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)

Although thrombopoietin (TPO) is known to play a fundamental role in both megakaryopoiesis and thrombopoiesis, the molecular mechanism of TPO-induced megakaryocytic differentiation is not known. In a human megakaryoblastic leukemia cell line, CMK, that showed some degree of megakaryocytic differentiation after culture with TPO, the cyclin-dependent kinase (Cdk) inhibitor p21(WAF1/Cip1), but not p27(Kip1), p16(INK4A), p15(INK4B), or p18(INK4C), was found to be upregulated in an immediately early response to TPO. The expression of p21 was found to be sustained over a period of 5 days by treatment with TPO in large polyploid cells that developed in response to TPO, but not in small undifferentiated cells, indicating a close correlation between the ligand-induced differentiation and p21 induction in CMK cells. To examine potential roles of Cdk inhibitors in megakaryocytic differentiation, CMK cells were transfected with the p21, p27, or p16 gene, together with a marker gene, beta-galactosidase, and were cultured with medium alone for 5 days. The ectopic expression of p21 or p27 but not of p16 led to induction of megakaryocytic differentiation of CMK cells. Overexpression of the N-terminal domain (amino acids [aa] 1 to 75) of p21 was sufficient to induce megakaryocytic differentiation, whereas that of the C-terminal domain (aa 76 to 164) had little or no effect on morphological features. Furthermore, we found that although TPO induced tyrosine phosphorylation of both STAT3 and STAT5 in CMK cells, only STAT5 showed binding activities to potential STAT-binding sites that locate in the promoter region of p21 gene (p21-SIE sites), thereby leading to transactivation of p21. These results suggested that p21 induction, possibly mediated through activated STAT5, could play an important role in TPO-induced megakaryocytic differentiation.
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PMID:Thrombopoietin-induced differentiation of a human megakaryoblastic leukemia cell line, CMK, involves transcriptional activation of p21(WAF1/Cip1) by STAT5. 911 65

The herpes simplex virus type 1 (HSV-1) mutant in 1814 contains an insertion mutation in the coding sequence for the virion transactivator protein VP16 and is thus impaired for the activation of immediate early (IE) gene expression. This virus was modified further by introducing the Moloney murine leukemia virus LTR promoter in place of the upstream sequences controlling expression of the IE regulatory protein ICPO, to yield mutant in 1820. In almost all cell types tested, in 1820 initiated infection less efficiently than in 1814, behaving as if lacking both VP16 and ICPO functions, but in BHK cells in 1820 was less impaired than in 1814. A rescuant of in 1820 at the VP16 locus, in 1825, also exhibited a host range phenotype, initiating replication as efficiently as wild-type HSV-1 in BHK cells but inefficiently in other cell types. In 1825 was unable to complement an ICPO null mutant in restricted cells, demonstrating that the promoter exchange prevented the expression of ICPO protein in functionally significant amounts. The novel host range properties of in 1820 provided a basis for the construction of additional viruses conditionally impaired for IE gene expression and assessment of their value as prototype vectors. Production of an HSV-1 mutant multiply defective in the expression of IE gene products was achieved by introduction of the temperature-sensitive mutation of HSV-1 tsK, which inactivates the IE transcription activator ICP4 at nonpermissive temperatures, into in 1820 to produce in 1820K. This mutant could be propagated effectively in BHK cells at 31 degrees but was effectively devoid of the major regulators ICPO, ICP4, and VP16 in other cells types at 38.5 degrees. Cultures could withstand infection with 5 PFU of in 1820K per cell without detectable cytopathology and could be reseeded to form colonies at approximately 90% efficiency. A derivative of in 1820K containing the Escherichia coli lacZ gene controlled by the human cytomegalovirus (HCMV) major IE promoter expressed low but detectable levels of beta-galactosidase in almost all cells after infection of cultures at 5 PFU per cell and incubation at 38.5 degrees. Cultures infected with 5 PFU per cell of an in 1820K derivative expressing neomycin phosphotransferase (npt) controlled by the HCMV IE promoter were resistant to killing by the antibiotic G418 for up to 3 days, and cell survival correlated with the retention of functional levels of npt. Mutants based on in 1820K can thus express foreign gene products in virtually all cells in a culture under conditions in which cytotoxicity is eliminated, demonstrating that progressive reduction of IE gene expression is an important step in the design of HSV-1-derived vectors.
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PMID:Construction and characterization of herpes simplex virus type 1 mutants with conditional defects in immediate early gene expression. 912 65

