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At least 55 records · Page 3Linked to original sources
PubMed1976

Role of a lipopolysaccharide gene for immunogenicity of the enterobacterial common antigen.

It is known that only certain strains of the family of Enterobacteriaceae, notably rough (R) mutants with the type R1 or R4 core, evoked antibodies in high titers against the common enterobacterial antigen (CA) after immunization of rabbits with heated cell suspensions. The present investigation deals with genetic and immunochemical aspects of certain R1 and R4 mutants isolated from Escherichia coli 08 and various Shigella serotypes which, unexpectedly, do not induce CA antibody formation. Immunochemical and genetical (transduction and conjugation) experiments revealed that the rough phenotype of these special mutants was evoked by a mutation of pyrE-linked rfa gene, called rfaL, which is involved in translocation of O-specific polysaccharides onto the lipopolysaccharide core. The transduction of the defective rfaL, allele into appropriate rough recipients results in transductants which have simultaneously lost the ability to evoke CA antibodies. This finding suggests that a close connection exists between the function of the rfaL gene and the expression of CA immunogenicity in R1 and R4 mutants. One of the strains synthesized neither O-hapten nor CA, suggesting a mutation in a region equivalent to the rfe genes of Salmonella.

Animals↗
PubMed1997

Purification and characterization of VSH-1, a generalized transducing bacteriophage of Serpulina hyodysenteriae.

Serpulina hyodysenteriae B204 cells treated with mitomycin (20 microg of mitomycin/ml of culture broth) lysed and released bacteriophages. Bacteriophage particles, precipitated by using polyethylene glycol and purified by CsC1 density gradient ultracentrifugation, had a buoyant density of 1.375 g/cm3 and consisted of a head (45-nm diameter) and an ultrastructurally simple (noncontractile) tail (64 by 9 nm) composed of at least 13 proteins with molecular masses ranging between 13 and 101 kDa. The purified bacteriophage has been designated VSH-1 (VSH for virus of S. hyodysenteriae). VSH-1 was incapable of lytic growth on any of five intestinal spirochete strains, representing three Serpulina species. VSH-1 nucleic acid was determined to be approximately 7.5 kb in size and to be linear, double-stranded DNA based on differential staining with acridine orange, DNase I sensitivity, electrophoretic mobility, and contour length as measured by electron microscopy. Phage DNA digested by the restriction enzymes SspI, AseI, EcoRV, and AflII gave electrophoretic banding patterns nearly identical to those of digested chromosomal DNA from S. hyodysenteriae. Additionally, VSH-1 DNA fragments hybridized with probes complementary to S. hyodysenteriae chromosomal genes nox and flaA1. When purified bacteriophages induced from cultures of S. hyodysenteriae A203 (deltaflaA1 593-762::cat) were added to growing cells of strain A216 (deltanox 438-760::kan), transductants (Cmr Kmr) were obtained at a frequency of 1.5 x l0(-6) per phage particle (enumerated by electron microscopy). These findings indicate that induced VSH-1 virions package DNA of S. hyodysenteriae and are capable of transferring host genes between cells of that spirochete. To our knowledge, this is the first report of genetic transduction of a spirochete.

Bacteriophages↗
PubMed2000

Direct intra-cardiomuscular transfer of beta2-adrenergic receptor gene augments cardiac output in cardiomyopathic hamsters.

In chronic heart failure, down-regulation of beta-adrenergic receptor (beta-AR) occurs in cardiomyocytes, resulting in low catecholamine response and impaired cardiac function. To correct the irregularity in the beta-AR system, beta-AR gene was transduced in vivo into failing cardiomyocytes. The Epstein-Barr virus (EBV)-based plasmid vector carrying human beta2-AR gene was injected into the left ventricular muscle of Bio14.6 cardiomyopathic hamsters whose beta-AR is down-regulated in the cardiomyocytes. The echocardiographic examinations revealed that stroke volume (SV) and cardiac output (CO) were significantly elevated at 2 to 4 days after the beta2-AR gene transfer. Systemic loading of isoproterenol increased the cardiac parameters more significantly on day 2 to day 7, indicating that the adrenergic response was augmented by the genetic transduction. The same procedure did not affect the cardiac function of normal hamsters. Immunohistochemical examinations demonstrated human beta2-AR expression in failing cardiomyocytes transduced with the gene. RT-PCR analysis detected mRNA for the transgene in the heart but not in the liver, spleen, or kidney. The procedures may provide a feasible strategy for gene therapy of severe heart failure. Gene Therapy (2000) 7, 2087-2093.

