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At least 685 records · Page 38Linked to original sources
PubMed2005

Infection of human hepatocyte chimeric mouse with genetically engineered hepatitis B virus.

Studies of hepatitis B virus (HBV) mutants have been hampered by the lack of a small animal model with long-term infection of cloned HBV. Using a mouse model in which liver cells were highly replaced with human hepatocytes that survived over a long time with mature human hepatocyte function, we performed transmission experiments of HBV. Human serum containing HBV and the virus produced in HepG2 cell lines that transiently or stably transfected with 1.4 genome length HBV DNA were inoculated. Genetically modified e-antigen-negative mutant strain also was produced and inoculated into the mouse model. A high-level (approximately 10(10) copies/mL) viremia was observed in mice inoculated with HBV-positive human serum samples. The level of viremia tended to be high in mice with a continuously high human hepatocyte replacement index. High levels and long-lasting viremia also were observed in mice injected with the in vitro generated HBV. The viremia continued up to 22 weeks until death or killing. Passage experiments showed that the serum of these mice contained infectious HBV. Genetically engineered hepatitis B e antigen-negative mutant clone also was shown to be infectious. Lamivudine effectively reduced the level of viremia in these infected mice. In conclusion, this mouse model of HBV infection is a useful tool for the study of HBV virology and evaluation of anti-HBV drugs. Our results indicate that HBeAg is dispensable for active viral production and transmission.

Animals↗
PubMed1989

Are the PR1 proteins of tobacco involved in genetically engineered resistance to TMV?

Transgenic tobacco plants constitutively expressing the coat protein (CP) of tobacco mosaic virus (TMV) exhibit enhanced resistance ot TMV (P. Powell Abel, R. S. Nelson, B. De, N. Hoffman, S. G. Rogers, R. T. Fraley, and R. N. Beachy, Science, 232, 738-743, 1986; R. S. Nelson, P. Powell Abel, and R. N. Beachy, Virology 158, 128-132, 1987). To determine if this enhanced resistance might be mediated through the PR1 family of pathogenesis-related (PR) proteins, their synthesis was examined. In transgenic plants derived from NN genotypic tobacco, a high proportion (approximately 80%) of those producing CP also expressed the PR1 genes at low levels. However, this correlation between TMV CP and PR1 gene expression was not observed in similarly transformed nn genotypic tobacco plants. Therefore, it appears unlikely that PR1 proteins play a critical role in genetically engineered resistance in transgenic plants producing TMV CP.

Gene Expression Regulation↗
PubMed2002

Coating with genetic engineered hydrophobin promotes growth of fibroblasts on a hydrophobic solid.

Class I Hydrophobins self-assemble at hydrophilic-hydrophobic interfaces into a highly insoluble amphipathic film. Upon self-assembly of these fungal proteins hydrophobic solids turn hydrophilic, while hydrophilic materials can be made hydrophobic. Hydrophobins thus change the nature of a surface. This property makes them interesting candidates to improve physio- and physico-chemical properties of implant surfaces. We here show that growth of fibroblasts on Teflon can be improved by coating the solid with genetically engineered SC3 hydrophobin. Either deleting a stretch of 25 amino acids at the N-terminus of the mature hydrophobin (TrSC3) or fusing the RGD peptide to this end (RGD-SC3) improved growth of fibroblasts on the solid surface. In addition, we have shown that assembled SC3 and TrSC3 are not toxic when added to the medium of a cell culture of fibroblasts in amounts up to 125 microg ml(-1).

Amino Acid Sequence↗
PubMed1997

HDL deficiency in genetically engineered mice requires elevated LDL to accelerate atherogenesis.

