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)

During embryogenesis, EndoB, the mouse form of human keratin 18 (K18), is expressed in a complex spatial and temporal pattern in various embryonic epithelia. We have compared the expression of transgenic human K18 to the endogenous mouse homolog and to the coexpressed, complementary keratin 8 homolog, EndoA, during postimplantation mouse embryogenesis and fetal development in order to determine the developmental expression pattern of the human gene in a mouse environment. The tissue distribution of K18 protein was identical to that of endogenous EndoB in both 7.5- and 13.5-day-old embryos, except for certain heart, eye, and extraembryonic mesodermal tissues in which K18 was not detected. These results indicate that the 10-kb K18 gene specifies appropriate developmental expression in the mouse and support previously reported differences in K18 expression in human and mouse fetal heart. We have also compared the expression patterns of K18 to a series of constructions that utilize the Escherichia coli gene for beta-galactosidase (lacZ) as a reporter gene. Some of these constructions were regulated correctly in embryos during development of the germ layers. However, none was expressed consistently in extraembryonic or in adult tissues. Analysis with methylation-sensitive restriction enzymes revealed that hypermethylation of the CpG-rich prokaryotic reporter gene was not the cause of its silence in adult transgenic liver. However, the repressed state of K18-LacZ transgenes in adult liver was correlated with a different chromatin state that lacked diagnostic DNase hypersensitive sites found in K18 transgenic liver. Expression of the lacZ reporter gene did not accurately reflect the developmental pattern of K18 even in constructions that used all available K18 sequences. We conclude that in these contexts, the lacZ gene was not a developmentally neutral reporter gene.
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PMID:Embryonic expression of human keratin 18 and K18-beta-galactosidase fusion genes in transgenic mice. 750 37

The cationic liposome DOTAP was complexed with plasmid DNA encoding beta-galactosidase in various ratios. As the concentration of DOTAP increased, the DNA became increasingly refractory to staining with ethidium bromide, presumably because the DNA was becoming condensed and being encapsulated by the liposomes. Transfection by DNA-DOTAP complexes at all ratios tested was unaffected by treatment of the complexes with DNase I. This finding has relevance to clinical trials for gene therapy of cystic fibrosis, in which patients are normally removed from treatment with DNase before receiving administration of DNA. We additionally tested the effect of aerosolisation of the liposome-DNA complex and of the DNA alone on the efficiency of in vitro transfection. Aerosolised DNA complexed with fresh DOTAP led to much lower reporter gene expression in Cos 7 cells than non-aerosolised complex, since aerosolisation appeared to destroy almost all of the plasmid. However, complexing the plasmid before passage through the nebuliser did protect most of the DNA from degradation, as reflected in the levels of transfection obtained. These findings contribute towards an overall understanding of both how DNA-cationic liposome complexes are formed and their fate following administration in vivo.
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PMID:Plasmid DNA molecules complexed with cationic liposomes are protected from degradation by nucleases and shearing by aerosolisation. 887 34

Trials of gene transfer for cystic fibrosis (CF) are currently underway. However, direct application to the airways may be impeded by the presence of airway secretions. We have therefore assessed the effect of CF sputum on the expression of the reporter gene beta-galactosidase complexed with the cationic liposome DC-Chol/DOPE in a number of cell lines in vitro. Transfection was markedly inhibited in the presence of sputum; the effect was concentration dependent and was only partially ameliorated by removal of sputum with phosphate-buffered saline (PBS) washing before gene transfer. However, treatment of the sputum-covered cells with recombinant human DNase (rhDNase, 50 micrograms/ml) but not with N-acetylcysteine, Nacystelyn, lysine (all 20 mM) or recombinant alginase (0.5 U/ml) significantly (P < 0.005) improved gene transfer. Adenovirus-mediated gene transfer efficiency in the presence of sputum was similarly inhibited, and again, treatment with rhDNase before transfection significantly improved gene transfer (P < 0.005). Transfection of Cos 7 cells in the presence of exogenous genomic DNA alone demonstrated similar inhibition to that observed with sputum and was also ameliorated by pre-treatment of DNA-covered cells with rhDNase. In a separate series of experiments performed in the absence of added sputum or genomic DNA, increasing concentrations of rhDNase resulted in a concentration-related decline in transfection efficiency. However, even at the highest concentration (500 micrograms/ml of rhDNase), transfection efficiency remained more than 50% of control. Thus, pre-treatment of CF airways with rhDNase may be appropriate before liposome or adenovirus-mediated gene therapy.
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PMID:The effect of mucolytic agents on gene transfer across a CF sputum barrier in vitro. 953 69

