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)

Vibrio cholerae neuraminidase (VCN) treatment of donor bone marrow cells results in a reduction in the number of hematopoietic colonies (CFUs) formed in the spleens of lethally irradiated mice. Treatment of marrow cells with sodium periodate under mild conditions, known to preferentially oxidize sialic acid, also reduced CFUs while subsequent potassium borohydride reduction restored CFUs to 80% of control levels. Innoculum viability as measured by in vitro incorporation of tritiated precursors into proteins, nucleic acids, and oligosaccharides was unaffected by VCN treatment. The ability of bone marrow cells in culture to respond to the hormone erythropoietin, as measured by the incorporation of 59Fe into cyclohexanone-extractable heme, was also not affected by neuraminidase, making a cytotoxic effect of the VCN preparation unlikely. Incubation of VCN-treated marrow with either beta-galactosidase or trypsin had no effect on the VCN-induced reduction in CFUs. These results are consistent with the idea that membrane sialic acid plays a direct and specific role in the implantation and development of CFUs.
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PMID:Sialic acid: a specific role in hematopoietic spleen colony formation. 73 8

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

A myoblast gene transfer approach was developed to deliver human erythropoietin (EPO) systemically. We created stable, high-level EPO-producing muscle cell clones by transfecting C2 myoblasts with a plasmid-bearing human EPO cDNA driven by cytomegalovirus enhancer/promoter and selection by G418. Eleven clones secreted EPO into the media as detected by radioimmunoassay. In vitro bioassay using the EPO-dependent human leukemic cell line UT-7/Epo confirmed the functional activity of the secreted EPO. After transplantation of 4 x 10(7) cells from C2-EPO9, the highest producing clone, the hematocrit increased from 43.4 +/- 2.8 to 56.1 +/- 2.7 (%) in 2 weeks in C3H mice that are syngeneic to C2 cells, and from 44.6 +/- 3.0 to 71.2 +/- 7.9 in nude mice. The increased hematocrit gradually returned to the basal level in 4-5 weeks in C3H mice, while it was sustained for at least 12 weeks in nude mice. Human EPO concentrations in the sera from transplanted nude mice were persistently high (31 +/- 24 mU/ml) at 12 weeks. C2 cells transduced with a retrovirus bearing beta-galactosidase gene were transplanted into nude mice, which showed X-Gal-positive myofibers in the transplanted area 3 months after the transplantation. These results demonstrate that myoblast gene transfer can successfully deliver functional human EPO capable of driving sustained erythropoiesis in mice. Thus, long-term EPO delivery for anemic patients may be feasible by myoblast gene transfer.
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PMID:Persistent erythropoiesis by myoblast transfer of erythropoietin cDNA. 789 6

Bicistronic retroviral vectors were constructed containing the foot-and-mouth disease virus (FMDV) internal ribosome entry site (IRES) followed by the coding region of beta-galactosidase (beta-gal) or therapeutic genes, with the selectable neomycin phosphotransferase gene under the control of the viral long terminal repeat (LTR) promoter. LNFX, a vector with a multiple cloning site 3' to foot-and-mouth disease virus IRES, was used to construct vectors encoding rat erythropoietin (EP), rat granulocyte colony-stimulating factor (G-CSF), human adenosine deaminase (ADA) and beta-gal. In transduced primary rat vascular smooth muscle cells the cytokines were expressed at high levels, similar to those obtained from vectors employing the viral LTR promoter. LNFZ, a vector encoding beta-gal, had a 10-fold increase in titer over that of LNPoZ, a comparable vector containing the poliovirus (Po) internal ribosome entry site. Primary canine vascular smooth muscle cells infected with LNFZ and LNPoZ expressed similar activities of beta-gal and neomycin phosphotransferase (NPT). Overall, these vectors had titers between 10(6) and 2 x 10(7) c.f.u./ml, indicating that foot-and-mouth disease virus IRES provides high-titer bicistronic vectors with high-level two gene expression.
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PMID:High-titer bicistronic retroviral vectors employing foot-and-mouth disease virus internal ribosome entry site. 875 98