To elucidate the cellular tropism of Moloney murine leukemia virus (MuLV), we have studied the transduction efficiency of a recombinant MuLV vector carrying the beta-galactosidase reporter gene on a variety of rodent cells. Under optimal conditions for in vitro cell transduction, primary cultures of adult rat fibroblasts derived from various organs were very poorly transduced by the ecotropic MuLV vector (0.02-0.12%) when compared to immortalized cells such as the F2408 (42%) and 3Y1 (defined as 100%) lines. Primary cultures of fibroblasts from neonatal (3.7%) or embryonic rat tissues (4.6%) and primary cultures of rat mammary epithelial cells (3-4%) were somewhat more susceptible. Immortalization of rodent fibroblasts with Polyomavirus Large T. SV40 Large T, and E6-E7 genes of human papilloma virus resulted in a modest or minimal increase in transduction efficiency, and introduction of the transforming genes v-Src, v-Ras, and v-Raf was in most instances associated with a decrease in MuLV vector entry. Variability of transduction efficiency was not related to differences in cellular growth rate and treatment of MuLV vectors in vitro with deoxyribonucleoside triphosphates and treatment of cells in culture with protease inhibitors failed to modify cellular entry of the MuLV vector. On the other hand, inhibition of cellular glycosylation with swansonine, 1-deoxymannojirimycin and, primarily, tunicamycin enhanced entry of the ecotropic vector by up to 220-fold, particularly into cells which were otherwise highly resistant. These findings demonstrate major differences in transduction efficiency of the ecotropic MuLV vector on rodent cells and indicate that cellular glycosylation plays a critical role in determining MuLV cellular tropism.
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PMID:Variable transduction efficiency of murine leukemia retroviral vector on mammalian cells: role of cellular glycosylation. 912 77

One problem in devising strategies of gene transfer to the nervous system is targeting specific neuronal populations. To evaluate the potential for using herpes simplex virus (HSV) as a vector for gene transfer to spinal cord motor neurons, the HSV-1 mutant LAT-LTR in which the E. coli beta-galactosidase gene is expressed under control of the HSV LAT core promoter (LAT) and the Moloney murine leukemia virus long terminal repeat (LTR) was inoculated unilaterally into the gastrocnemius muscle. Infectious virus was isolated from the spinal cord on days 3-7 post inoculation (PI). Immunocytochemical labeling of HSV antigen was detected in ipsilateral ventral horn neurons in the spinal cord at day 3 PI and had spread to contiguous spinal cord regions by day 6 PI. No viral antigen was detected at 14 or 28 DPI. beta-galactosidase expression (driven by the LAT-LTR promoter) was detected in neurons of the ventral horn on days 3, 6, 14, and 28 PI. Histological analysis showed mild lesions in the ventral horn on day 3 PI which progressed through days 6, 14 and 28 PI. This study demonstrates the feasibility of gene delivery into spinal motor neurons after injection of an HSV vector at a peripheral muscular site. This approach should prove useful in neurobiological investigations and it suggests a possible application to development of gene therapy for heritable diseases affecting motor neurons.
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PMID:Targeting and gene expression in spinal cord motor neurons following intramuscular inoculation of an HSV-1 vector. 922 64

Vascular cells are an important target for gene transfer because of their potential to deliver gene products both locally and systemically. Direct retroviral gene transfer to vascular cells in vivo has been limited by inefficient rates of transduction. We hypothesized that vascular cell transduction efficiency (TE), during short retroviral incubation periods, is significantly improved in vitro and in vivo using centrifugation to increase viral titer. Furthermore, we hypothesized a linear relationship between concentration of viable viral particles (measured as colony-forming units (CFUs)/cell) and retroviral TE during short incubation periods. Cultured rat pulmonary artery endothelial cells (RPAECs), rat aortic smooth muscle cells (RSMCs), and human iliac artery endothelial cells (HIAECs) demonstrated a strong correlation between TE and high concentrations of virus (> 100 CFU/cell) during retroviral incubation periods of 10 to 60 minutes. High titers, and thereby high concentrations, were achieved by centrifugation and resuspension in a fraction of the original volume. Titers was consistently increased tenfold, for a twentyfold increase in concentration by volume. A 20-minute incubation with a Moloney murine leukemia-derived retroviral vector coding for human placental alkaline phosphatase, pLJhpAP, at a concentration of 1150 CFU/cell yielded TEs of 10.6% +/- 0.7%, 40.4% +/- 1.6%, and 15.1% +/- 2.0% for RPAECs, RSMCs, and HIAECs, respectively. A similar effect was shown using the Moloney murine leukemia-derived MFGlacZ retroviral vector, coding for Escherichia coli beta-galactosidase. Increased titer and concentration had no effect on target cell viability, as shown by trypan blue exclusion. Although RSMCs had the most cells transduced in a given incubation period (p < 0.05), RPAECs had the highest replication rate (p < 0.05), suggesting the importance of factors other than cell cycle on retroviral TEs during short, clinically relevant incubation periods. In subsequent in vivo experiments, gene transfer was achieved in the rat carotid artery during a 20-minute incubation period infusing the concentrated pLJhpAP retrovirus after carotid balloon injury. Rats infused with virus 2 days after balloon injury exhibited hpAP activity (0 to 10 cells/section/rat) in the neointima of five out of six rats. Rats infused 4 days after balloon injury exhibited hpAP activity (0 to 25 cells/section/rat) in the media and adventitia of five out of five rats. Control rats that received the balloon injury alone or the balloon injury and unconcentrated retrovirus exhibited zero hpAP activity. We conclude that the TE of retroviral-mediated gene transfer to vascular cells in vitro and in vivo can be improved during short, clinically relevant incubation periods using centrifugation to increase retroviral titer, and thereby concentration of viable viral particles.
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PMID:Improved retroviral transduction efficiency of vascular cells in vitro and in vivo during clinically relevant incubation periods using centrifugation to increase viral titers. 924 Mar 30

