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)

We recently cloned biosynthesis genes for the O7-lipopolysaccharide (O7-LPS) side chain from the Escherichia coli K-1 strain VW187 (M. A. Valvano, and J. H. Crosa, Infect. Immun. 57:937-943, 1989). To characterize the O7-LPS region, the recombinant cosmids pJHCV31 and pJHCV32 were mutagenized by transposon mutagenesis with Tn3HoHo1, which carries a promoterless lac operon and can therefore generate lacZ transcriptional fusions with target DNA sequences. Cells containing mutated plasmids were examined for their ability to react by coagglutination with O7 antiserum. The LPS pattern profiles of the insertion mutants were also investigated by electrophoresis of cell envelope fractions, followed by silver staining and immunoblotting analysis. These experiments identified three phenotypic classes of mutants and defined a region in the cloned DNA of about 14 kilobase pairs that is essential for O7-LPS expression. Analysis of beta-galactosidase production by cells carrying plasmids with transposon insertions indicated that transcription occurs in only one direction along the O7-LPS region. In vitro transcription-translation experiments revealed that the O7-LPS region encodes at least 16 polypeptides with molecular masses ranging from 20 to 48 kilodaltons. Also, the O7-LPS region in VW187 was mutagenized by homologous recombination with subsets of the cloned O7-LPS genes subcloned into a suicide plasmid vector. O7-LPS-deficient mutants of VW187 were complemented with pJHCV31 and pJHCV32, confirming that these cosmids contain genetic information that is essential for the expression of the O7 polysaccharide.
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PMID:Genetic analysis of the O7-polysaccharide biosynthesis region from the Escherichia coli O7:K1 strain VW187. 216 82

It was possible to define the effects of trehalose dimycolate (TDM), a glycolipid extracted from Mycobacterium tuberculosis, on mouse peritoneal macrophages more precisely using endotoxin-free culture conditions. TDM-elicited macrophages, when assayed in vitro in the absence of endotoxin, were unable to limit tumor growth; however, after a short treatment (4 h) with low doses of lipopolysaccharide (LPS; 1-10 ng/ml), they exhibited a strong cytostatic capacity against P815 mastocytoma cells. Thus, TDM injected in vivo did not activate macrophages fully but it primed them to respond in vitro to low doses of LPS, which provided the final stimulus for activation to antitumor competence. Macrophages elicited by an injection of killed group C Streptococci were also in a primed state; in contrast, thioglycollate-elicited macrophages were in a nonreceptive state. Besides LPS, concanavalin A (5 micrograms/ml), MDP (0.2-1 microgram/ml) and the ionophore A23187 (5 microM) can deliver the activation signal to TDM-primed macrophages. Primed macrophages were found to express several biochemical markers previously described as specific for activated macrophages (low levels of alkaline phosphodiesterase and beta-galactosidase, for example) and, although they were not cytotoxic for tumor cells, they had the capacity to release large amounts of H2O2. However, when pulsed by LPS or MDP, primed macrophages responded by further modifications in their metabolism: the rate of glucose consumption and the labeling of glycoproteins by D-[2-3H]mannose were greatly increased and the secretion of a polypeptide of 22 kDa was enhanced. The activation-associated biochemical markers are thus acquired in two steps. The ability to produce activated oxygen species is expressed earlier than the antitumoral activity.
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PMID:Macrophage activation by trehalose dimycolate requirement for an expression signal in vitro for antitumoral activity; biochemical markers distinguishing primed and fully activated macrophages. 300 1

