic reversion was observed in null mutants inducibly expressing myosin II. RacB could also be inducibly expressed, whereas the protein could not be expressed from a constitutive promoter, presumably because expression at high levels is lethal. Therefore, the inducible tRNA system can be used to control expression of endogenous Dictyostelium genes.

Animals↗
PubMed2006

Wnt-10b promotes differentiation of skin epithelial cells in vitro.

To evaluate the role of Wnt-10b in epithelial differentiation, we investigated the effects of Wnt-10b on adult mouse-derived primary skin epithelial cells (MPSEC). Recombinant Wnt-10b protein (rWnt-10b) was prepared using a gene engineering technique and MPSEC were cultured in its presence, which resulted in morphological changes from cuboidal to spindle-shaped and inhibited their proliferation. Further, involvement of the canonical Wnt signal pathway was also observed. MPSEC treated with rWnt-10b showed characteristics of the hair shaft and inner root sheath of the hair follicle, in results of Ayoub Shklar staining and immunocytochemistry. Further, the cells expressed mRNA for differentiated epithelial cells, including keratin 1, keratin 2, loricrin, mHa5, and mHb5, in association with a decreased expression of the basal cell marker keratin 5. These results suggest that Wnt-10b promotes the differentiation of MPSEC.

Animals↗
PubMed2006

[Cloning, expression and identification of recombinant fusion protein GX1-rmhTNFalpha].

AIM: To construct prokaryotic vector GX1-rmhTNF containing neovascular-targeting peptide GX1 and human TNFalpha and produce the GX1-rmhTNF protein. METHODS: We have previously obtained a specific phage peptide CGNSNPKSC (GX1) binding to vasculature of human gastric cancer. The GX1-rmhTNFalpha vector was constructed by merging sequence of neovascular-targeting peptide GX1 with N-terminal of new recombined human TNFalpha (rmhTNFalpha) using gene engineering methods. The expression of the fusion protein GX1-rmhTNFalpha in E. coli was induced by temperature. The expression of GX1-rmhTNFalpha was detected by SDS-PAGE and Western blot. RESULTS: A novel protein with expected molecular mass about 18,000 was found after SDS-PAGE and gel staining. The expressed product showed a good binding ability to anti-TNFalpha monoclonal antibody. CONCLUSION: The prokaryotic vector GX1-rmhTNF was constructed and the expression of GX1-rmhTNF protein was successfully induced, which was helpful for further purification of GX1-rmhTNF protein.

Blotting, Western↗
PubMed1997

Expression of the Escherichia coli bo-type ubiquinol oxidase with a chimeric subunit II having the CuA-cytochrome c domain from the thermophilic Bacillus caa3-type cytochrome c oxidase.

The C-terminal periplasmic domain of subunit II of the Escherichia coli bo-type ubiquinol oxidase was replaced with the counterpart of the thermophilic Bacillus caa3-type cytochrome c oxidase containing the CuA-cytochrome c domain by means of gene engineering techniques. The chimeric terminal oxidase was expressed by a pBR322 derivative in a terminal oxidase deficient mutant of E. coli, although the amount of the chimeric enzyme was smaller than that of the Escherichia coli bo-type ubiquinol oxidase expressed by the original cytochrome bo-expressing plasmid. The chimeric enzyme showed much higher TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) oxidase activity than the wild-type cytochrome bo, but lower activity than the thermophilic Bacillus caa3-type cytochrome c oxidase. The chimeric subunit II was confirmed to bind to heme C. These results suggest that the CuA-cytochrome c domain grafted to this membrane anchor can facilitate electron transfer from reduced TMPD to low-spin protoheme b in subunit I.

Bacillus↗
PubMed2006

[Vascularization in transplantation of gene modified tissue engineered bone for repairing bone defect].

