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)

One major concern about using adenoviral vectors for repetitive gene delivery to lung epithelial cells is the induction of an immune response to the vector, thus, impeding effective gene transduction. To assess the immune response to the adenoviral vector, repetitive intratracheal (i.t.) gene dosing was performed in CD-1 mice using the replication-deficient adenovirus 5 (Ade5) vector carrying the lacZ gene, and compared to the antibody responses induced by conventional intranasal (i.n.) and intraperitoneal (i.p.) routes of immunization. Kinetics of serum IgG, IgA, and IgM antibody responses to the adenoviral vector and to beta-galactosidase (beta-Gal) were evaluated. Two or three adenoviral vector doses given by i.t., i.n., or i.p. routes resulted in serum IgG titers in excess of 1:200,000, whereas serum IgM and IgA were moderately induced. Analysis of the predominant murine IgG subclass was determined to be IgG2b and IgG2a. To determine the localization of this antibody response, the ELISPOT assay was employed. Lymphocytes were isolated from the lung, the lower respiratory lymph nodes (LRLN), the nasal passages (NP), and the spleen. For i.t- and i.n.-administered mice, the highest IgA spot-forming cell (SFC) response to Ade5 and beta-Gal was located in the NP and in the lung. Both the lung and the LRLN showed elevated numbers of IgG SFCs (4- to 12-fold greater than splenic IgG SFC response) for Ade5 and beta-Gal. This evidence suggests that the lung and associated lymphoid tissues were the source for serum antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Intratracheal gene delivery with adenoviral vector induces elevated systemic IgG and mucosal IgA antibodies to adenovirus and beta-galactosidase. 757 8

The haematopoietic development of embryonic stem (ES) cell injection chimaeras was analysed using beta-galactosidase expression from an X-linked transgene as a marker to distinguish the ES-derived cell population from the host cells. The number of cells in the different haematopoietic cell subpopulations was determined by flow cytometry. When the proportions of ES-derived cells in the antigen-positive lineages were compared to the ES cell contribution to all cells in teh organs, we found an unexpected bias in the haematopoietic differentiation of ES-derived cells. ES descendants were overrepresented in the bone marrow B lymphoid cell population and the splenic myeloid cells but were underrepresented in the CD4-positive T lymphoid cells in the spleen. These results were obtained by comparison with control female animals that were X chromosome mosaic for beta-galactosidase expression. These findings of uneven contribution to haematopoietic development by ES cells indicate that the commitment of ES cell descendants may be different from that of the host cells.
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PMID:The development of haematopoietic cells is biased in embryonic stem cell chimaeras. 764 91

A 100-kDa DNA binding protein was found to be dramatically up-regulated upon the mitogenic stimulation of murine splenocytes with bacterial lipopolysaccharide (LPS). The induced DNA binding protein was also found to exhibit moderate binding specificity for the immunoglobulin isotype switch DNA repeats. Furthermore, the induction of the 100-kDa protein by LPS was found to be mediated by both an increase in the protein's stability and an increase in the synthesis of the protein. In vitro phosphorylation experiments revealed that the 100-kDa DNA binding protein was one of the most heavily phosphorylated proteins in both lymphoid and nonlymphoid nuclear extracts. Although this in vitro phosphorylation initially appeared to be mediated by a potent nuclear kinase activity, it was later determined that a significant part of the detected labeling was due to the direct binding of ATP by the 100-kDa protein. Antibodies raised to the 100-kDa DNA binding protein were used to isolate cDNA clones from a lymphocyte cDNA lambda gt11 expression library. Nucleotide sequence analysis revealed that the cloned cDNAs were identical to the mouse nucleolin gene. The beta-galactosidase fusion proteins (encoded by exons 3-14 of nucleolin) and a more severely truncated 45-kDa protein (encoded by exons 5-14 of nucleolin) were both found to bind strongly to DNA and ATP. Furthermore, the strength of DNA binding was found to be highly dependent on the overall dG content of the DNA probes. Our experiments also revealed that apart from binding ATP and G-rich DNA, nucleolin directly bound GTP, dATP, and dGTP, but not dCTP, dTTP, or dUTP. Computer analysis revealed that the putative ATP binding domains appear to fall within two of the phylogenetically conserved RNA binding domains of nucleolin.
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PMID:The murine nucleolin protein is an inducible DNA and ATP binding protein which is readily detected in nuclear extracts of lipopolysaccharide-treated splenocytes. 769 29

The quantitation of intracellular beta-galactosidase activity has been described for viable cells. By using the fluorogenic substrate fluorescein-di-beta-D-galactopyranoside (FDG) in conjunction with flow cytometry, the proportion of positive cells as well as the level of expression can be determined. In this paper we describe beta-galactosidase expression in lymphoid and myeloid cells from transgenic mice that widely express beta-galactosidase from an inserted lacZ transgene. Both foetal and adult haematopoietic tissues are able to express beta-galactosidase. The intracellular fluorescence reflecting beta-galactosidase activity can be readily combined with fluorescently labelled antibodies against cell surface antigens. Thus, beta-galactosidase can be used as a marker in transplantation experiments to study the development of lymphoid and myeloid precursor cells.
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PMID:Simultaneous detection of beta-galactosidase activity and surface antigen expression in viable haematopoietic cells. 785 Nov 57