To evaluate the role of leukaemia inhibitory factor (LIF) for maintaining pluripotent embryonic stem (ES) cells in culture, we established several exogenous LIF-independent ES cell lines by continuous passaging in culture. The newly established ES cells, Kli and CBli, sustained their growth and remained undifferentiated in LIF-deficient medium. Analysis of chimaeric animals, produced with the beta-galactosidase transgenic Kli ES cells, revealed that LIF-independent ES cells can contribute to all embryonic germ layers. There was no detectable LIF protein in ES cell conditioned medium, and no upregulation of LIF mRNA was found. The addition of neutralising anti-LIF antibodies was not sufficient to abrogate the self renewal of the Kli ES cells. These studies suggest that the signalling pathway involving diffusible LIF can be bypassed for maintaining the pluripotency in culture, and indicate a considerable heterogeneity in growth factor dependence and differentiation of different ES cells.
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PMID:Self renewal of embryonic stem cells in the absence of feeder cells and exogenous leukaemia inhibitory factor. 925 6

Ballistic transfection, based on cell and tissue bombardment by the tungsten and gold microparticles covered with the gene DNA, was used for the delivery of a bacterial beta-galactosidase and a full-length cDNA copy of the human dystrophin genes into mouse skeletal muscles. CMV-lacZ, SV40-lacZ, LTR-lacZneo and full-length cDNA dystrophin (pDMD-1, approximately 16 kb) in eukaryotic expression vector pJ OMEGA driven by mouse leukaemia virus promotor (pMLVDy) were used throughout the studies. Musculus glutaeus superficialis of C57BL/6J and quadriceps femoris of mdx male mice were opened surgically under anesthesia and bombarded by means of the gene-gun technique originally developed by us. Different mixtures of gold and tungsten particles at ratios of 4:1, 1:1, 1:4 were applied. X-gal assay revealed marked beta-gal activity, both in total muscles and whole muscle fibers on histological sections, up to three months after transfection. The most intensive staining was observed after SV40-lacZ delivery. No staining was detected with LTR-lacZneo DNA as well as in untreated muscles. The higher tungsten particle concentration in the bombardment mixture correlated with more intense X-gal staining. At the gold/tungsten ratio of 1:4 the microparticles penetrated the musculus glutaeus superficialis and transfected the underlying musculus glutaeus medius as well. Immuno-cytochemical assay for human dystrophin revealed dystrophin positive myofibers (DPM) in the bombarded area up to two months after transfection. The proportion of DMP varied from 2.5% on day 17 up two 5% on day 60 after bombardment compared to only 0.5% in the control mdx mice. These results suggest the applicability of particle bombardment for gene delivery into muscle fibers.
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PMID:Bacterial beta-galactosidase and human dystrophin genes are expressed in mouse skeletal muscle fibers after ballistic transfection. 931 10