Somatic gene therapy has been proposed as a means to achieve systemic delivery of therapeutic proteins. However, there is limited evidence that current methods of gene delivery can practically achieve this goal. In this study, we demonstrate that, following a single intramuscular administration of a recombinant adeno-associated virus (rAAV) vector containing the beta-galactosidase (AAV-lacZ) gene into adult BALB/c mice, protein expression was detected in myofibers for at least 32 weeks. A single intramuscular administration of an AAV vector containing a gene for human erythropoietin (AAV-Epo) into mice resulted in dose-dependent secretion of erythropoietin and corresponding increases in red blood cell production that persisted for up to 40 weeks. Primary human myotubes transduced in vitro with the AAV-Epo vector also showed dose-dependent production of Epo. These results demonstrate that rAAV vectors are able to transduce skeletal muscle and are capable of achieving sustained expression and systemic delivery of a therapeutic protein following a single intramuscular administration. Gene therapy using AAV vectors may provide a practical strategy for the treatment of inherited and acquired protein deficiencies.
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PMID:Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein. 894 64

RAS mutations arise at high frequency (20-40%) in both acute myeloid leukemia and myelodysplastic syndrome (which is considered to be a manifestation of preleukemic disease). In each case, mutations arise predominantly at the N-RAS locus. These observations suggest a fundamental role for this oncogene in leukemogenesis. However, despite its obvious significance, little is known of how this key oncogene may subvert the process of hematopoiesis in human cells. Using CD34+ progenitor cells, we have modeled the preleukemic state by infecting these cells with amphotropic retrovirus expressing mutant N-RAS together with the selectable marker gene lacZ. Expression of the lacZ gene product, beta-galactosidase, allows direct identification and study of N-RAS-expressing cells by incubating infected cultures with a fluorogenic substrate for beta-galactosidase, which gives rise to a fluorescent signal within the infected cells. By using multiparameter flow cytometry, we have studied the ability of CD34+ cells expressing mutant N-RAS to undergo erythroid differentiation induced by erythropoietin. By this means, we have found that erythroid progenitor cells expressing mutant N-RAS exhibit a proliferative defect resulting in an increased cell doubling time and a decrease in the proportion of cells in S + G2M phase of the cell cycle. This is linked to a slowing in the rate of differentiation as determined by comparative cell-surface marker analysis and ultimate failure of the differentiation program at the late-erythroblast stage of development. The dyserythropoiesis was also linked to an increased tendency of the RAS-expressing cells to undergo programmed cell death during their differentiation program. This erythroid lineage dysplasia recapitulates one of the most common features of myelodysplastic syndrome, and for the first time provides a causative link between mutational activation of N-RAS and the pathogenesis of preleukemia.
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PMID:Mutant N-RAS induces erythroid lineage dysplasia in human CD34+ cells. 910 20

Aryl hydrocarbon receptor nuclear translocator (ARNT) is a component of the transcription factors, aryl hydrocarbon receptor (AhR) and hypoxia-inducible factor 1, which transactivate their target genes, such as CYP1A1 and erythropoietin, in response to xenobiotic aromatic hydrocarbons and to low O2 concentration, respectively. Since ARNT was isolated as a factor required for the nuclear translocation of AhR from the cytoplasm in response to xenobiotics, the subcellular localization of ARNT has been of great interest. In this investigation, we analyzed the subcellular distribution of ARNT using transient expression of a fusion gene with beta-galactosidase and microinjection of recombinant proteins containing various fragments of ARNT in the linker region of glutathione S-transferase/green fluorescent protein. We found a clear nuclear localization of ARNT in the absence of exogenous ligands to AhR, and identified the nuclear localization signal (NLS) of amino acid residues 39-61. The characterized NLS consists of 23 amino acids, and can be classified as a novel variant of the bipartite type on the basis of having two separate regions responsible for efficient nuclear translocation activity, but considerable deviation of the sequence from the consensus of the classical bipartite type NLSs. Like the well characterized NLS of the SV40 T-antigen, this variant bipartite type of ARNT NLS was also mediated by the two components of nuclear pore targeting complex, PTAC58 and PTAC97, to target to the nuclear rim in an in vitro nuclear transport assay.
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PMID:A nuclear localization signal of human aryl hydrocarbon receptor nuclear translocator/hypoxia-inducible factor 1beta is a novel bipartite type recognized by the two components of nuclear pore-targeting complex. 921 13