In humans, a low HDL concentration is one of the strongest indicators of increased risk for coronary heart disease. Apolipoprotein A-I (apo A-I) synthetic defects results in extremely low HDL levels and are frequently although not invariably associated with premature atherosclerosis. To investigate atherosclerosis susceptibility associated with HDL deficiency alone and in combination with other risk factors, such as high levels of LDL, we have quantified diet-induced atherogenesis in a series of genetically engineered mice, including mice with low HDL levels due to targeted disruption of both apo A-I alleles (AI KO mice), mice with high LDL levels due to expression of a human apolipoprotein B transgene (Btg mice), and mice with combined high LDL and low HDL levels due to the presence of the human apo B transgene and apo A-I knockout alleles, respectively (AI KO/Btg mice). After exposure to an atherogenic diet, AI KO and control mice had negligible lesions. All mice expressing the apo B transgene developed extensive lesions, but AI KO/Btg mice developed significantly larger lesions than Btg mice: 56, 260 +/- 4630 micron 2 for AI KO/Btg (n = 27) versus 38, 120 +/- 3350 micron 2 for Btg mice (n = 19) (P < .02). Results of this study, consistent with several human epidemiological studies, indicate that HDL deficiency in the mouse does not by itself lead to the development of atherosclerosis but does increase atherosclerosis susceptibility when accompanied by other risk factors, in this case elevated LDL.

Animals↗
PubMed2002

[Isolation and characterization of a p-nitrophenol degradation Pseudomonas sp. strain P3 and construction of a genetically engineered bacterium].

A Pseudomonas strain P3 was isolated in this work. P3 can grow with p-nitrophenol as the sole carbon and nitrogen source. Growth in media with nitrogen, P3 can degrade p-nitrophenol to accumulate nitrite in the culture media. P3 can utilize a series of aromatic compounds as sole carbon sources. Different heavy metal ions have different effects on the degradation of p-Nitrophenol by P3. Glucose had no effect on the degradation of p-nitrophenol, while trace yeast extract greatly increased the degradation rate. Methyl parathion hydrolase gene mpd was clone into P3 by conjugation and genetically engineered bacterium PM was obtained. Methyl parathion hydrolase was expressed by PM. PM could grew on methyl parathion as sole carbon source. PM could degrade methyl parathion with relatively high activity and stability.

Biodegradation, Environmental↗
PubMed2005

Probing the interface between biomolecules and inorganic materials using yeast surface display and genetic engineering.

Although promising for biomimetic materials applications, polypeptides binding inorganic material surfaces and the mechanism of their function have been difficult to characterize. This paper reports sequence-activity relationships of peptides interfacing with semiconductor CdS, and presents methodologies broadly applicable to studying peptide-solid surface interactions. We first employed yeast surface display with a human repertoire antibody library and identified rarely-occurring scFv fragments as CdS-binding polypeptides. Using our semi-quantitative cell-surface binding assay, site-directed mutational analysis, and genetic engineering we defined short distal regions of the displayed polypeptides necessary and sufficient for CdS binding. Alanine scanning mutagenesis in combination with a series of engineered polyhistidine peptides elucidated a direct relationship between histidine number and binding strength, which appeared to be further modulated by arginine and basic residues. The minimum strength of interaction was established by competition studies using soluble synthetic peptide analogs, which showed half-maximal inhibition of yeast binding to CdS at approximately 2 microM peptide. We then showed the ability of cells displaying material-specific polypeptides to form self-healing biofilms and discriminate between materials of fabricated heterostructure surfaces. Furthermore, we demonstrated the synthetic potential of the selected soluble CdS peptide in mediating aqueous synthesis of fluorescent CdS nanoparticles at room temperature. This platform may be further applied to elucidate mechanisms governing interfacial interactions and to generate material-specific reagents useful in medicine, biosensors, and bioproduction of high value inorganic materials.

Amino Acid Sequence↗
PubMed2003

Versatile method employing basic techniques of genetic engineering to study the ability of low-molecular-weight compounds to bind covalently with DNA in cell-free systems.