OBJECTIVE: To study the vascularization of the composite of bone morphogenetic protein 2 (BMP-2) gene transfected marrow mesenchymal stem cells (MSCs) and biodegradable scaffolds in repairing bone defect. METHODS: Adenovirus vector carrying BMP-2 (Ad-BMP-2) gene transfected MSCs and gene modified tissue engineered bone was constructed. The 1.5 cm radial defect models were made on 60 rabbits, which were evenly divided into 4 groups randomly (n= 15, 30 sides). Different materials were used in 4 groups: Ad-BMP-2 transfected MSCs plus PLA/PCL (group A), Ad-Lacz transfected MSCs plus PLA/PCL (group B), MSCs plus PLA/PCL (group C) and only PLA/PCL scaffolds (group D). The X-ray, capillary vessel ink infusion, histology, TEM, VEGF expression and microvascular density counting (MVD) were made 4, 8, and 12 weeks after operation. RESULTS: In group A after 4 weeks, foliated formed bones image was observed in the transplanted bones, new vessels grew into the bones, the pores of scaffolds were filled with cartilage callus, osteoblasts with active function grew around the microvessels, and VEGF expression and the number of microvessels were significantly superior to those of other groups, showing statistically significant difference (P<0.01); after 8 weeks, increasingly more new bones grew in the transplanted bones, microvessels distended and connected with each other, cartilage callus changed into trabecular bones; after 12 weeks, lamellar bone became successive, marrow cavity recanalized, microvessels showed orderly longitudinal arrangement. In groups B and C, the capability of bone formation was weak, the regeneration of blood vessels was slow, after 12 weeks, defects were mostly repaired, microvessels grew among the new trabecular bones. In group D, few new vessels were observed at each time, after 12 weeks, broken ends became hardened, the defected area was filled with fibrous tissue. CONCLUSION: BMP-2 gene therapy, by up-regulating VEGF expression, indirectly induces vascularization of grafts, promotes the living of seed cells, and thus accelerates new bone formation.

Animals↗
PubMed1991

Analyses of the requirements for the synthesis of virus-like particles by feline immunodeficiency virus gag using baculovirus vectors.

Feline immunodeficiency virus (FIV) gag gene was expressed in baculovirus vectors to investigate its potential for the assembly of virus-like particles. The unprocessed 50-kDa FIV gag precursor made in infected insect cells by recombinant AcFIVGAG-1 was myristoylated, assembled at the cell surface into virus-like particles (with diameters of approximately 100 nm), and efficiently released into the culture supernatant fluids. The presence of the complete viral-coded protease component of the FIV pol gene engineered into a second expression vector (AcFIVGAG-P5) resulted in the efficient processing of the gag precursor to its component proteins and abolished particle formation and secretion. Insertion of a stop codon in this vector upstream of the putative gag-pol frameshift site (GGGAAAC) resulted in the derivation of an expression vector (AcFIVGAG-R) that made a truncated, unprocessed 46-kDa FIV gag precursor lacking some 34 amino acids in the p10 carboxy-proximal coding region of gag. This vector synthesized tubular structures in the cytoplasm of infected cells and released them into the cell supernatant. The results demonstrate that the FIV gag precursor can spontaneously assemble into virus-like particles without any other virus proteins and that the carboxy-terminal part of the precursor gag protein is essential for such assembly.

Animals↗
PubMed1996

Inhibitory effect of double transfection to xenoendothelial cells using both decay accelerating factor and homologous restriction factor 20 genes on complement dependent cytolysis.

Hyperacute rejection in discordant xenotransplantation occurs due to the complement activation via classical and/or alternative pathway. Regulator of complement activation (RCA) molecules inhibit species-specific complement dependent cytolysis. To confirm the effect of gene engineering using double RCA molecules on xenogeneic cells, the inhibitory effect on complement dependent cytolysis was compared between bovine aortic endothelial cells (BAEC) doubly transfected with both decay accelerating factor (DAF) and homologous restriction factor (HRF) 20 cDNA and BAEC singly transfected with DAF alone using retroviral vector. The positive percent expression of DAF and HRF20 antigen on double transfectant (BAEC/D+H) was 97.0% and 95.0%, respectively, whereas that of DAF antigen on single transfectant (BAEC/D) was 97.0%. After incubated with 25% human serum and anti-BAEC antibody, the viability of double transfectant (BAEC/ D+H) was significantly preserved, compared with that of single transfectant (BAEC/D). These findings demonstrated that xenoendothelial cells doubly transfected with both DAF and HRF20 cDNA could be protected from complement dependent cytolysis more effectively than those singly transfected with DAF alone in the presence of antixenoendothelial antibody.