Animals↗
PubMed2000

Repulsive axon guidance: Abelson and Enabled play opposing roles downstream of the roundabout receptor.

Drosophila Roundabout (Robo) is the founding member of a conserved family of repulsive axon guidance receptors that respond to secreted Slit proteins. Little is known about the signaling mechanisms which function downstream of Robo to mediate repulsion. Here, we present genetic and biochemical evidence that the Abelson (Abl) tyrosine kinase and its substrate Enabled (Ena) play direct and opposing roles in Robo signal transduction. Genetic interactions support a model in which Abl functions to antagonize Robo signaling, while Ena is required in part for Robo's repulsive output. Both Abl and Ena can directly bind to Robo's cytoplasmic domain. A mutant form of Robo that interferes with Ena binding is partially impaired in Robo function, while a mutation in a conserved cytoplasmic tyrosine that can be phosphorylated by Abl generates a hyperactive Robo receptor.

Animals↗
PubMed1994

Genetics of signal transduction: tales from the mouse.

Recent progress in understanding signal transduction owes much to new genetic approaches, first by unraveling the molecular basis of classic mutations, and then by the use of gene targeting. Recent studies have examined mammalian signal transduction from cell surface to nucleus, especially ligand-receptor systems and cytosolic signal transducers.

Animals↗
PubMed1995

Blood stem cell transplantation and gene therapy of cancer.

Based on the concept of circulating hematopoietic stem cells with indefinite self-renewal capacity that gives rise to all three cell lineages, peripheral blood progenitor cells (PBPCs) have widely replaced the use of bone marrow (BM) progenitors for autologous transplantation purposes in patients with malignant hematological disorders and selected solid tumors. Ex vivo purification of normal CD34+ cell subsets contained in the patient's apheresis product possibly eliminates clonogenic tumor cells, but also serves as a target cell population for gene transduction. Genetic tagging of PBPC autografts has proven that: 1) NEOR gene expression is sustained for more than 18 months and 2) clonogenic tumor cells contaminating the autograft contribute to relapse. A second generation of gene transduction studies includes new treatment strategies such as the induction of chemoprotection (multidrug resistance gene-1), chemotherapy sensitization (p53), cancer vaccination and genetic chemosensitization. Most recently allogeneic PBPC transplantation has successfully been introduced with the intention of improving the graft-versus-leukemia effect without inducing a higher incidence or more severe graft-versus-host disease (GVHD) than what is expected after BM transplantation. Introducing the herpes virus thymidine kinase cDNA into activated donor T cells makes them susceptible to gangciclovir, thus allowing the in vivo inactivation of GVHD-inducing T cells. With the close interaction of molecular genetics and clinical oncology/hematology, genetic engineering of stem cell grafts will lead into a new stage of stem cell transplantation technology.

Genetic Therapy↗
PubMed1999

Retroviral-fibronectin interactions in transduction of mammalian cells.

Hematopoiesis occurs in a complex environment in the medullary cavity in close proximity to stromal cells, fibroblasts, endothelial cells and matrix molecules. Hematopoietic cell interactions in this environment appear to involve both integrin and proteoglycan-mediated cell-cell and cell-matrix interactions. Genetic transduction of hematopoietic stem cells via retroviral vectors has been hampered by low efficiency of gene transfer. Recently, hematopoietic stem cell adhesion to the extracellular matrix molecule fibronectin has been shown to increase transduction of these target cells using retrovirus vectors. The mechanism of increased transduction appears to involve colocalization of virus particles and target cells. These data are reviewed in this paper.