Numerous antitumor and carcinogenic compounds and free radicals are able to modify DNA by forming covalent bonds, mainly with nucleophilic centers in nucleobases. Such a binding is usually of utmost importance for the biological outcome. The level of DNA adducts formed by a given agent is in most cases extremely low; hence their detection is very difficult. Here we propose a simple approach, exploiting techniques widely used in genetic engineering, to demonstrate and characterize the covalent modification of a DNA fragment by any low-molecular-weight compound of interest in a cell-free system. The specifically designed, several-hundred-base-pairs-long double-stranded deoxyoligonucleotide (PCR amplified)--subject to modification--includes two restriction sites: one containing only GC base pairs recognized by restriction endonuclease MspI and the other including only AT base pairs recognized by restriction endonuclease Tru1I. The covalent modification of the restriction sites abolishes their recognition and thus cleavage by the endonucleases applied. The formation of DNA adducts is induced by incubating the oligonucleotide with increasing concentrations of a studied compound, in the appropriate activating system if required. Then, the modified oligonucleotide is submitted to digestion by the above-mentioned restriction endonucleases and the DNA fragments are separated by polyacrylamide gel electrophoresis. The inhibition of cleavage indicates the occurrence of covalent modification of the restriction site(s) while simultaneously pointing at the kind of base pairs involved in DNA adduct formation. The validation of the method was performed for two DNA binding antitumor compounds, cisplatin and CC-1065, which form adducts preferentially with guanine and adenine, respectively.

Base Sequence↗
PubMedDate not supplied

Genetic engineering as an approach to xenotransplantation.

A discordant organ xenograft, such as a heart or kidney transplanted from pig to human, is rejected within 1 to 2 hours by a process called hyperacute rejection (HAR) caused in large or full measure by preexisting host xenoreactive natural antibodies (XNAs) and complement. HAR can be averted by blocking either complement or XNAs; but the xenograft is then rejected at 3 to 4 days by a process called delayed xenograft rejection (DXR), which can occur without the presence of T cells. DXR involves type II endothelial cell (EC) activation (the phase of activation that includes up-regulation of proinflammatory genes in the EC), infiltration into the graft of host monocytes and natural killer cells (both of which appear to be activated), and a complex cytokine picture. We discuss in this paper various approaches that might help prevent DXR, after which we assume that one would still have to deal with a xenograft counterpart of the T cell-mediated allograft rejection response. The approach to avoiding DXR focuses largely on genetic engineering of the ECs in the graft.

Animals↗
PubMed2004

A novel immobilised design for the production of the heterologous protein lysozyme by a genetically engineered Aspergillus niger strain.

A novel immobilisation design for increasing the final concentration of the heterologous protein lysozyme by a genetically engineered fungus, Aspergillus niger B1, was developed. A central composition design was used to investigate different immobilised polymer types (alginate and pectate), polymer concentration [24% and 4% (w/v)], inoculum support ratios (1:2 and 1:4) and gel-inducing agent concentration [CaCl(2), 2% and 3.5% (w/v)]. Studies of the kinetics of production showed that optimum lysozyme productivity occurred after 10 days. Lysozyme production was significantly affected by polymer type, polymer concentration, and inoculum support ratio. Overall, immobilisation in Ca-pectate resulted in higher lysozyme production compared to that in Ca-alginate. Similar effects were observed when the polymer concentration was reduced. Regardless of polymer type and concentration, increasing the fungal inoculum level increased lysozyme production. A significantly higher lysozyme yield was achieved with Ca-pectate in comparison to Ca-alginate (approximately 20-23 mg l(-1) and 0.5-2 mg l(-1), respectively). The maximum lysozyme yield achieved was about 23 mg l(-1) by immobilisation in Ca-pectate 2% (w/v) with 33% (v/v) mycelium and 3.5% (w/v) gel-inducing agent (CaCl(2)). Response surface methodology was used to investigate the effect of pH and water activity (a(w)). The best medium pH was 4.5-5.0, and bead a(w) for optimum lysozyme yield was 0.94, regardless of polymer type.

Alginates↗
PubMed1995

A genetically engineered V79 cell line SD1 expressing rat CYP2B1 exhibits chromosomal instability at the integration site of the transfected DNA.