Somatic mutations and drugs that either reduce beta 1-6GlcNAc-branching of N-linked oligosaccharides or block the addition of terminal sequences containing galactose and sialic acid have been shown to inhibit tumour growth and metastasis. In an attempt to further define the oligosaccharide sequences that contribute to the malignant phenotype, we have selected spontaneous wheat germ agglutinin-resistant (WGAR) mutants from highly metastatic murine lymphoid tumour cells and characterized four mutant phenotypes. Mutants were selected from VM4, a clone of the MDAY-D2 tumour cell line which had been transfected with the bacterial beta-galactosidase gene (LacZ). VM4 cells retained the malignant phenotype of MDAY-D2 and the cells expressed LacZ, which facilitated the counting of metastases as the tumour cells stained blue when incubated with 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal). The most frequently isolated mutant was defective in the transport of UDP-Gal into the Golgi, and as previously observed for this mutation, the cells were non-metastatic and produced very slow-growing solid tumours. Mutants expressing CMP-SA hydroxylase, and consequently glycoconjugates with N-glycolylneuraminic acid (NeuNGc), remained highly metastatic, but grew more slowly than VM4 cells as s.c. tumours in mice. A novel WGAR mutant showing a large increase in Gal beta 1-4GlcNAc:alpha 2-6 sialyltransferase (SA-T) mRNA levels (ST6N) and enzyme activity was observed to be less metastatic and also grew more slowly at the s.c. site of inoculation. Finally, a fourth phenotypic class of WGAR mutants showed a complex phenotype including expression of a beta Gal-binding cell surface lectin and reduced sialylation of glycoconjugates. These results suggest that changes in either the amount, the type or linkage of sialic acid in tumour cell glycoconjugates can affect tumour growth and metastasis.
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PMID:Sialylation and malignant potential in tumour cell glycosylation mutants. 788 Nov 81

Cell-surface sugar receptors may participate in interactions of lymphoid cells that influence their adhesive properties and proliferation. Their expression on cells of the pre-B line BLIN-I, the B-lymphoblastoid line Croco II, the myeloma line RPMI 8226 and the T-lymphoblastoid line CCRF-CEM was monitored with a panel of 14 types of chemically glycosylated E. coli beta-galactosidase at a non-saturating ligand concentration. Quantitative differences were determined for the capacity of the different cell types to bind constituents of the carbohydrate part of glycoconjugates. They were corroborated by analyses of binding for lactose-, beta-N-acetylgalactosamine-, beta-N-acetylglucosamine- and fucose-exposing neoglycoenzymes up to saturation levels. Values of dissociation constants of the tetrameric enzyme were in the range of 3-300 nM. Several types of sugar receptor led to carbohydrate-inhibitable adhesion of cells to 6 types of nitrocellulose-immobilized neoglycoprotein, their effectiveness being most obvious for the myeloma cells. Analyses of the carbohydrate-ligand-mediated adhesion of the other cell types revealed a comparatively decreased response. Only a few carbohydrates among the 7 types tested were effective in reducing cell adhesion to a far more complex ligand-bearing matrix than immobilized neoglycoproteins, namely bone-marrow stromal cell layers: sialic acid and N-acetylgalactosamine for B-lymphoblastoid cells and rhamnose for pre-B cells. These cellular interactions may encompass sugar receptors on the stromal cells and other types of molecular recognition in addition to the detected activities on the lymphoid cells.
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PMID:Adhesion of human lymphoid cell lines to immobilized carbohydrates and to bone-marrow stromal cell layers by surface sugar receptors. 839 77

Myxoma virus is a leporipoxvirus of New World rabbits (Sylvilagus sp.) that induces a rapidly lethal infection known as myxomatosis in the European rabbit (Oryctolagus cuniculus). Like all poxviruses, myxoma virus encodes a plethora of proteins to circumvent or inhibit a variety of host antiviral immune mechanisms. M-T7, the most abundantly secreted protein of myxoma virus-infected cells, was originally identified as an interferon-gamma receptor homolog (Upton, Mossman, and McFadden, Science 258, 1369-1372, 1992). Here, we demonstrate that M-T7 is dispensable for virus replication in cultured cells but is a critical virulence factor for virus pathogenesis in European rabbits. Disruption of both copies of the M-T7 gene in myxoma virus was achieved by the deletion of 372 bp of M-T7 coding sequences, replacement with a selectable marker, p7.5Ecogpt, and selection of a recombinant virus (vMyxlac-T7gpt) resistant to mycophenolic acid. vMyxlac-T7gpt expressed no detectable M-T7 protein and infected cells supernatants were devoid of any detectable interferon-gamma binding activities. Immunohistochemical staining with anti-beta-galactosidase and anti-CD43 antibodies demonstrated that in vMyxlac-T7gpt-infected rabbits the loss of M-T7 not only caused a dramatic reduction in disease symptoms and viral dissemination to secondary sites, but also dramatically influenced host leukocyte behavior. Notably, primary lesions in wild-type virus infections were generally underlayed by large masses of inflammatory cells that did not effectively migrate into the dermal sites of viral replication, whereas in vMyxlac-T7gpt infections this apparent block to leukocyte influx was relieved. A second major phenotypic distinction noted for the M-T7 knockout virus was the extensive activation of lymphocytes in secondary immune organs, particularly the spleen and lymph nodes, by Day 4 of the infection. This is in stark contrast to infection by wild-type myxoma virus, which results in relatively little, if any, cellular activation of germinal centers of spleen and lymph node by Day 4. We conclude that M-T7 functions early in infection to (1) retard inflammatory cell migration into infected tissues and (2) disrupt the communication between sentinel immune cells at the site of primary virus infection in the subdermis and lymphocytes in the secondary lymphoid organs, thereby disabling the host from mounting an effective cellular immune response. To summarize, in addition to neutralizing host interferon-gamma at infected sites, we propose that M-T7 protein also modifies leukocyte traffic in the vicinity of virus lesions, thus effectively severing the link between antigen presenting cells of the infected tissue and the effector lymphocytes of the peripheral immune organs.
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PMID:Myxoma virus M-T7, a secreted homolog of the interferon-gamma receptor, is a critical virulence factor for the development of myxomatosis in European rabbits. 855 83