Animals↗
PubMedDate not supplied

Presentation of tumor antigens by dendritic cells genetically modified with viral and nonviral vectors.

Genetic modification of dendritic cells (DCs) with recombinant vectors encoding tumor antigens may aid in developing new immunotherapeutic treatments for patients with cancer. Here, we characterized antigen presentation by human DCs genetically modified with plasmid cDNAs, RNAs, adenoviruses, or retroviruses, encoding the melanoma antigen gp100 or the tumor-testis antigen NY-ESO-1. Monocyte-derived DCs were electroporated with cDNAs or RNAs, or transduced with adenoviruses. CD34+ hematopoietic stem cell-derived DCs were used for retroviral transduction. Genetically modified DCs were coincubated with CD8+ and CD4+ T cells that recognized major histocompatibility complex class I- and class II-restricted epitopes from gp100 and NY-ESO-1, and specific recognition was evaluated by interferongamma secretion. Cytokine release by both CD8+ and CD4+ T cells was consistently higher in response to DCs modified with adenoviruses than cDNAs or RNAs, and maturation of DCs after genetic modification did not consistently alter patterns of recognition. Also, retrovirally transduced DCs encoding gp100 were well recognized by both CD8+ and CD4+ T cells. These data suggest that DCs transduced with viral vectors may be more efficient than DCs transfected with cDNAs or RNAs for the induction of tumor reactive CD8+ and CD4+ T cells in vitro and in human vaccination trials.

Adenoviridae↗
PubMed1998

Molecular, genetic and physiological analysis of Cladosporium resistance gene function in tomato.

Characterization of the DNA sequence of 4 tomato leaf mould disease resistance genes (Cf-2, Cf-4, Cf-5 and Cf-9) leads to the prediction that they encode C-terminally membrane anchored glycopeptides with many extracytoplasmic leucine rich repeats (LRRs). The N terminal LRRs are variable between the Cf-genes, suggesting a role in specificity, and the C terminal LRRs are more conserved, suggesting a role in signal transduction. Genetic analysis has revealed several Rcr genes that are required for Cf-gene function; their isolation will help us understand how Cf-genes work. Cf-9 confers responsiveness to pathogen-encoded Avr9 peptide on introduction to tobacco. Tobacco suspension cultures carrying the Cf-9 gene produce reactive oxygen species in response to Avr9 peptide, whereas untransformed cultures do not. The significance of these observations is discussed.

Cladosporium↗
PubMed2005

Functions and mechanisms of receptor tyrosine kinase Torso signaling: lessons from Drosophila embryonic terminal development.

The Torso receptor tyrosine kinase (RTK) is required for cell fate specification in the terminal regions (head and tail) of the early Drosophila embryo. Torso contains a split tyrosine kinase domain and belongs to the type III subgroup of the RTK superfamily that also includes the platelet-derived growth factor receptors, stem cell or steel factor receptor c-Kit proto-oncoprotein, colony-stimulating factor-1 receptor, and vascular endothelial growth factor receptor. The Torso pathway has been a model system for studying RTK signal transduction. Genetic and biochemical studies of Torso signaling have provided valuable insights into the biological functions and mechanisms of RTK signaling during early Drosophila embryogenesis.

Animals↗
PubMed1993

Adenoviral-mediated gene transfer to rabbit synovium in vivo.

Currently, treatment for rheumatoid arthritis and other inflammatory arthropathies is often ineffective in ameliorating the progression of the disease, particularly the invasive destruction of cartilage and bone by rheumatoid synovium. Multiple aspects of this inflammatory process are mediated by the synovial lining cells (synoviocytes). Genetic modification of these cells in vivo represents a potential method for the treatment of these conditions. In this report, we describe a novel technique for the genetic transduction of synovial lining cells in vivo using recombinant adenoviral vectors and intraarticular injection techniques. Purified high titer suspensions of a recombinant adenoviral vector containing the gene for Escherichia coli beta-galactosidase (AdCMVlacZ) were directly injected into the hind knees of New Zealand white rabbits. Synovial tissues were then examined for transgenic lacZ expression using a combination of in situ staining for beta-galactosidase activity, immunohistochemical staining, and transmission electron microscopy. High efficiency gene transfer and lacZ expression was observed in both type A and type B synoviocytes throughout the articular and periarticular synovium of the rabbit knee, with continued expression of transgenic lacZ detected for > or = 8 wk after infection.