The genetically engineered cell line SD1 was constructed by co-transfection of V79 Chinese hamster cells with two plasmids: one containing a full-length cDNA encoding rat CYP2B1 and the second incorporating a selective marker gene. This cell line has been used in gene mutation tests and in cytokinesis-block micronucleus assays to identify procarcinogens which are metabolized by CYP2B1 to reactive metabolites. An elevated frequency of spontaneous micronuclei was recorded in SD1 cells compared to parental V79 cultures. Karyotypic analysis revealed a chromosomal instability which was manifested by amplification of the p-arms of a chromosome designated 'n' (derived from chromosome 8). This chromosome was variable in length and sometimes exhibited a telomeric fusion which led to the formation of a dicentric chromosome. Fluorescence in situ hybridization with digoxigenin-labelled plasmid DNA showed the presence of pSV450 plasmid DNA coamplified with genomic DNA sequences located in the terminal region of chromosome 'n'.

Animals↗
PubMed2004

Production of 3-hydroxy-n-phenylalkanoic acids by a genetically engineered strain of Pseudomonas putida.

Overexpression of the gene encoding the poly-3-hydroxy-n-phenylalkanoate (PHPhA) depolymerase (phaZ) in Pseudomonas putida U avoids the accumulation of these polymers as storage granules. In this recombinant strain, the 3-OH-acyl-CoA derivatives released from the different aliphatic or aromatic poly-3-hydroxyalkanoates (PHAs) are catabolized through the beta-oxidation pathway and transformed into general metabolites (acetyl-CoA, succinyl-CoA, phenylacetyl-CoA) or into non-metabolizable end-products (cinnamoyl-CoA). Taking into account the biochemical, pharmaceutical and industrial interest of some PHA catabolites (i.e., 3-OH-PhAs), we designed a genetically engineered strain of P. putida U (P. putida U DeltafadBA-phaZ) that efficiently bioconverts (more than 80%) different n-phenylalkanoic acids into their 3-hydroxyderivatives and excretes these compounds into the culture broth.

Acids, Acyclic↗
PubMed1983

Construction of live vaccines by using genetically engineered poxviruses: biological activity of recombinant vaccinia virus expressing influenza virus hemagglutinin.

Recombinant vaccinia viruses containing the cloned hemagglutinin (HA) gene from influenza virus were constructed. The biological activity of these poxvirus vectors was demonstrated both in vitro and in vivo. Expression of HA in cells infected with recombinant vaccinia was detected by using specific anti-HA antiserum and 125I-labeled protein A, showing that HA synthesized under the regulation of vaccinia virus was antigenic. Immunization of rabbits with these recombinant poxviruses resulted in the production of antibodies reactive with authentic influenza HA as detected by radioimmunoassay, by inhibition of HA erythrocyte agglutination, and by neutralization of influenza virus infectivity. The production of antibodies directed against influenza HA suggested that the HA gene expressed in vaccinia is immunogenic. These data indicate the potential of genetically engineered poxviruses for use as generic live vaccine vehicles that have both human and veterinary applications.

Animals↗
PubMed1994

Metabolism of polyhalogenated compounds by a genetically engineered bacterium.

The decomposition of organic compounds by bacteria has been studied for almost a century, during which time selective enrichment culture has generated microorganisms capable of metabolizing thousands of organic compounds. But attempts to obtain pure cultures of bacteria that can metabolize highly halogenated compounds, a large and important class of pollutants, have been largely unsuccessful. Polyhalogenated compounds are most frequently metabolized by anaerobic bacteria as a result of reductive dehalogenation reactions, the products of which are typically substrates for bacterial oxygenases. Complete metabolism of polyhalogenated compounds therefore necessitates the sequential use of anaerobic and aerobic bacteria. Here we combine seven genes encoding two multi-component oxygenases in a single strain of Pseudomonas which as a result metabolizes polyhalogenated compounds by means of sequential reductive and oxidative reactions to yield non-toxic products. Cytochrome P450cam monooxygenase reduces polyhalogenated compounds, which are bound at the camphor-binding site, under subatmospheric oxygen tensions. We find that these reduction products are oxidizable substrates for toluene dioxygenase. Perhalogenated chlorofluorocarbons also act as substrates for the genetically engineered strain.

Camphor 5-Monooxygenase↗
PubMed1987

Genetically engineered antibody molecules and their application.