The DNase of Epstein-Barr virus (EBV) is a 470-amino-acid protein which possesses both endonuclease and exonuclease activities and accepts both double-stranded DNA and single-stranded DNA as substrates. It has been reported that this protein may be found in the nucleus and/or cytoplasm of infected cells. In this study, using cell fractionation and immunoblotting to determine the distribution of EBV DNase in Akata cells stimulated with anti-human immunoglobulin G antibody (anti-IgG), the DNase was found to be located predominantly in the nucleus. To map the signals in DNase which mediate its nuclear localization, we monitored the nuclear transport of fusion proteins consisting of various fragments of EBV DNase linked to a cytoplasmic protein, beta-galactosidase (beta-Gal). The results demonstrated that two regions of the DNase with nuclear localization signal (NLS) activity, designated NLS-A (amino acids 239-266) and NLS-B (amino acids 291-306), were able independently to localize the beta-Gal to the nuclei of HEp-2 and HeLa cells. Five basic residues (R or K) were found in each NLS and distributed differently in primary structure. The basic domains and flanking residues of NLS-A and NLS-B are 250YKRPCKRSFIRFI262 and 294LKDVRKRKLGPGH306, respectively. Further examination of these sequences revealed that NLS-A contains bulky aromatic amino acids (Y and F) which may diminish its capacity to act as a strong NLS and lacks the typical proline and glycine helix-breakers. However, NLS-B contains typical proline and glycine helix-breakers and the histidine residue at amino acid 306 is required for NLS activity. In addition, two hydrophobic regions within the DNase were found to inhibit the function of NLS-A but not NLS-B, suggesting that these two domains are different types of NLSs and differ in their sensitivity to hydrophobic regions in the context of protein structure.
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PMID:Epstein-Barr virus DNase contains two nuclear localization signals, which are different in sensitivity to the hydrophobic regions. 968 72

Cellular nuclease activity is a potential barrier to the successful delivery of foreign genes to mammalian cells. We tested the hypothesis that transfection in the presence of a specific DNase inhibitor can enhance the expression of foreign gene products. We have used DMI-2, a polyketide metabolite of Streptomyces sp. strain 560 to enhance the expression of bacterial chloramphenicol acetyltransferase (CAT) in the human lung adenocarcinoma cell line H441. DMI-2 has been shown previously to inhibit porcine DNase II, an acid pH nuclease contained in the endosomal/lysosomal compartment. Transfection of H441 cells in the presence of 0.1-1 microgram/ml DMI-2 caused: (1) 10-fold enhancement of CAT activity when the bacterial plasmid was complexed with either surfactant protein A-poly-lysine or transferrin-poly-lysine; (2) 1.5- to two-fold enhancement of CAT activity in cells exposed to lipofectin-DNA complexes: (3) no effect on transfection via calcium phosphate co-precipitation. DMI-2 alone showed no inherent transfection activity. In experiments using SP-A-poly-lysine and plasmid containing the beta-galactosidase reporter gene, DMI-2 increased the number of transfected cells. Methanolysis products of DMI-2 did not inhibit DNase II and did not enhance transfection efficiency. Taken together, the data support the hypothesis that nuclease action is a significant barrier to expression of foreign genes and inhibition of specific nucleases may facilitate transfection.
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PMID:Enhanced reporter gene expression in cells transfected in the presence of DMI-2, an acid nuclease inhibitor. 993 Mar 26