Animals↗
PubMed2005

The unexpected structural role of glutamate synthase [4Fe-4S](+1,+2) clusters as demonstrated by site-directed mutagenesis of conserved C residues at the N-terminus of the enzyme beta subunit.

Azospirillum brasilense glutamate synthase (GltS) is a complex iron-sulfur flavoprotein whose catalytically active alphabeta protomer (alpha subunit, 162kDa; beta subunit, 52.3 kDa) contains one FAD, one FMN, one [3Fe-4S](0,+1), and two [4Fe-4S](+1,+2) clusters. The structure of the alpha subunit has been determined providing information on the mechanism of ammonia transfer from L-glutamine to 2-oxoglutarate through a 30 A-long intramolecular tunnel. On the contrary, details of the electron transfer pathway from NADPH to the postulated 2-iminoglutarate intermediate through the enzyme flavin co-factors and [Fe-S] clusters are largely indirect. To identify the location and role of each one of the GltS [4Fe-4S] clusters, we individually substituted the four cysteinyl residues forming the first of two conserved C-rich regions at the N-terminus of GltS beta subunit with alanyl residues. The engineered genes encoding the beta subunit variants (and derivatives carrying C-terminal His6-tags) were co-expressed with the wild-type alpha subunit gene. In all cases the C/A substitutions prevented alpha and beta subunits association to yield the GltS alphabeta protomer. This result is consistent with the fact that these residues are responsible for the formation of glutamate synthase [4Fe-4S](+1,+2) clusters within the N-terminal region of the beta subunit, and that these clusters are implicated not only in electron transfer between the GltS flavins, but also in alphabeta heterodimer formation by structuring an N-terminal [Fe-S] beta subunit interface subdomain, as suggested by the three-dimensional structure of dihydropyrimidine dehydrogenase, an enzyme containing an N-terminal beta subunit-like domain.

Alanine↗
PubMedDate not supplied

[Cytotoxic properties of a recombinant hybrid of the A protein of Staphylococcus aureus with a fragment of exotoxin A of Pseudomonas aeruginosa].

By gene-engineering technique a chimeric protein made up of fragments of Staphylococcus aureus protein A and . Pseudomonas aeruginosa exotoxin A has been constructed. The chimeric protein was shown to preserve features characteristic of its both constituents--it ADP-ribosylates elongation factor 2 and binds to Ig. Cytotoxic properties of the chimeric protein were studied in two model systems. Treatment of target cells in both systems was performed successively with antibodies against corresponding antigens and after washing--with recombinant chimeric toxin which bound to antibodies on the surface of target cells. In the first model system human B-lymphoma cells (Daudi line) carrying Ig molecules on their surface were treated with polyclonal antibodies against human Ig L-chains. In the other system, human T-lymphoma cells (Jurkat line) were treated successively with monoclonal antibodies against cell surface CD5 antigen and further on--with polyclonal antibodies against mouse Ig. In both systems, only a slight inhibition of the target cells' growth was registered. The probable reasons of low cytotoxic activity of the chimeric protein and prospects of increasing it are discussed.

ADP Ribose Transferases↗
PubMed2003

Expression of a bifunctional fusion of the Escherichia coli genes for trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase in transgenic rice plants increases trehalose accumulation and abiotic stress tolerance without stunting growth.

