Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A cloned cDNA that encodes human reticulocyte 15-lipoxygenase was characterized by Northern blot analysis and heterologous expression in bacteria. The 2.7 kb cDNA specifically hybridizes to reticulocyte RNA from anemic rabbits. The RNA levels correlate with the appearance of enzymatic activity in anemia. The cDNA was subcloned into an inducible bacterial expression vector in frame with the amino terminal ten amino acids of beta-galactosidase (pUCLOX). The soluble fraction of the cell lysate of E. coli transformed with pUCLOX contained a protein that has the same catalytic and antigenic characteristics as reticulocyte 15-lipoxygenase.
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PMID:The expression of active human reticulocyte 15-lipoxygenase in bacteria. 169 10

Anemia is an invariable consequence of end-stage renal failure (ESRF) and recombinant erythropoietin has dramatically improved the quality of life of patients with ESRF. As an alternative approach, we developed a myoblast gene transfer system for the systemic delivery of human erythropoietin (EPO). We recently reported that transplantation of 4 x 10(7) cells of a C2 myoblast cell clone that stably secretes high level of functional human EPO, increased hematocrit from 44.6 +/- 3.0 to 71.2 +/- 7.9(%) in 2 wk, and the increase was sustained for at least 12 wk in nude mice. A renal failure model was created by a two-step nephrectomy in nude mice, and myoblasts were transplanted 3 wk after the second nephrectomy, when mean blood urea nitrogen level had increased from 26.3 +/- 6.1 to 85.4 +/- 24.0 (mg/dl) and the hematocrit had decreased from 45.2 +/- 2.7 to 33.9 +/- 3.7(%). After transplantation, the hematocrit markedly increased to 68.6 +/- 4.2(%) 2 wk, and to 68.5 +/- 4.0(%) 7 wk after the transplantation. Serum human EPO concentration determined by ELISA indicated a persistent steady EPO production from the transplanted muscle cells 8 wk after the transplantation. The fate of transplanted myoblasts in uremic mice was monitored by transplanting the EPO-secreting clone which had also been transduced with BAG retrovirus bearing the beta-galactosidase gene. 8 wk later, X-gal positive myofibers were detected in the entire transplanted area. The results demonstrate that myoblasts can be transplanted in uremic mice, and that myoblast gene transfer can achieve sufficient and sustained delivery of functionally active EPO to correct anemia associated with renal failure in mice.
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PMID:Myoblast transfer of human erythropoietin gene in a mouse model of renal failure. 770 87

The genomic DNA of chicken anemia virus (CAV) was cloned and sequenced from a Japanese isolate CAA82-2. The nucleotide sequence of CAA82-2 isolate was 98% identical with that of the European Cuxhaven-1 strain (Noteborn et al., J. Virol. 65, 3131-3139, 1991). Nine open reading frames (ORFs) consisting of more than 100 nucleotides were found, i.e., four ORFs (CA1-CA4) on the plus strand and five ORFs (CA1R-CA5R) on the minus strand. These ORFs with the exception of CA4 are conserved between the two CAV isolates. All of these ORFs were expressed in Escherichia coli as fusion proteins with beta-galactosidase. By Western blot analysis, the CA2 and CA3 fusion proteins were found to react with CAV-infected chicken sera. Rabbit hyperimmune sera against the CA1, CA2, and CA3 fusion proteins were produced and tested their reactivity to CAV-infected cells. Two viral proteins with the apparent size of 54 and 16 kDa reacted with the antibodies against CA1 and CA3 fusion proteins, respectively. The 16-kDa protein, CA3, was suggested to be a major immunogen on CAV infection.
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PMID:Gene organization of chicken anemia virus. 777 81