The effect of bacterial endotoxin (LPS) on lysosomal enzyme activities of fibroblasts from normals and mucolipidosis III patients was investigated. Exposure of normal fibroblasts to LPS for 24 hours resulted in enhanced intracellular activities of beta-glucuronidase, beta-galactosidase, alpha-L-iduronidase and beta-N-acetylglucosaminidase. Endotoxin also led to an increased extracellular activity of beta-N-acetylglucosaminidase. In contrast, mucolipidosis III fibroblasts did not show either intracellular or extracellular increase of lysosomal hydrolases after LPS treatment. Difference in cellular responsiveness to LPS may be related to the mechanism of LPS-cell interaction.
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PMID:Effect of bacterial endotoxin on lysosomal enzyme activities of normal and mucolipidosis III fibroblasts. 726 Feb 37

We have been developing both local and systemic gene therapy approaches to treat inflammatory and autoimmune diseases. To determine if systemic, constitutive expression of biologically active anti-inflammatory agents is therapeutic and/or has associated toxicity, mouse hematopoietic stem cells were infected with retroviral vectors carrying the genes for human IL-1 receptor antagonist (IL-1Ra), human soluble TNF receptor p75 (sTNFR), or the beta-galactosidase (lacZ) gene, and transplanted into lethally irradiated recipients. The serum levels of human IL-1Ra and human sTNFR in the long-term reconstituted mice, 2-7 months after transplantation, were 596 and 158 ng/ml respectively. The long-term expression of human IL-1Ra had minimal effects on the PBMC profile whereas human sTNFR expression increased the percentage of B220 and Mac.1 stained cells and decreased slightly the specific T cell subsets. The ability of these proteins to protect the transplanted mice from endotoxin treatment was determined by measuring serum interleukin-6 (IL-6) and interleukin-10 (IL-10) responses after LPS injection at 1.5, 3, 4.5 and 24 h after treatment. The IL-1Ra group showed diminished IL-10 levels and less mortality after injection of LPS. These results demonstrate that constitutive, systemic expression of IL-1Ra and sTNFR is able to confer partial protective effects following treatment with endotoxin. These results further demonstrate that gene transfer methods which result in systemic, long-term expression of immunodulatory proteins could be applied to the treatment of inflammatory diseases.
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PMID:Constitutive systemic expression of IL-1Ra or soluble TNF receptor by genetically modified hematopoietic cells suppresses LPS induction of IL-6 and IL-10. 913 39

Antiphospholipid syndrome (APS) is an autoimmune disease characterized by the persistent presence of antiphospholipid antibodies (aPLs) and recurrent thrombosis or fetal loss. The thrombophilic state has been partially related to the induction of a proinflammatory and procoagulant endothelial cell (EC) phenotype induced by anti-beta(2)-glycoprotein I (beta(2)-GPI) antibodies that bind beta(2)-GPI expressed on the EC surface. Anti-beta(2)-GPI antibody binding has been shown to induce nuclear factor-kappa B (NF-kappa B) translocation leading to a proinflammatory EC phenotype similar to that elicited by interaction with microbial products (lipopolysaccharide [LPS]) and proinflammatory cytokines (interleukin 1 beta [IL-1 beta], tumor necrosis factor alpha [TNF-alpha]). However, the upstream signaling events are not characterized yet. To investigate the endothelial signaling cascade activated by anti-beta(2)-GPI antibodies, we transiently cotransfected immortalized human microvascular endothelial cells (HMEC-1) with dominant-negative constructs of different components of the pathway (Delta TRAF2, Delta TRAF6, Delta MyD88) together with reporter genes (NF-kappa B luciferase and pCMV-beta-galactosidase). Results showed that both human anti-beta(2)-GPI IgM monoclonal antibodies as well as polyclonal affinity-purified anti-beta(2)-GPI IgG display a signaling cascade comparable to that activated by LPS or IL-1. Delta TRAF6 and Delta MyD88 significantly abrogate antibody-induced as well as IL-1- or LPS-induced NF-kappa B activation, whereas Delta TRAF2 (involved in NF-kappa B activation by TNF) does not affect it. Moreover, anti- beta(2)-GPI antibodies and LPS followed the same time kinetic of IL-1 receptor-activated kinase (IRAK) phosphorylation, suggesting an involvement of the toll-like receptor (TLR) family. Our findings demonstrate that anti-beta(2)-GPI antibodies react with their antigen likely associated to a member of the TLR/IL-1 receptor family on the EC surface and directly induce activation.
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PMID:Role of the MyD88 transduction signaling pathway in endothelial activation by antiphospholipid antibodies. 1253 7