Immunoglobulin genes can be efficiently expressed following transfection into myeloma cells. Using protoplast fusion, transfection frequencies greater than 10(-3) can be achieved. Compatible plasmids containing two different selectible markers are used to simultaneously deliver heavy and light chain genes to the same cell. To produce molecules with differing specificities the rearranged and expressed variable regions can be cloned from the appropriate hybridoma. In some cases, variable regions from cDNAs can be inserted into the expression vectors. It is possible to manipulate the immunoglobulin genes and produce novel antibody molecules. Antibodies have been produced in which the variable regions from mouse antibodies have been joined to human constant regions. In addition, antibodies with altered constant regions have been produced. These genetically engineered antibodies provide a unique set of reagents to study structure-function relationships within the molecule. They also can potentially be used in the diagnosis and therapy of human disease.

Animals↗
PubMed2006

Misacylation of yeast amber suppressor tRNA(Tyr) by E. coli lysyl-tRNA synthetase and its effective repression by genetic engineering of the tRNA sequence.

Through an exhaustive search for Escherichia coli aminoacyl-tRNA synthetase(s) responsible for the misacylation of yeast suppressor tRNA(Tyr), E. coli lysyl-tRNA synthetase was found to have a weak activity to aminoacylate yeast amber suppressor tRNA(Tyr) (CUA) with L-lysine. Since our protein-synthesizing system for site-specific incorporation of unnatural amino acids into proteins is based on the use of yeast suppressor tRNA(Tyr)/tyrosyl-tRNA synthetase (TyrRS) pair as the "carrier" of unusual amino acid in E. coli translation system, this misacylation must be repressed as low as possible. We have succeeded in effectively repressing the misacylation by changing several nucleotides in this tRNA by genetic engineering. This "optimized" tRNA together with our mutant TyrRS should serve as an efficient and faithful tool for site-specific incorporation of unnatural amino acids into proteins in a protein-synthesizing system in vitro or in vivo.

Amino Acyl-tRNA Synthetases↗
PubMed1989

[Study on Bacillus pumilus as a recipient strain for genetic engineering of Bacillus].

Bacillus pumilus 289 can be transformed easily by plasmid pUB110 through protoplast transformation with the frequency of 10(-5)--10(-3), similar to B. subtilis AS 1.1176, a derivative strain of B. subtilis 168. The regeneration frequency of its protoplast is only slightly lower than B. subtilis AS1.1176 (0.3-12.0% compare to 1.53--24.16%). Plasmid pUB110 can be maintained in both bacterial strains stably. The frequency of loss of the plasmid in both strains is lower than 3% after 45 generations in LB medium. But it is quite different that the hybrid plasmid (pUB110 with 3.9 kb foreign DNA fragment) can be maintained much more stably in B. pumilus 289 than in B. subtilis AS1.1176. The frequency of loss of the plasmid is lower than 5% in B. pumilus 289 and 24% in B. subtilis AS1.1176 after 25 generations when they grown in SH medium. The expression level of foreign gene in B. pumilus 289 is also much higher than that in B. subtilis AS1.1176. Therefore B. pumilus 289 is valuable to be exploited as recipient strain for genetic engineering of Bacillus in the future.

Bacillus↗
PubMed2003

Genetically engineered corn rootworm resistance: potential for reduction of human health effects from pesticides.

OBJECTIVE AND METHODS: Insecticide use, grower preferences regarding genetically engineered (GE) corn resistant to corn rootworm (CRW), and the health effects of using various CRW insecticides (organophosphates, pyrethroids, fipronil and carbamates) are reviewed for current and future farm practices. RESULTS: Pest damage to corn has been reduced only one-third by insecticide applications. Health costs from insecticide use appear significant, but costs attributable to CRW control are not quantifiable from available data. Methods reducing health-related costs of insecticide-based CRW control should be evaluated. As a first step, organophosphate insecticide use has been reduced as they have high acute toxicity and risk of long-term neurological consequences. A second step is to use agents which more specifically target the CRW. CONCLUSION: Whereas current insecticides may be poisonous to many species of insects, birds, mammals and humans, a protein derived from Bacillus thurigiensis and produced in plants via genetic modification can target the specific insect of CRW (Coleoptra), sparing other insect and non-insect species from injury.

Animals↗