Differentiation of hematopoietic precursor cells results in the formation of clonally related descendent cells. Using the mosaic expression of beta-galactosidase in female mouse fetuses heterozygous for an X-linked lacZ transgene, we analyzed the clonal relationship of the hematopoietic progeny. The proportion of beta-galactosidase positive cells for different T- and B-lymphoid and myeloid cell populations was determined at different stages of fetal development. We found excellent correlations of the proportion of beta-galactosidase expressing cells for all hematopoietic lineages confirming that they share a common ancestry. Therefore, it was possible to estimate the number of common precursor cells (PC) based on binomial distribution and covariance analysis of pairs of different hematopoietic cell populations. Our results obtained from hematopoietic cells at 15.5 to 18.5 days of gestation indicated the presence of 15 to 18 lymphoid and 18 to 22 myeloid/lymphoid specific precursor cells. Statistical analysis of the precursor cell numbers showed a trend of increasing numbers that was highly significant. The precursor cell number was inversely related to maturity of the cell populations analyzed; ie, the lowest number of lymphoid and lymphoid/myeloid precursors was calculated when the most mature CD3+ T-cell population was used for comparison. Determination of PC numbers can therefore be used to assess the relative maturity and developmental potential of individual cell populations.
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PMID:Estimation of the number of hematopoietic precursor cells during fetal mouse development by covariance analysis. 883 42

A method to concentrate drugs, DNA, or other materials with target cells in two-phase polymer systems for high-efficiency electroloading is described. The two-phase polymer system is utilized for cell and loading material selection, as well as for cell aggregation before electrofusion. The phase mixing of several water-soluble polymers is characterized, and the polyethylene glycol-Dextran (PEG m.w. 8,000 + Dextran m.w. 71,000) mixture is selected to illustrate the advantage of the two-phase systems. Fluorescently labeled Dextran or DNA is loaded into Chinese hamster ovary (CHO) and JTL cells, using electroporation in either the two-phase polymer system or the conventional single-phase suspension. The loading efficiency is 4 to 30 times higher for the two-phase system, with the best advantage at lower applied field range. Transfections of CHO, COS, Melan C, and JTL lymphoid cells using pSV-beta-galactosidase (for CHO and COS), pBK-RSV-tyrosinase, and pCP4-fucosidase plasmids, respectively, by electroporation in the two-phase polymer system and the conventional single-phase electroporation method, are compared. The former method is far superior to the latter in terms of efficiency. The threshold and optimal field strengths for the former are significantly lower than those for the latter method, so the former method is more favorable in terms of equipment requirement and safety. Electrofusion efficiency in the two-phase system is comparable to that in polyethylene glycol suspension alone and is a significant improvement from the conventional electrofusion method with dielectrophoresis. The two-phase polymer method is, therefore, a valuable technique for gene delivery to a limited cell source, as in ex vivo gene therapy.
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PMID:High-efficiency loading, transfection, and fusion of cells by electroporation in two-phase polymer systems. 884 49

We have developed a simple, safe and versatile method, termed antifection, by which antibodies are used as delivery vehicles to introduce genes into cells expressing specific surface antigens. Antibodies directed against CD3, CD34 or surface immunoglobulins were covalently coupled to plasmids containing marker genes (neoR, beta-galactosidase). Such conjugates were used in vitro and/or in vivo to antifect (transfect using antifection) cells bearing the respective targeted epitope on either normal splenic B lymphocytes or lymphoid-related cell lines. In these conditions the expression of the protein encoded by the marker gene was readily detected. Antifection is a method of delivering genes through a physiological cellular pathway, receptor-mediated endocytosis, into specific cell types, and thus may be considered as an alternative for gene therapy strategy.
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PMID:Antifection: an antibody-mediated method to introduce genes into lymphoid cells in vitro and in vivo. 885 99


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