A multi-epitope antigen gene of hepatitis C virus(HCV) was fused to beta-galactosidase gene and introduced into attenuated Salmonella typhimurium SL3261 to construct HCV recombined live vaccine candidate SL3261 (pWR/PCX). when the oral live bacteria were used to immunize mice or rabbits, specific anti-GZ-PCX IgG was detected at week 6 and the strongest antibody responses happened at week 12 at a titer of 1:800 and 1:25,600 in mice and rabbits, respectively, which showed significant difference compared with those of SL3261 and blank controls. Anti-GZ-PCX sIgA in mice's intestine and anti-LPS antibody in sera were also detected. The oral live bacteria elicited obvious DTH reaction and proliferation response of peripheral lymphocytes by GZ-PCX antigen. The body weight of immunized mice slightly decreased but no other toxic effects was observed, which showed the safety of oral immunization. The study of oral live HCV multi-epitope vaccine might be able to provide a new route for the researches of HCV vaccines.
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PMID:[Immune responses of a recombined live Salmonella typhimurium SL3261 expressing a multi-epitope antigen of HCV]. 1254 60

We here describe an assay for the detection of interferon-like activity in Atlantic salmon based on the transient transfection of chinook salmon embryo cells (CHSE-214 cells) with a rainbow trout Mx1 promoter linked to a luciferase reporter. A beta-galactosidase gene under the control of a constitutively expressed beta-actin promoter was used as a transfection standard, and luciferase and beta gal expression were measured by a commercially available kit. Interferon containing supernatants from poly I:C- or CpG-stimulated leucocytes added to transfected CHSE-cells induced high luciferase expression (>60-fold induction compared to supernatants from non-stimulated cells). There was no response to supernatants from LPS- and ConA/PMA-stimulated leucocytes, demonstrating the specificity for type I interferon-like activity. Duplicate samples analysed using a cell protection assay for detection of antiviral activity correlated well with levels obtained by the Mx1 promoter reporter gene assay (R2=0.97), confirming the reporter assay as a reliable substitute for the standard antiviral assay. The Mx reporter gene assay also has advantages in terms of sensitivity, high dynamic range and reliability over the conventional cell protection assay.
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PMID:Quantification of Atlantic salmon type-I interferon using an Mx1 promoter reporter gene assay. 1512 21

Although several observations show local T cell recognition of retinal Ag, there has been no direct demonstration that the APC were retinal derived, rather than recruited. In this study, CD45(+) cells isolated from immunologically quiescent murine retina were tested in vitro for functional evidence of Ag presentation to naive and Ag-experienced CD4 T cells specific for beta-galactosidase. Because CD45(+) cells from brain have been reported to be efficient APC, they were included for comparison. Measures of activation included changes in CD4, CD25, CD44, CD45RB, CD62L, CD69, caspase-3 activation, CFSE dilution, size, number of cells recovered, and cytokine production. Retinal CD45(+) cells gave no evidence of Ag-dependent TCR ligation in naive T cells, unlike splenic APC and CD45(+) cells from brain, which supported potent responses. Instead, addition of retinal CD45(+) cells to cocultures of naive 3E9 T cells plus splenic APC reduced the yield of activated T cells and cytokine production by limiting T cell activation at early time points. Ag-experienced T cells responded weakly to Ag presented by retinal CD45(+) cells. Activating the retinal cells with IFN-gamma, anti-CD40, or LPS incrementally increased their APC activity. Addition of neutralizing Abs to TGF-beta did not reveal suppressed retinal APC activity. Because retina lacks tissue equivalents of meninges and choroid plexus, rich sources of dendritic cells in brain, cells from retina may better represent the APC activity of fresh, adult CNS parenchymal and perivascular cells. The activity of the retinal CD45(+) cells appears to be directed to limiting T cell responses.
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PMID:The antigen-presenting activity of fresh, adult parenchymal microglia and perivascular cells from retina. 1515 73