Trehalose plays an important role in stress tolerance in plants. Trehalose-producing, transgenic rice (Oryza sativa) plants were generated by the introduction of a gene encoding a bifunctional fusion (TPSP) of the trehalose-6-phosphate (T-6-P) synthase (TPS) and T-6-P phosphatase (TPP) of Escherichia coli, under the control of the maize (Zea mays) ubiquitin promoter (Ubi1). The high catalytic efficiency (Seo et al., 2000) of the fusion enzyme and the single-gene engineering strategy make this an attractive candidate for high-level production of trehalose; it has the added advantage of reducing the accumulation of potentially deleterious T-6-P. The trehalose levels in leaf and seed extracts from Ubi1::TPSP plants were increased up to 1.076 mg g fresh weight(-1). This level was 200-fold higher than that of transgenic tobacco (Nicotiana tabacum) plants transformed independently with either TPS or TPP expression cassettes. The carbohydrate profiles were significantly altered in the seeds, but not in the leaves, of Ubi1::TPSP plants. It has been reported that transgenic plants with E. coli TPS and/or TPP were severely stunted and root morphology was altered. Interestingly, our Ubi1::TPSP plants showed no growth inhibition or visible phenotypic alterations despite the high-level production of trehalose. Moreover, trehalose accumulation in Ubi1::TPSP plants resulted in increased tolerance to drought, salt, and cold, as shown by chlorophyll fluorescence and growth inhibition analyses. Thus, our results suggest that trehalose acts as a global protectant against abiotic stress, and that rice is more tolerant to trehalose synthesis than dicots.

Adaptation, Physiological↗
PubMed1999

Gene-enhanced tissue engineering: applications for wound healing using cultured dermal fibroblasts transduced retrovirally with the PDGF-B gene.

The treatment of difficult wounds remains a considerable clinical challenge. The goal of this study was to determine whether genetic augmentation of dermal cells on resorbable matrices can stimulate the healing process, leading to increased tissue repair in a rat full-thickness excisional wound repair model. The human platelet-derived growth factor B (PDGF-B) gene was the initial gene chosen to test this hypothesis. The human PDGF-B gene was obtained from human umbilical vein endothelial cells (HUVEC) by reverse transcriptase-polymerase chain reaction, cloned into retroviral vectors under control of either the cytomegalovirus promoter or the rat beta-actin promoter, and introduced into primary rat dermal cells. In vitro results demonstrate that rat dermal cells are transduced and selected readily using retroviral vectors, and engineered to secrete PDGF-B at a steady-state level of approximately 2 ng per milliliter culture per 1 million cells per 24 hours. Seeding of the gene-modified cells onto polyglycolic acid (PGA) scaffold matrices and introduction into the rat model resulted in substantially increased fibroblast hypercellularity over control wounds at both 7 and 14 days posttreatment. Our results demonstrate that gene augmentation of rat dermal fibroblasts with the PDGF-B gene introduced into this animal model via PGA matrices modulates wound healing and suggests that experimentation with additional genes for use separately or in combination with PDGF-B for additional, improved wound healing is warranted.

Animals↗
PubMed1998

Detection of hepatitis C virus core protein circulating within different virus particle populations.

Progress in studying pathogenesis and increasing the reliability of hepatitis C diagnosis can be achieved by analysis of different forms of virus particles circulating in blood of both patients and infected persons. Detection of hepatitis C virus (HCV) proteins faces two basic difficulties: low concentration of HCV proteins, and their blocking by antibodies. The aim of this work was to develop a method for the detection of nucleocapsid (core) protein in the plasma of HCV-infected persons using monoclonal antibodies (MABs). Twenty-seven anti-HCV-positive donor plasmas were studied of which 21 contained HCV RNA and 6 were negative. The plasmas were centrifuged for 3 hr at 143,000 g and the antigenic activity of core-protein was studied in the pellets by EIA using four MABs able to recognize four nonoverlapping determinants, two at N-terminus and two at C-terminus of recombinant core (1-150 aa). The determinants detected were present in the natural core protein of at least two genotypes (1b and 3a). Maximal efficiency of recombinant protein detection was achieved with 2 MABs, whereas a combination of 4 MABs was necessary for optimal detection of natural core protein. This is indicative of different conformational structures of natural protein and its gene-engineered analog. The sensitivity of core detection by monoclonal sandwich assay was 1 ng/ml in the pellet or 5 pg/ml after normalization to the initial plasma volume. To dissociate immune complexes, the pellet was treated with 2.5 M KBr after first treating the pellet with the nonionic detergent Tween 80 to remove the virus lipid envelope. Using this treatment protocol, core protein was found in 19 of 21 RNA positive plasmas.

Animals↗