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At least 1,099 records · Page 61Linked to original sources
PubMed2003

Complete tumor prevention by engineered tumor cell vaccines employing nonviral vectors.

We report that 100% mice survival after tumor challenge is achieved with cytokine-engineered cells employing nonviral lipoplexes and without using viral vectors. We describe this effect with cytokine-secreting tumor cell vaccines, based on cell clones or fresh transfected cells. Tumor cells were transfected with murine granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-4 plasmids employing the cationic lipid DOTAP, were irradiated (150 Gy) and kept frozen until use. The transfection efficacy was analyzed by qRT-PCR and flow cytometry. Vaccination induced potent antitumor rejection, resulting in 100% mice survival. Furthermore, the antitumor immunity was long lasting, since a two-fold survival delay was observed in mice after tumor rechallenge (6 months later). While cell clones secreting GM-CSF were the most effective in wild-type tumor cell rejection, little or no effect was observed with clones secreting IL-4. We found similar antitumor efficacy employing fresh transfected cells by nonviral procedures, demonstrating that cells genetically modified by nonviral vectors (both clones and fresh transfected cells) are a safe and efficient tool for antitumor vaccines. These vaccines allow us to achieve the highest antitumor efficacy based on nonviral gene therapy techniques. In addition, the vaccination success with fresh transfected cells simplifies the procedure and provides new insights into the clinical application of nonviral gene therapy procedures.

Animals↗
PubMed2003

Calmodulin-dependent regulation of mammalian nitric oxide synthase.

The nitric oxide synthases are large, modular, dimeric enzymes composed of a reductase domain, which is related to cytochrome P450 reductase, and a structurally unique oxygenase domain containing a Cys-ligated haem. Both the neuronal and endothelial isoforms are activated by the reversible binding of calmodulin (CaM) at elevated intracellular Ca(2+) levels to produce NO as part of a number of cell signalling pathways. CaM binds to the linker region between the two domains and activates the enzyme by inducing intramolecular electron transfer. Protein-engineering experiments have shown that a series of unusual autoinhibitory inserts found only in the CaM-dependent NOS isoforms control both CaM binding and the structural rearrangement it induces. These lie in the reductase domain of the enzyme and include a 40-amino-acid autoinhibitory loop in the FMN-binding module, a 30-amino-acid extension to the C-terminus and the CaM-binding site itself. The substrate (NADPH) also plays an important role in defining the CaM-dependence of the reductase domain by inducing a tight conformational lock in the absence of CaM. Both the substrate and the conformational lock appear to be released on CaM binding; the resultant domain mobility leads to activation.

Animals↗
PubMed2005

Cell-replacement therapy for diabetes: Generating functional insulin-producing tissue from adult human liver cells.

Shortage in tissue availability from cadaver donors and the need for life-long immunosuppression severely restrict the large-scale application of cell-replacement therapy for diabetic patients. This study suggests the potential use of adult human liver as alternate tissue for autologous beta-cell-replacement therapy. By using pancreatic and duodenal homeobox gene 1 (PDX-1) and soluble factors, we induced a comprehensive developmental shift of adult human liver cells into functional insulin-producing cells. PDX-1-treated human liver cells express insulin, store it in defined granules, and secrete the hormone in a glucose-regulated manner. When transplanted under the renal capsule of diabetic, immunodeficient mice, the cells ameliorated hyperglycemia for prolonged periods of time. Inducing developmental redirection of adult liver offers the potential of a cell-replacement therapy for diabetics by allowing the patient to be the donor of his own insulin-producing tissue.

Adenoviridae↗
PubMed1992

Expression of murine interleukin 7 in a murine glioma cell line results in reduced tumorigenicity in vivo.