Gene transfer to provide long-term expression of a therapeutic product, without introducing unwelcome genetic information, is a goal being sought for therapy of both hereditary and acquired diseases. Polyoma virus pseudocapsids, generated from a VP1-expressing recombinant baculovirus, lack viral DNA and have been successfully used to introduce small exogenous genes stably into cells in vitro by a process designated 'pseudofection'; although pseudocapsids protect only about 3 kbp of exogenous DNA, low efficiency transfer of a larger fragment (6.2 kbp) has been observed. Here, expression of a 7.2 kbp plasmid (pCMV beta) encoding the beta-galactosidase gene was assessed to monitor not only efficiency, but the ability of pseudocapsids to transfer larger-sized DNA on their own, or in the presence of the polycation, poly-L-lysine, added to protect nonencapsidated DNA. When complexed to pseudocapsids only, the efficiency of expression of the transferred beta-galactosidase gene (in human or rodent cells), although low, appeared to stabilise with time. In the presence of polylysine, unencapsidated DNA was shown to be protected against DNase activity, but electron microscopy (EM) revealed the formation of large mixed aggregates. The addition of pseudocapsids to these aggregates, and measurement of mobilities of the complexes in CsCl equilibrum centrifugation, indicated that they contained negligible amounts of VP1. For subsequent pseudofection experiments, DNA was complexed first with pseudocapsids, then polylysine was added. The latter did not appear to displace pseudocapsids from DNA, and was found to increase the efficiency of short-term expression both in in vitro and in vivo experiments. Gene expression, analysed histochemically or by the polymerase chain reaction, revealed transcriptional activity of the input gene, with expression first diminishing, then stabilising over time. The presence of pseudocapsids, in complexes with DNA with or without polylysine, allowed for stable and persistent gene expression.
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PMID:Enhancement by polylysine of transient, but not stable, expression of genes carried into cells by polyoma VP1 pseudocapsids. 993 Mar 47

Complexes composed of peptide ligand for the serpin enzyme complex receptor covalently coupled to poly-L-lysine condensed by charge interaction with plasmid DNA direct gene transfer into receptor bearing cells. We compared intensity and duration of reporter gene expression in vitro and in vivo from serpin-enzyme receptor-directed gene transfer complexes prepared with poly-L-lysine of different chain lengths. When substituted with linker and ligand to comparable extents, DNA complexes containing short chain poly-L-lysine were larger and gave higher peak expression but significantly shorter duration of expression than those containing long chain poly-L-lysine. Both peak expression and duration of expression exceeded that observed with Lipofectin. Neither naked DNA nor DNA complexed with unsubstituted polylysine was effective in gene transfer. For in vivo experiments, complexes containing optimal ligand and degree of substitution (based on in vitro data, peptide C105Y, 11 ligands/plasmid DNA molecule) were prepared with either short chain or long chain polylysine and a beta-galactosidase expression plasmid. Following injection into the tail veins of mice, longer chain complexes gave significantly higher expression of reporter gene in lung and spleen that lasted for a significantly longer period of time than the shorter chain complexes. The short chain poly-L-lysine-DNA complexes were larger in diameter, as assessed by electron microscopy or atomic force microscopy, and gave less protection against DNase digestion in vitro than longer chain complexes. Thus, for gene transfer complexes directed at the serpin enzyme complex receptor, longer chain poly-L-lysine gave a much longer duration of expression both in vitro and in vivo. We speculate that this may be due to protection against degradation afforded the plasmid DNA by the tighter compaction produced by long chain poly-L-lysine.
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PMID:Chain length of the polylysine in receptor-targeted gene transfer complexes affects duration of reporter gene expression both in vitro and in vivo. 998 33