We studied the efficiency of plasmid/liposome complexes, Moloney murine leukemia virus-derived (MMLV) retroviruses, pseudotyped vesicular stomatitis virus protein-G (VSV-G)-containing retroviruses, and adenoviruses in delivering genes into the rabbit carotid artery using a silastic collar applied to the adventitia. This method was used for gene transfer because (a) it provides a gene delivery reservoir; (b) no intraluminal manipulations are performed; (c) installation of the collar induces arterial smooth muscle cell (SMC) proliferation and enhances retroviral gene transfer efficiency where target cell proliferation is required. The transfer of the beta-galactosidase (lacZ) marker gene to the adventitia and media occurred with all gene transfer systems. Adenoviruses also transferred the beta-galactosidase gene to some endothelial cells. After 5 days, adenoviral vectors produced the highest gene transfer efficiency with up to 10%+/-6% of cells showing beta-galactosidase activity. Pseudotyped VSV-G retroviruses were also effective in achieving gene transfer in 0.05%+/-0.03% of cells in the adventitia and media. Plasmid/liposome complexes and MMLV retroviruses infected 0.05%+/-0.03% and <0.01%+/-0.01% of cells, respectively. It is concluded that replication-deficient adenoviruses, VSV-G pseudotyped retroviruses, and plasmid/liposome complexes can be used for gene transfer to the arterial wall using the collar method. Because the endothelium remains anatomically present throughout the experiments, the model may be useful for the gene transfer studies involving diffusible or secreted gene products that primarily act on the endothelium. Effects on medial SMC and even endothelium can be achieved from the adventitial side, suggesting an alternative route for the delivery of therapeutically useful genes into the arterial wall.
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PMID:Gene transfer into the carotid artery using an adventitial collar: comparison of the effectiveness of the plasmid-liposome complexes, retroviruses, pseudotyped retroviruses, and adenoviruses. 932 66

We have investigated the feasibility of using high-titer murine leukemia virus-based retroviral vectors to deliver exogenous genes to naive and chronically inflamed knee joints of rabbits in vivo. Intraarticular injection of retrovirus encoding beta-galactosidase (beta-gal or lacZ) was found to transduce synoviocytes in both naive and inflamed joints, but a significantly higher number of lacZ+ cells were found in inflamed knees. Using a retrovirus encoding a secretable marker, human growth hormone (hGH), quantitative comparison of ex vivo and in vivo gene delivery methods demonstrated that transgene expression following in vivo gene transfer was at least equivalent to that of the ex vivo method in inflamed knees. In addition, hGH transgene expression was maintained for at least 4 weeks. These experiments suggest that high-titer retroviral vector could be used for efficient in vivo gene transfer to inflamed joints in patients with rheumatoid arthritis (RA).
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PMID:Direct retrovirus-mediated gene transfer to the synovium of the rabbit knee: implications for arthritis gene therapy. 934 35

Pantropic retroviral vectors were used to introduce transgenes into Japanese medaka (Oryzias latipes). These vectors contain the long terminal repeat (LTR) sequence of Moloney murine leukemia virus (Mo-MLV) and a reporter gene (neo or lacZ) regulated by the LTR sequence of rous sarcoma virus (RSV). Because these pseudotyped retroviral vectors contain the vesicular stomatitis virus envelope glycoprotein (VSV-G), they have an extremely broad host cell range and can infect many no mammalian species. Newly fertilized medaka eggs (intact or dechorionated) were electroporated at different voltage settings in the presence of 4 x 10(4) cfu of pantropic retroviral vector. The survival rates of the pantropic retroviral vector-treated embryos ranged from 65% to 20% with increasing amplitude of electroporation. Dechorionation did not substantially affect the survival rate of embryos. PCR amplification demonstrated proviral sequences in up to 60% of the 2-month-old fish. The efficiency of gene transfer was enhanced by dechorionation. Furthermore, overnight incubation of dechorionated embryos with pantropic retroviral vectors without electroporation also resulted in proviral integration in 60% of the embryos without compromising survival rate. Southern blot analysis of DNA samples isolated from polymerase chain reaction (PCR) as positive F1 reaction animals confirmed the integration of a single copy of the provirus into the host genome. Three P1 transgenic females transmitted the proviral sequence to 50% of their F1 progeny in a back cross with wild-type males, suggesting that the entire germline of these P1 fish was transformed by the pantropic retroviral vector. Expression of the neomycin phosphotranferase transgene in F1 transgenic individuals was detected by reverse transcription (RT)-PCR amplification of the neo mRNA sequence. Furthermore, expression of a beta-galactosidase transgene was also observed in 4-day-old F1 transgenic individuals. Thus, pantropic retroviral vectors provide a convenient method to stably introduce and express foreign genes in medaka.
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PMID:Pantropic retroviral vector integration, expression, and germline transmission in medaka (Oryzias latipes). 941 87


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