Adenoviridae↗
PubMed1999

Transfer of lacZ marker gene to the meniscus.

BACKGROUND: Lesions in the avascular two-thirds of the meniscus do not heal well and are of concern clinically. Various growth factors promote the synthesis of matrix by meniscal cells and thus have the potential to augment healing. However, their clinical application is severely hindered by problems with delivery. An attractive approach to overcoming such problems is to transfer genes that encode the growth factors in question to the site of the injury. As a prelude to this, we evaluated methods for delivering genes to the meniscus. METHODS: Gene transfer was evaluated in vitro and in vivo with a lacZ marker gene, which expresses the enzyme beta-galactosidase. Two types of vectors were tested: an adenovirus and a retrovirus. Monolayers of lapine, canine, and human meniscal cells, as well as intact lapine and human menisci, were used for the in vitro studies. Lesions were created in the menisci of rabbits and dogs for the in vivo studies. Gene transfer to the sites of the experimental meniscal lesions in vivo was accomplished in two ways. In the lapine model, a suspension of adenovirus carrying the lacZ marker gene was mixed with whole blood and the clot was inserted into the lesion. In the canine model, retrovirally transduced allogenic meniscal cells carrying the lacZ marker gene were embedded in collagen gels and transferred to the defects. The animals were killed at various time-points, and gene expression was evaluated by histological examination of sections stained with 5-bromo-4-chloro-indolyl-beta-D-galactose (X-gal), from which a blue chromagen is released in the presence of beta-galactosidase. RESULTS: Monolayer cultures of lapine, canine, and human meniscal cells were susceptible to genetic transduction by both adenoviral and retroviral vectors. In vitro gene transfer to intact human and lapine menisci proved possible both by direct, adenoviral, delivery and indirect, retroviral, delivery. Gene expression persisted for at least twenty weeks under in vitro conditions. With regard to the in vivo studies, gene expression persisted within the clot and in some of the adjacent meniscal cells for at least three weeks in the lapine defect model. In the canine defect model, gene expression persisted within the transplanted, transduced meniscal cells for at least six weeks. CONCLUSIONS: It is possible to transfer genes to sites of meniscal damage and to express them locally within the lesion for several weeks.

Animals↗
PubMed1996

Intensified antitumor immunity by a cancer vaccine that produces granulocyte-macrophage colony-stimulating factor plus interleukin 4.

Vaccination with irradiated tumor cells genetically modified to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF tumor vaccine) induces a potent systemic antitumor immunity. To develop a protocol for cancer therapy to further augment the host immune response, we examined the effects of the GM-CSF tumor vaccines simultaneously producing additional cytokines. We prepared cancer vaccines expressing double cytokines by sequential recombinant retrovirus-mediated genetic transductions. We then used a murine intracerebral tumor model in which the GM-CSF tumor vaccine was less effective in immunopotentiation and evaluated tumor vaccines producing various cytokines in conjunction with GM-CSF. The cytokine combination of GM-CSF and interleukin 4 induced more potent antitumor immunity than GM-CSF alone. An in vivo depletion test showed that CD4+, CD8+, and asialoGM1+ cells were required for the optimum function of the GM-CSF plus interleukin 4 tumor vaccine. Histological examinations revealed infiltration of inflammatory cells at the site of tumor cell challenge as well as at the site of vaccination, indicating the induction of a systemic antitumor immune response which reached the central nervous system. Our findings suggest the feasibility of applying the intensified vaccination strategy to treat human cancers including malignant brain tumors.

3T3 Cells↗
PubMed1997

Pleiotropic expression of heterologous cytokine/receptor genes in HTLV-1 associated diseases: candidate TRS for chimeric gene therapy.