Woodchucks infected with the woodchuck hepatitis virus (WHV) is the best available animal model for testing the immunotherapeutic effects of dendritic cells (DCs) in the setting of a chronic infection, as woodchucks develop a persistent infection resembling that seen in humans infected with the hepatitis B virus. In the present study, DCs were generated from woodchuck peripheral blood mononuclear cells (wDCs) in the presence of human granulocyte macrophage colony-stimulating factor (hGM-CSF) and human interleukin 4 (hIL-4). After 7 days of culture, cells with morphology similar to DCs were stained positively with a cross-reactive anti-human CD86 antibody. Functional analysis showed that uptake of FITC-dextran by wDCs was very efficient and was partially inhibited after LPS-induced maturation. Furthermore, wDCs stimulated allogenic lymphocytes and induced proliferation. Moreover, wDCs were transduced efficiently with a human adenovirus serotype 5 for the expression of beta-galactosidase. Following transduction and in vivo administration of such DCs into woodchucks, an antigen-specific cellular immune response was induced. These results demonstrate that wDCs can be generated from the peripheral blood. Following transfection with a recombinant adenovirus wDCs can be used as a feasible and effective tool for eliciting WHV-specific T-cell responses indicating their potential to serve as prophylactic and therapeutic vaccines.
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PMID:Woodchuck dendritic cells generated from peripheral blood mononuclear cells and transduced with recombinant human adenovirus serotype 5 induce antigen-specific cellular immune responses. 1738 94

A common gene variant in the heparin-binding domain (HBD) of extracellular superoxide dismutase (ECSOD) may predispose human carriers to ischaemic heart disease. We have demonstrated that the HBD of ECSOD is important for ECSOD to restore vascular dysfunction produced by endotoxin. The purpose of this study was to determine whether the gene variant in the HBD of ECSOD (ECSOD(R213G)) protects against endothelial dysfunction in a model of inflammation. We constructed a recombinant adenovirus that expresses ECSOD(R213G). Adenoviral vectors expressing ECSOD, ECSOD(R213G) or beta-galactosidase (LacZ, a control) were injected i.v. in mice. After 3 days, at which time the plasma SOD activity is maximal, vehicle or endotoxin (lipopolysaccharide or LPS, 40 mg kg(-1)) was injected i.p. Vasomotor function of aorta in vitro was examined 1 day later. Maximal relaxation to sodium nitroprusside was similar in aorta from normal and LPS-treated mice. Maximal relaxation to acetylcholine (10(-5)) was impaired after LPS and LacZ (63 +/- 3%, mean +/- s.e.m.) compared to normal vessels (83 +/- 3%) (P < 0.05). Gene transfer of ECSOD improved (P < 0.05) relaxation in response to acetylcholine (76 +/- 5%) after LPS, whereas gene transfer of ECSOD(R213G) had no effect (65 +/- 4%). Superoxide was increased in aorta (measured using lucigenin and hydroethidine) after LPS, and levels of superoxide were significantly reduced following ECSOD but not ECSOD(R213G). Thus, ECSOD reduces superoxide and improves relaxation to acetylcholine in the aorta after LPS, while the ECSOD variant R213G had minimal effect. These findings suggest that, in contrast to ECSOD, the common human gene variant of ECSOD fails to protect against endothelial dysfunction produced by an inflammatory stimulus.
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PMID:Effects of a common human gene variant of extracellular superoxide dismutase on endothelial function after endotoxin in mice. 1771 13


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