We have examined the immunoregulatory effect of local and continuous secretion of interleukin 7 (IL-7) from murine glioma cells (203-glioma) engineered by murine IL-7 gene transfection. Secretion of IL-7 from glioma cells did not result in morphology or growth rate changes but did reduce tumorigenicity in vivo in proportion to the amount of IL-7 produced. This reduction in tumorigenicity could be reversed in a dose-dependent fashion by injection of anti-IL-7 neutralizing monoclonal antibody at the tumor site. Mice immunized with IL-7-producing glioma cells showed a specific immune response to 203-glioma but not to two other syngeneic cell lines (B-16, a melanoma, and YM-12, a fibrosarcoma). IL-7-producing glioma cells were not rejected in mice depleted of CD8+ cells but were rejected in mice depleted of CD4+ or NK1.1+ cells. These results suggest that CD8+ T cells may play an important role in tumor rejection.

Animals↗
PubMed2001

Treatment of ischemic wounds using cultured dermal fibroblasts transduced retrovirally with PDGF-B and VEGF121 genes.

The healing of ischemic wounds is a particularly difficult clinical challenge. In this study, rabbit dermal fibroblasts transduced retrovirally with human platelet-derived growth factor B (PDGF-B) and human vascular endothelial growth factor 121 (VEGF121) genes were used to treat wounds in a rabbit ischemic ear model. The PDGF-B and VEGF121 genes were obtained from human umbilical vein endothelial cells (HUVECs) by reverse transcription-polymerase chain reaction, cloned into retroviral vectors under control of the beta-actin promoter, and introduced into primary rabbit dermal fibroblast cells. In vitro results demonstrated that rabbit dermal fibroblasts are transduced and selected readily using retroviral vectors, and are engineered to secrete PDGF-B and VEGF121 at steady-state levels of 150 ng per 10(6) cells per 24 hours and 230 ng per 10(6) cells per 24 hours respectively. These cells were then seeded onto polyglycolic acid (PGA) scaffold matrices and used to treat ischemic rabbit ear wounds. Immunohistochemistry showed intense staining for PDGF-B and VEGF121 in the wounds treated with these transduced cells compared with the control treatment groups. For the relatively more ischemic distal ear wounds, granulation tissue deposition was increased significantly in the wounds treated with PDGF-B- and VEGF121-transduced cells compared with wounds treated with PGA alone. These results demonstrate that gene augmentation of rabbit dermal fibroblasts with the PDGF-B and VEGF121 genes introduced into this ischemic wound model via PGA matrices modulates wound healing, and may have clinical potential in the treatment of ischemic wounds.

Animals↗
PubMed2006

Genetic modification of cassava for enhanced starch production.

To date, transgenic approaches to biofortify subsistence crops have been rather limited. This is particularly true for the starchy root crop cassava (Manihot esculenta Crantz). Cassava has one of the highest rates of CO(2) fixation and sucrose synthesis for any C3 plant, but rarely reaches its yield potentials in the field. It was our hypothesis that starch production in cassava tuberous roots could be increased substantially by increasing the sink strength for carbohydrate. To test this hypothesis, we generated transgenic plants with enhanced tuberous root ADP-glucose pyrophosphorylase (AGPase) activity. This was achieved by expressing a modified form of the bacterial glgC gene under the control of a Class I patatin promoter. AGPase catalyses the rate-limiting step in starch biosynthesis, and therefore the expression of a more active bacterial form of the enzyme was expected to lead to increased starch production. To facilitate maximal AGPase activity, we modified the Escherichia coli glgC gene (encoding AGPase) by site-directed mutagenesis (G336D) to reduce allosteric feedback regulation by fructose-1,6-bisphosphate. Transgenic plants (three) expressing the glgC gene had up to 70% higher AGPase activity than control plants when assayed under conditions optimal for plant and not bacterial AGPase activity. Plants having the highest AGPase activities had up to a 2.6-fold increase in total tuberous root biomass when grown under glasshouse conditions. In addition, plants with the highest tuberous root AGPase activity had significant increases in above-ground biomass, consistent with a possible reduction in feedback inhibition on photosynthetic carbon fixation. These results demonstrate that targeted modification of enzymes regulating source-sink relationships in crop plants having high carbohydrate source strengths is an effective strategy for increasing carbohydrate yields in sink tissues.