DNA motifs that encode for specific transcriptional regulatory sequences (TRS) when engineered adjacent to the structural protein coding domain of a suicide enzyme can provide cell-lineage specific protein expression. The disparate up-regulation of several genes in adult T-cell leukemia (ATL) versus HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP), seropositive carriers (SPC) and uninfected normals may reflect events at the molecular level related to leukemogenesis or to processes maintaining the heme-oncologic phenotype. Further, the genetic transduction of cytokine and receptor genes uniquely associated with ATL may provide targets for the development of leukemia-specific gene therapies aimed at exploiting differences in the production of certain growth factors and growth factor receptors. Comparisons of the transcriptional and translational levels of interleukin-2 receptor alpha chain (IL-2R alpha), transforming growth factor-beta 1 (TGF-beta 1) and intracellular adhesion molecule-1 (ICAM-1) in ATL, HAM/TSP, and SPC and in several control populations revealed selectively up-regulated expression in ATL. We evaluated the feasibility of using lymphoid-specific TRS to activate herpes simplex virus thymidine kinase (HSVtk) to achieve selective cytotoxicity in leukemias expressing terminal deoxynucleotidyl transferase (TdT). Selective and efficient leukemic cell killing was produced and suggests that similar chimeric gene constructs containing TRS elements for IL-2R alpha, TGF-beta 1, or ICAM-1 may prove useful in designing gene therapies to treat ATL.

Adult↗
PubMed1997

In situ gene transfer into rat auxiliary liver transplant.

BACKGROUND: A replication-defective retrovirus BAG vector was tested for in situ delivery of the beta-galactosidase gene to auxiliary liver transplant in a rat model. METHODS: The BAG vector, which was shown to be effective in genetic transduction of cultured NIH/3T3 cells, was produced in a psi2 packaging cell and later amplified in a selected PA317 clone. Hepatocyte replication was induced by one-third hepatectomy of the donor liver, and the procedure was followed by auxiliary partial liver transplantation. Twenty-four hours after hepatic induction or transplantation, viral supernatant at 37 degrees C was perfused into the liver graft via the portal vein during a temporary occlusion of the graft portal vein. RESULTS: All animals survived the transplantation procedures and were killed at specified time intervals. Histochemical staining of the liver graft specimens indicated the expression of beta-galactosidase in the gene transferred group but not in the control animals. As demonstrated by polymerase chain reaction assay, the proviral beta-galactosidase sequence was present in the graft specimens, but absent from all other tissues tested. CONCLUSIONS: In short, the retrovirus BAG vector can be useful for in situ delivery of foreign genes to liver graft in transplantation and other clinical settings, providing a simple, consistent, and reliable alternative in hepatic gene therapy experiments.

3T3 Cells↗
PubMed1998

Glutamate-modulated production of GABA in immortalized astrocytes transduced by a glutamic acid decarboxylase-expressing retrovirus.

Replication-defective Moloney murine leukemia virus expressing the GAD67 gene under the control of the GFAP promoter was produced using selected clones of a fibroblast-packaging cell line. A spontaneously immortalized astrocyte cell line was infected with this virus and cellular clones expressing GAD67 selected. Astrocyte and fibroblast clones expressed functional GAD (detected by glutamic acid decarboxylation), but only fibroblasts were able to also produce GABA in the extracellular medium. When exposed to 200 microM glutamate, despite an observed difference in the rates of glutamate accumulation in control and GAD67-expressing astrocytes, similar proportions of glutamate taken up were detected. In GAD67-expressing astrocytes, the glutamate was mainly converted into GABA, suggesting GAD transgene activity to be dominant over other glutamate metabolic pathways, such as glutamine synthetase and glutamate dehydrogenase. Moreover, rapid GABA release into the cell medium was also observed, suggesting the involvement of reverse GABA transporters. The use of the GFAP promoter might be able to take advantage of its activation in response to factors inducing reactive gliosis observed in pathological insults. GAD67-expressing astrocytes might therefore be used for future grafting in pathological situations in which an excess of glutamate results in neuronal dysfunction or cell death.

4-Aminobutyrate Transaminase↗