Recombinant adeno-associated virus (rAAV) vectors were evaluated for gene transfer into the skeletal muscle of adult immunocompetent mice. A study using a vector encoding nuclear localized beta-galactosidase (rAAV-nls-lacZ) examined: (i) the efficiency and duration of transgene expression; (ii) the status of the AAV genome in the transduced fibers; and (iii) the possibility of improving gene transfer by inducing muscle regeneration. In the absence of regeneration, the injection of 1.7 x 10(7) particles in the quadriceps resulted in gene transfer to 10-70% of myofibers. Histological analysis indicated that the vector was able to reach myofiber nuclei distant from the injection point. Cellular infiltrates were absent at early time points but became conspicuous in the vicinity of some positive fibers at 4-8 weeks and subsided by 26 weeks. Southern analysis indicated that one to three copies of the vector genome were present per cell genome equivalent. They were associated with high-molecular-weight DNA in the form of tandem oligomers or interlocked circles. Gene transfer was not facilitated in the regenerating muscle. Rather, an early inflammatory response resulted in the elimination of most positive fibers after 8 weeks. The presence of regenerated fibers with beta-galactosidase-positive nuclei suggested that myoblasts had been transduced and were able to fuse to form new fibers. Gene transfer in the absence of immune reactions against the transgene product was studied by injecting mice with a rAAV carrying the murine erythropoietin (mEpo) cDNA. Dose-dependent elevation in the hematocrit was measured for over 200 days and corresponded to 5- to 20-fold increases in plasma Epo levels. We conclude that AAV vectors efficiently and stably transduce post-mitotic muscle fibers and myoblasts in vivo.
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PMID:Efficient and stable adeno-associated virus-mediated transduction in the skeletal muscle of adult immunocompetent mice. 938 55

Hypoxia-inducible factor 1 alpha (HIF1alpha) and its related factor, HLF, activate expression of a group of genes such as erythropoietin in response to low oxygen. Transfection analysis using fusion genes of GAL4DBD with various fragments of the two factors delineated two transcription activation domains which are inducible in response to hypoxia and are localized in the C-terminal half. Their sequences are conserved between HLF and HIF1alpha. One is designated NAD (N-terminal activation domain), while the other is CAD (C-terminal activation domain). Immunoblot analysis revealed that NADs, which were rarely detectable at normoxia, became stabilized and accumulated at hypoxia, whereas CADs were constitutively expressed. In the mammalian two-hybrid system, CAD and NAD baits enhanced the luciferase expression from a reporter gene by co-transfection with CREB-binding protein (CBP) prey, whereas CAD, but not NAD, enhanced beta-galactosidase expression in yeast by CBP co-expression, suggesting that NAD and CAD interact with CBP/p300 by a different mechanism. Co-transfection experiments revealed that expression of Ref-1 and thioredoxin further enhanced the luciferase activity expressed by CAD, but not by NAD. Amino acid replacement in the sequences of CADs revealed a specific cysteine to be essential for their hypoxia-inducible interaction with CBP. Nuclear translocation of thioredoxin from cytoplasm was observed upon reducing O2 concentrations.
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PMID:Molecular mechanisms of transcription activation by HLF and HIF1alpha in response to hypoxia: their stabilization and redox signal-induced interaction with CBP/p300. 1020 54

Intramuscular administration of plasmid DNA is a promising strategy to express therapeutic genes, however, it is limited by a relatively low level of gene expression. We report here that a non-ionic carrier, SP1017, composed of two amphiphilic block copolymers, pluronics L61 and F127, also known as poloxamers, significantly increases intramuscular expression of plasmid DNA. Two reporter genes, luciferase and beta-galactosidase, and one therapeutic gene, erythropoietin, were injected intramuscularly with and without SP1017 into C57Bl/6 and Balb/C mice and Sprague-Dawley rats. SP1017 increased gene expression by about 10-fold and maintained higher gene expression compared with naked DNA. Comparison of SP1017 with polyvinyl pyrrolidone (PVP) showed that SP1017 exhibited a significantly higher efficacy and its optimal dose was 500-fold lower. Experiments with beta-galactosidase using X-gal staining suggested that SP1017 considerably increased plasmid DNA diffusion through the tissue. SP1017 also improved expression of the erythropoietin gene leading to an increase in its systemic level and hematocrits. Previous toxicity studies have suggested that SP1017 has over a 1000-fold safety margin. Poloxamers used in SP1017 are listed in the US Pharmacopeia as inactive excipients and are widely used in a variety of clinical applications. We believe that the described system constitutes a simple and efficient gene transfer method to achieve local or systemic production of therapeutic proteins.
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PMID:A combination of poloxamers increases gene expression of plasmid DNA in skeletal muscle. 1084 59


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