Biomass↗
PubMed2005

Molecular cloning and in vitro evaluation of an infectious simian-human immunodeficiency virus containing env of a primary Chinese HIV-1 subtype C isolate.

Human immunodeficiency virus (HIV) clade C is the most prevalent subtype and accounts for approximately 50% of all HIV infections worldwide. In China, the prevalent HIV strains are B'/C subtypes, in which the envelope belongs to subtype C. To evaluate potential AIDS vaccines targeting Chinese viral strains in non-human primate models, we constructed an infectious simian-human immunodeficiency virus (SHIV) that expresses most of the envelope of a primary HIV strain, which was isolated from a HIV-positive intravenous drug user from XinJiang province in China. The resulting chimeric SHIV-XJ02170 was infectious in human, rhesus monkey and cynomolgus monkey peripheral blood mononuclear cells (PBMC) and used CCR5 exclusively as coreceptor.

Animals↗
PubMed1993

A vector for facile PCR product cloning and modification generating any desired 4-base 5' overhang: pRPM.

A pair of plasmids were developed for cloning PCR products in order to facilitate the preparation of products with 5' overhangs consisting of any desired 4-nucleotide sequence. These vectors allow DNA to be cloned into a unique restriction site by blunt-end ligation or by AT-cloning. The cloned DNA is subsequently excised using class IIS restriction enzyme sites flanking the insert yielding a fragment that is entirely free of vector sequences. These enzymes recognize sequences in the vector but "reach-over" the junction to cut within the insert thereby generating a 4-base 5' overhang sequence determined by the 5' sequences in the insert. Thus, in cases where the insert was originally generated by PCR, the overhangs are specified by the primer. More generally, these vectors offer a unique capability for the reversible cloning of any blunt DNA fragment because excision and fill-in reactions precisely regenerate the original blunt fragment regardless of its sequence. These "reach-over" product modification vectors represent general and flexible tools for the generation of fragments for use in engineering DNA constructs.

Base Sequence↗
PubMed1998

A novel point mutation at position 156 of reverse transcriptase from feline immunodeficiency virus confers resistance to the combination of (-)-beta-2',3'-dideoxy-3'-thiacytidine and 3'-azido-3'-deoxythymidine.

Mutants of feline immunodeficiency virus (FIV) resistant to (-)-beta-2',3'-dideoxy-3'-thiacytidine (3TC) were selected by culturing virus in the presence of increasing stepwise concentrations of 3TC. Two plaque-purified variants were isolated from the original mutant population, and both of these mutants were resistant to 3TC. Surprisingly, these mutants were also phenotypically resistant to 3'-azido-3'-deoxythymidine (AZT) and to the combination of 3TC and AZT. Purified reverse transcriptase (RT) from one of these plaque-purified mutants was resistant to the 5'-triphosphates of 3TC and AZT. DNA sequence analysis of the RT-encoding region of the pol gene amplified from the plaque-purified mutants revealed a Pro-to-Ser mutation at position 156 of RT. A site-directed mutant of FIV engineered to contain this Pro-156-Ser mutation was resistant to 3TC, AZT, and the combination of 3TC and AZT, confirming the role of the Pro-156-Ser mutation in the resistance of FIV to these two nucleoside analogs. This represents the first report of a lentiviral mutant resistant to the combination of AZT and 3TC due to a single, unique point mutation.

Amino Acid Sequence↗
PubMed2004

Human-compatible collagen matrix for prolonged and reversible systemic delivery of erythropoietin in mice from gene-modified marrow stromal cells.