The hematopoietic stem cell has long been considered an ideal target for the introduction of therapeutic genes to treat human disorders such as Fanconi anemia (FA). Although recent progress in large animal models is encouraging, application to nonmalignant conditions is limited by the perceived necessity of myeloablative conditioning. We and others have shown that very low irradiation doses are sufficient to allow significant hematopoietic engraftment in murine hosts even after the introduction of xenogeneic genes. To determine the degree of engraftment of genetically modified cells attainable with very low irradiation doses in larger animals, we employed the rhesus macaque competitive repopulation model. Four animals underwent mobilization with stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) followed by apheresis. The apheresis product was enriched for the CD34-positive fraction by immunomagnetic selection and split equally for transduction with either G1FC26, a retroviral vector carrying the Fanconi anemia complementation group C gene, or PLII, a nonexpression control retroviral vector carrying both neomycin and beta-galactosidase gene sequences modified to prevent translation. Transductions were performed daily in the presence of fresh IL-3, IL-6, SCF, and Flt-3 ligand on fibronectin-coated plates over 96 h. Animals were conditioned with a single dose of either 100 (n = 2) or 200 (n = 2) cGy and received the combined products of transduction on the following day. None of the animals experienced clinically significant neutropenia nor required the use of central line placement, transfusional support with blood products, or intravenous antibiotics. Using real-time PCR, circulating levels of genetically modified cells as high as 1% were initially detected. Stable, albeit, significantly lower levels from both vector-transduced aliquots (<0.1%) persisted beyond 12 months posttransplant in all four animals. Although not sufficient to correct the phenotype in many human disorders, stable low-level engraftment by genetically modified cells following low-intensity conditioning may prove adequate in disorders such as FA due to the selective advantage conferred upon corrected cells.
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PMID:Persistent low-level engraftment of rhesus peripheral blood progenitor cells transduced with the fanconi anemia C gene after conditioning with low-dose irradiation. 1140 5

Inefficient gene transfer, inaccessibility of stem cell compartments, transient gene expression, and adverse immune and inflammatory reactions to vector and transgenic protein are major barriers to successful in vivo application of gene therapy for most genetic diseases. Prenatal gene therapy with integrating vectors may overcome these problems and prevent early irreparable organ damage. To this end, high-dose attenuated VSV-G pseudotyped equine infectious anaemia virus (EIAV) encoding beta-galactosidase under the CMV promoter was injected into the fetal circulation of immuno-competent MF1 mice. We saw prolonged, extensive gene expression in the liver, heart, brain and muscle, and to a lesser extent in the kidney and lung of postnatal mice. Progressive clustered hepatocyte staining suggests clonal expansion of cells stably transduced. We thus provide proof of principle for efficient gene delivery and persistent transgene expression after prenatal application of the EIAV vector and its potential for permanent correction of genetic diseases.
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PMID:Long-term transgene expression by administration of a lentivirus-based vector to the fetal circulation of immuno-competent mice. 1285 88

Gene therapy for Duchenne muscular dystrophy has so far not been successful because of the difficulty in achieving efficient and permanent gene transfer to the large number of affected muscles and the development of immune reactions against vector and transgenic protein. In addition, the prenatal onset of disease complicates postnatal gene therapy. We have therefore proposed a fetal approach to overcome these barriers. We have applied beta-galactosidase expressing equine infectious anaemia virus (EIAV) lentiviruses pseudotyped with VSV-G by single or combined injection via different routes to the MF1 mouse fetus on day 15 of gestation and describe substantial gene delivery to the musculature. Highly efficient gene transfer to skeletal muscles, including the diaphragm and intercostal muscles, as well as to cardiac myocytes was observed and gene expression persisted for at least 15 months after administration of this integrating vector. These findings support the concept of in utero gene delivery for therapeutic and long-term prevention/correction of muscular dystrophies and pave the way for a future application in the clinic.
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PMID:Highly efficient EIAV-mediated in utero gene transfer and expression in the major muscle groups affected by Duchenne muscular dystrophy. 1514 Nov 56

Nutrient deprivation during ischemia leads to severe insult to neurons causing widespread excitotoxic damage in specific brain regions such as the hippocampus. One possible strategy for preventing neurodegeneration is to express therapeutic proteins in the brain to protect against excitotoxicity. We investigated the utility of equine infectious anemia virus (EIAV)-based vectors as genetic tools for delivery of therapeutic proteins in an in vivo excitotoxicity model. The efficacy of these vectors at preventing cellular loss in target brain areas following excitotoxic insult was also assessed. EIAV vectors generated to overexpress the human antiapoptotic Bcl-2 or growth factor glial-derived neurotrophic factor (GDNF) genes protected against glutamate-induced toxicity in cultured hippocampal neurons. In an in vivo excitotoxicity model, adult Wistar rats received a unilateral dose of the glutamate receptor agonist N-methyl-D-aspartate to the hippocampus that induced a large lesion in the CA1 region. Neuronal loss could not be protected by prior transduction of a control vector expressing beta-galactosidase. In contrast, EIAV-mediated expression of Bcl-2 and GDNF significantly reduced lesion size thus protecting the hippocampus from excitotoxic damage. These results demonstrate that EIAV vectors can be effectively used to deliver putative neuroprotective genes to target brain areas and prevent cellular loss in the event of a neurological insult. Therefore these lentiviral vectors provide potential therapeutic tools for use in cases of acute neurotrauma such as cerebral ischemia.
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PMID:Lentiviral-mediated delivery of Bcl-2 or GDNF protects against excitotoxicity in the rat hippocampus. 1558 9