Membrane vesicles released by Escherichia coli O157:H7 into culture medium were purified and analyzed for protein and DNA content. Electron micrographs revealed vesicles that are spherical, range in size from 20 to 100 nm, and have a complete bilayer. Analysis of vesicle protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrates vesicles that contain many proteins with molecular sizes similar to outer membrane proteins and a number of cellular proteins. Immunoblot (Western) analysis of vesicles suggests the presence of cell antigens. Treatment of vesicles with exogenous DNase hydrolyzed surface-associated DNA; PCR demonstrated that vesicles contain DNA encoding the virulence genes eae, stx1 and stx2, and uidA, which encodes for beta-galactosidase. Immunoblot analysis of intact and lysed, proteinase K-treated vesicles demonstrate that Shiga toxins 1 and 2 are contained within vesicles. These results suggest that vesicles contain toxic material and transfer experiments demonstrate that vesicles can deliver genetic material to other gram-negative organisms.
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PMID:Export of virulence genes and Shiga toxin by membrane vesicles of Escherichia coli O157:H7. 1022 67

Clostridium septicum is responsible for several diseases in humans and animals. The bacterium is capable of a simple kind of multicellular behavior known as swarming. In this investigation, environmental and physiologic factors affecting growth and swarm cell formation in C. septicum were studied over a range of dilution rates (D = 0.02 to 0.65 h(-1)) in glucose-limited, glucose-excess, and mucin-limited chemostats. Cellular differentiation was observed at low specific growth rates, irrespective of the carbon and energy source, showing that swarming occurred in response to nutrient depletion. Differential expression of virulence determinants was detected in swarm cells. Hemolysin was secreted by short motile rods but not swarm cells, whereas in cultures grown with glucose, only swarm cells formed DNase, hyaluronidase, and neuraminidase. However, neuraminidase and, to a lesser degree, hyaluronidase were induced in short motile rods in mucin-limited cultures. Both swarm cells and short rods were cytotoxic to Vero cells. Mucin was chemotaxic to C. septicum, and large amounts of mucin-degrading enzymes (beta-galactosidase, N-acetyl beta-glucosaminidase, glycosulfatase, and neuraminidase) were produced. Synthesis of these enzymes was catabolite regulated. In chemostat experiments, glycosulfatase secretion occurred only in swarm cells at low dilution rates in mucin-limited cultures. Determinations of oligosaccharide utilization demonstrated that N-acetylglucosamine, galactose, and N-acetylgalactosamine were the main carbon sources for C. septicum in mucin. Neuraminic acid was not assimilated, showing that neuraminidase does not have a direct nutritional function in this pathogen.
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PMID:Toxin synthesis and mucin breakdown are related to swarming phenomenon in Clostridium septicum. 1116 9

On the basis of phenotypic and genotypic characteristics, a novel species belonging to the genus Pedobacter is described. A facultatively psychrophilic, Gram-negative, aerobic, rod-shaped strain, A37(T), was isolated from alpine glacier cryoconite. The non-flagellated and non-spore-forming isolate grew over a temperature range of 1-25 degrees C, showed activities of oxidase, catalase, DNase, protease (gelatin, casein), amylase, beta-glucosidase, beta-galactosidase and beta-lactamase and degraded oil hydrocarbons. A distinct optimum temperature of 15 degrees C was observed for both protease production and oil hydrocarbon biodegradation. Analysis of 16S rDNA revealed that strain A37(T) represents a distinct taxon within PEDOBACTER: DNA from strain A37(T) showed only 19.7 % genetic relatedness to the DNA of Pedobacter piscium. The DNA G+C content was 43.4 mol%. Dominant fatty acids (51 %) were iso-15 : 0 2-OH and 16 : 1omega7c. The strain is assigned to a novel Pedobacter species, for which the name Pedobacter cryoconitis sp. nov. is proposed, with A37(T) (=DSM 14825(T)=LMG 21415(T)) as the type strain.
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PMID:Pedobacter cryoconitis sp. nov., a facultative psychrophile from alpine glacier cryoconite. 1313 9


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