Bone marrow stromal cells (MSCs) can be exploited therapeutically in transgenic cell therapy approaches. Our aim was to determine if gene-modified MSCs sequestered within a clinically approved, bovine type I collagen-based viscous bulking material could serve as a retrievable implant for systemic delivery of erythropoietin (Epo). To test this hypothesis, we embedded Epo-secreting MSCs in viscous collagen (Contigen) and determined the pharmacological effect following implantation in normal mice. Primary MSCs from C57Bl/6 mice were retrovirally engineered to express murine Epo (mEpo) and 10(7) cells of a clonal population secreting 3 U of mEpo/10(6) cells/24 h were implanted subcutaneously in normal C57Bl/6 mice with and without viscous collagen. Without matrix support, Hct rose to >70% for <25 days and returned to baseline by 60 days. However, in mice implanted with viscous collagen-embedded MSCs, the Hct rose to >70% up to 203 days postimplantation (P < 0.0001). In parallel, plasma Epo concentration was significantly increased (P < 0.05) for >145 days. Moreover, surgical removal of the viscous collagen organoid 24 days after implantation led to reduction of Hct to baseline levels within 14 days. In conclusion, this investigation demonstrates that mEpo(+) MSCs embedded in a human-compatible viscous collagen matrix offers a potent, durable, and reversible approach for delivery of plasma-soluble therapeutic proteins.

Animals↗
PubMed2003

Complete reversal of coenzyme specificity by concerted mutation of three consecutive residues in alcohol dehydrogenase.

Gastric tissues from amphibian Rana perezi express the only vertebrate alcohol dehydrogenase (ADH8) that is specific for NADP(H) instead of NAD(H). In the crystallographic ADH8-NADP+ complex, a binding pocket for the extra phosphate group of coenzyme is formed by ADH8-specific residues Gly223-Thr224-His225, and the highly conserved Leu200 and Lys228. To investigate the minimal structural determinants for coenzyme specificity, several ADH8 mutants involving residues 223 to 225 were engineered and kinetically characterized. Computer-assisted modeling of the docked coenzymes was also performed with the mutant enzymes and compared with the wild-type crystallographic binary complex. The G223D mutant, having a negative charge in the phosphate-binding site, still preferred NADP(H) over NAD(H), as did the T224I and H225N mutants. Catalytic efficiency with NADP(H) dropped dramatically in the double mutants, G223D/T224I and T224I/H225N, and in the triple mutant, G223D/T224I/H225N (kcat/KmNADPH = 760 mm-1 min-1), as compared with the wild-type enzyme (kcat/KmNADPH = 133330 mm-1 min-1). This was associated with a lower binding affinity for NADP+ and a change in the rate-limiting step. Conversely, in the triple mutant, catalytic efficiency with NAD(H) increased, reaching values (kcat/KmNADH = 155000 mm-1 min-1) similar to those of the wild-type enzyme with NADP(H). The complete reversal of ADH8 coenzyme specificity was therefore attained by the substitution of only three consecutive residues in the phosphate-binding site, an unprecedented achievement within the ADH family.

Alcohol Dehydrogenase↗
PubMed2006

Engineered superoxide dismutase monomers for superoxide biosensor applications.

Because of its high reaction rate and specificity, the enzyme superoxide dismutase (SOD) offers great potential for the sensitive quantification of superoxide radicals in electrochemical biosensors. In this work, monomeric mutants of human Cu,Zn-SOD were engineered to contain one or two additional cysteine residues, which could be used to bind the protein to gold surfaces, thus making the use of promotor molecules unnecessary. Six mutants were successfully designed, expressed, and purified. All mutants bound directly to unmodified gold surfaces via the sulfur of the cysteine residues and showed a quasi-reversible, direct electron transfer to the electrode. Thermodynamic and kinetic parameters of the electron transfer were characterized and showed only slight variations between the individual mutants. For one of the mutants, the interaction with the superoxide radical was studied in more detail. For both partial reactions of the dismutation, an interaction between protein and radical could be shown. In an amperometric biosensorial approach, the SOD-mutant electrode was successfully applied for the detection of superoxide radicals. In the oxidation region, the electrode surpassed the sensitivity of the commonly used cytochrome c electrodes by approximately 1 order of magnitude while not being limited by interferences, but the electrode did not fully reach the sensitivity of dimeric Cu,Zn-SOD immobilized on MPA-modified gold.