Hematopoietic cells are often exposed to transient hypoxia and reoxygenation as they develop and migrate. Given that bone marrow (BM) failure occurred in patients with Fanconi anemia (FA), we reason that hypoxia-then-reoxygenation represents a physiologically relevant stress for FA hematopoietic progenitor/stem cells. Here we show that expansion of Fancc-/- BM cells enriched for progenitor and stem cells was significantly decreased after 2 continuous cycles of hyperoxic-hypoxic-hyperoxic treatments compared with wild-type (WT) BM cells. This inhibition was attributable to a marked decrease of lineage-depleted (Lin-) ScaI- c-kit+ cells and more primitive Lin- ScaI+ c-kit+ cells in Fancc-/- BM cells following reoxygenation. Evaluation of the cell-cycle profile of long-term BM culture (LTBMC) revealed that a vast majority (70.6%) of reoxygenated Fancc-/- LTBMC cells was residing in the G0 and G1 phases compared with 55.8% in WT LTBMC cells. Fancc-/- LTBMC cells stained intensely for SA-beta-galactosidase activity, a biomarker for senescence; this was associated with increased expression of senescence-associated proteins p53 and p21(WAF1/CIP1). Taken together, these results suggest that reoxygenation induces premature senescence in Fancc-/- BM hematopoietic cells by signaling through p53, up-regulating p21, and causing senescent cell-cycle arrest. Thus, reoxygenation-induced premature senescence may be a novel mechanism underlying hematopoietic cell depletion and BM failure in FA.
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PMID:Hypoxia-reoxygenation induces premature senescence in FA bone marrow hematopoietic cells. 1576 96

The present study examines gene delivery to cultured motor neurons (MNs) with the Rabies G protein (RabG)-pseudotyped lentiviral equine infectious anemia virus (RabG.EIAV) vector. RabG.EIAV-mediated beta-galactosidase (RabG.EIAV-LacZ) gene expression in cultured MNs plateaus 120 h after infection. The rate and percent of gene expression observed are titer-dependent (P < 0.001). The rat IGF-I cDNA sequence was then cloned into a RabG.EIAV vector (RabG.EIAV-IGF-I) and was shown to induce IGF-I expression in HEK 293 cells. MNs infected with RabG.EIAV-IGF-I demonstrate enhanced survival compared to MNs infected with RabG.EIAV-LacZ virus (P < 0.01). In addition, IGF-I expression in cultured MNs induced profound MN axonal elongation compared to control virus (P < 0.01). The enhanced motor neuron tropism of RabG.EIAV previously demonstrated in vivo, together with the trophic effects of RabG.EIAV-IGF-I MN gene expression may lend this vector to therapeutic application in motor neuron disease.
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PMID:Trophic activity of Rabies G protein-pseudotyped equine infectious anemia viral vector mediated IGF-I motor neuron gene transfer in vitro. 1600 36

Klotho, an antiaging gene with restricted organ distribution, is mainly expressed in the kidney tubules; the mutant mice have shortened life span, arteriosclerosis, anemia, and osteoporesis, features common to patients with chronic renal failure. Conceivably, the reduction of the Klotho gene expression may contribute to the development of kidney failure; alternatively, its overexpression may lead to the amelioration of renal injury in an ICR-derived glomerulonephritis (ICGN) mouse model with subtle immune complex-mediated disease. To address this issue, four different strains of mice were generated by cross-breeding: ICGN mice without the Klotho transgene (ICGN), ICGN mice with the Klotho transgene (ICGN/klTG), wild-type mice with the Klotho transgene (klTG), and wild-type mice without the Klotho transgene (control). At 40 weeks old, the survival rate was approximately 30% in ICGN mice, and approximately 70% in the ICGN/klTG group. This improvement was associated with dramatic improvement in renal functions, morphological lesions, and cytochrome c oxidase activity but a reduction in beta-galactosidase activity (a senescence-associated protein), mitochondrial DNA fragmentation, superoxide anion generation, lipid peroxidation, and Bax protein expression and apoptosis. Interestingly, improvement was seen in both the tubular and glomerular compartments of the kidney, although Klotho is exclusively confined to the tubules, suggesting that its gene product has a remarkable renoprotective effect by potentially serving as a circulating hormone while mitigating the mitochondrial oxidative stress.
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PMID:Amelioration of progressive renal injury by genetic manipulation of Klotho gene. 1728 45


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