Biosensing Techniques↗
PubMed2003

Generating MHC Class II+/Ii- phenotype after adenoviral delivery of both an expressible gene for MHC Class II inducer and an antisense Ii-RNA construct in tumor cells.

Tumor cells engineered by gene transduction to be MHC Class II+/Ii- are novel APCs capable of presenting endogenous tumor antigen epitopes to activate T helper cells. The MHC Class II+/Ii- tumor cell phenotype is created by transfecting genes for either CIITA or IFN-gamma, and inhibiting induced Ii mRNA by an Ii reverse gene construct (Ii-RGC). Adenoviral vectors are preferred for the delivery of such genes because of high infection efficiency and ubiquity of the adenoviral receptor on many cell types and tumors. Here we show that at 5 MOI (multiplicity of infection), recombinant adenoviruses with CIITA or IFN-gamma genes converted virtually all MC-38 colon adenocarcinoma cells and Renca renal carcinoma cells in culture to MHC Class II+/Ii+ cells. A single recombinant adenovirus with both genes for IFN-gamma and Ii-RGC (rAV/IFN-gamma/Ii-RGC) efficiently induced the MHC Class II+/Ii- phenotype. Injection of tumor nodules with rAV/Ii-RGC and rAV/CIITA/IFN-gamma combined with a suboptimal dose of rAV/IL-2 induced a potent antitumor immune response. The methods are adaptable for producing enhanced genetic vaccines, attenuated virus vaccines (eg, vaccinia), and ex vivo cell-based vaccines (dendritic and tumor cells).

Adenoviridae↗
PubMed1996

Engineering the Drosophila genome: chromosome rearrangements by design.

We show that site-specific recombination can be used to engineer chromosome rearrangements in Drosophila melanogaster. The FLP site-specific recombinase acts on chromosomal target sites located within specially constructed P elements to provide an easy screen for the recovery of rearrangements with breakpoints that can be chosen in advance. Paracentric and pericentric inversions are easily recovered when two elements lie in the same chromosome in opposite orientation. These inversions are readily reversible. Duplications and deficiencies can be recovered by recombination between two elements that lie in the same orientation on the same chromosome or on homologues. We observe that the frequency of recombination between FRTs at ectopic locations decreases as the distance that separates those FRTs increases. We also describe methods to determine the absolute orientation of these P elements within the chromosome. The ability to produce chromosome rearrangements precisely between preselected sites provides a powerful new tool for investigations into the relationships between chromosome arrangement, structure, and function.

Animals↗
PubMed2001

[Preparation of genetically engineered retinal pigment epithelial cells capable of secreting nerve growth factor].

OBJECTIVE: To develop genetically engineered retinal pigment epithelial (RPE) cells which are able to express bioactive nerve growth factor (NGF) steadily and efficiently and to combine gene therapy with retinal transplantation techniques to treat retinal degeneration. METHODS: Replication-deficient retrovirus-plasmid recombinant pLXSN-NGF was constructed via subclone and clone, and then packaged into pT67 cells to obtain virion containing a secretable form of NGF gene. We evaluated titer of the retroviral recombinant with NIH 3T3 cells and then transduced NGF gene into cultured RPE cells by the retroviral recombinant. We also analyzed the expression of NGF gene in transduced and untransduced RPE cells via reverse transcription (RT)-PCR and Western blot. The bioactivity of expressed NGF were evaluated by stimulating PC 12 cells with conditioned medium from transduced and untransduced RPE cells. RESULTS: The titer of replication-deficient retroviral recombinant reached 1.2 x 10(7) cfu/ml. NGF protein and mRNA were detected in the conditioned medium from transduced RPE cells, but not from the normal RPE cells. Marked neurite process outgrowth from PC 12 cells was observed after being stimulated with the conditioned medium from transduced RPE cells. CONCLUSIONS: RPE cells are able to be transduced by replication-deficient retrovirus with high efficiency, and secret bioactive NGF at a high level, which might be an ideal candidate for engineered cells.

3T3 Cells↗
PubMed1999

PCR-mediated recombination: a general method applied to construct chimeric infectious molecular clones of plasma-derived HIV-1 RNA.

A PCR-based approach was developed that provides a powerful tool for engineering recombinant molecules without reliance on restriction sites. DNA sequences were first amplified by high-fidelity PCR using Pfu polymerase; they were then used both as 'megaprimers' and templates in subsequent asymmetric long PCR amplifications to form chimeric clones. To demonstrate the technique, we constructed chimeric full-length HIV-1 clones derived from reverse-transcribed plasma viral RNA and proviral LTRs. Biologic characterization of these clones showed that most were infectious in tissue culture and sequence analysis demonstrated an error rate of only one base change in 20 kb of DNA sequence. For PCR-mediated recombination, it is necessary to know the sequence of the 3' and 5' overlapping regions of the desired PCR products. This method may be extended to include construction of chimeras between any DNA fragments lacking sequence homology. Such chimeras may be constructed by introducing overlapping sequences to one of the fragments. To ensure that unwanted mutations have not been introduced into the clones constructed by this method, each clone should be sequenced. Our results demonstrate that by using a high-fidelity polymerase and highly controlled PCR conditions, the PCR-introduced error rate can be greatly minimized. This new procedure may be used to construct infectious chimeras of HIV or SIV for studies of vaccines and pathogenesis. Moreover, the method is designed to exchange viral genes at precise boundaries to study individual gene products from different HIV genomes. It can also be used to construct expression vectors for production of specific proteins or delivery vectors for gene transfer and gene therapy. Finally, the technique described here provides a versatile tool to transfer genes or gene fragments from different sources for genetic investigation and engineering.

Chimera↗
PubMed2005

Exploring the charge space of protein-protein association: a proteomic study.

The rate of association of a protein complex is a function of an intrinsic basal rate and of the magnitude of electrostatic steering. In the present study we analyze the contribution of electrostatics towards the association rate of proteins in a database of 68 transient hetero-protein-protein complexes. Our calculations are based on an upgraded version of the computer algorithm PARE, which was shown to successfully predict the impact of mutations on k(on) by calculating the difference in Columbic energy of interaction of a pair of proteins. HyPare (http://bip.weizmann.ac.il/HyPare), automatically calculates the impact of mutations on a per-residue basis for all residues of a protein-protein interaction, achieving a precision similar to that of PARE. Our calculations show that electrostatics play a marginal role (<10 fold) in determining the rate of association for about half of the complexes in the database. Strong electrostatic steering, which results in an increase of over 100-fold in k(on), was calculated for about 25% of the complexes. Applying HyPare to all 68 complexes in the database shows that a small number of residues are hotspots for association. About 40% of the hotspots are calculated to increase the rate of association upon mutation, and thus increase binding affinity. This is a much higher ratio than found for hotspots for dissociation, where the large majority cause weaker binding. About 40% of the hotspots are located outside the physical boundary of the binding site, making them ideal candidates for protein engineering. Our data shows that a majority of protein-protein complexes are not optimized for fast association. Hotspots are not evenly distributed between all types of amino acids. About 75% of all hotspots are of charged residues. This is understandable, as a charge-reverse mutant changes the total charge by 2. The small number of hydrophobic residues that are hotspots upon mutation probably relates to their location and surrounding. For 18 out of the 68 complexes in the database, experimental values of k(on) are available. For these, a basal rate of association was calculated to be in the range of 10(4)M(-1)s(-1) to 10(7)M(-1)s(-1). Some of these rates were verified independently from experimental mutant data. The basal rates were correlated with the size of the proteins and the shape of the interface.

Algorithms↗