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 investigated whether CD4 gene regulatory sequences might be useful for developing transcriptionally targeted Moloney murine leukemia virus (Mo-MLV)-based retroviral vectors for gene expression specifically in CD4(+) cells. We could modulate Mo-MLV long terminal repeat (LTR) activity by inserting a 438-bp-long fragment containing the murine CD4 silencer in the LTR of the vector; both beta-galactosidase and green fluorescent protein reporter gene activities were strongly down-regulated in both murine and human CD8(+) cells, but not in CD4(+) lymphoid cell lines and freshly isolated lymphocytes transduced with this vector, compared with the findings using a control vector carrying wild-type LTRs. Titration experiments on NIH-3T3 cells revealed that inclusion of the CD4 silencer in the LTRs did not reduce the titer of the vectors. These findings indicate that a cellular silencer can be successfully included in retroviral vectors, where it maintains its transcription-regulatory function, thus suggesting a novel approach to transcriptional targeting.
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PMID:Modulation of Moloney leukemia virus long terminal repeat transcriptional activity by the murine CD4 silencer in retroviral vectors. 1102 97

We have used a simple, single-gene retrovirus carrying the Escherichia coli beta-galactosidase reporter gene (lacZ), termed LlacZ. This virus was found to infect immortalized myeloid and lymphoid precursor/leukemic cell lines efficiently as well as primary murine bone marrow clonogenic progenitors, without apparent modulation of growth or phenotype. Following infection of bone marrow cells, a significant proportion of progenitors--36% of lineage-negative cells with low levels of c-kit expression (lin-/c-kit(lo)) known to be enriched with pluripotent hemopoietic stem cells, and 19% of Sca1-positive cells known to be enriched with transplantable cells with lymphomyeloid-reconstituting ability--were shown to express lacZ. Use of an LlacZ-infected population of post 5-fluorouracil bone marrow cells to reconstitute lethally irradiated mice demonstrated the presence of lacZ-expressing cells in the spleen at day 12 post-transplantation with provirus detected in individual spleen colonies (CFU-S). In the long term (3-6 months following transplantation), lacZ expression was detected in hematopoietic tissues of all recipient mice. The use of two-color in situ and flow cytometry analysis combined with lineage-specific antibodies showed lacZ expression in both myeloid and lymphoid cells in spleen and bone marrow. In addition, lacZ-expressing cells were detected in secondary recipient mice injected with bone marrow cells derived from primary LlacZ recipients. Overall, these data show the efficacy of a single gene vector for stem cell transduction, the utility of beta-galactosidase as a single cell marker for stem cell transduction and reconstitution ability, and the need for protocol optimization to see high-level multilineage gene expression.
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PMID:The beta-galactosidase gene as a marker for hematopoietic reconstitution: individual cell analysis in a murine bone marrow transplantation model. 1109 90

The Sca-1 surface glycoprotein is used routinely as a marker for haematopoietic stem cell enrichment. Two allelic genes, Ly-6A and Ly-6E, encode this marker and appear to be differentially regulated in haematopoietic cells and haematopoietic stem cells. The Sca-1 protein has been shown to be expressed at a greater frequency in these cells from Ly-6A strains of mice. To study the specific expression pattern and haematopoietic regulation of the Ly-6A gene, we constructed a 14 kb cassette from a genomic Ly-6A fragment, inserted a lacZ reporter gene and created transgenic mice. We found that the Ly-6A lacZ transgene was expressed in the haematopoietic tissues and predominantly in the T-lymphoid lineage. Some expression was also found in the B-lymphoid and myeloid lineages. We demonstrated functional haematopoietic stem cell enrichment by sorting for beta-galactosidase-expressing cells from the bone marrow. In addition, we found an interesting embryonic expression pattern in the AGM region, the site of the first haematopoietic stem cell generation. Surprisingly, when compared with data from Ly-6E lacZ transgenic mice, our results suggest that the Ly-6A cassette does not improve lacZ marker gene expression in haematopoietic cells.
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PMID:Expression of the Ly-6A (Sca-1) lacZ transgene in mouse haematopoietic stem cells and embryos. 1184 45

The adjuvanticity of MALP-2, a 2-kDa synthetic lipopeptide with macrophage-stimulatory activity, was evaluated in BALB/c mice using beta-galactosidase (beta-gal) as model antigen. When co-administered with beta-gal by either the intranasal (i.n.) or i.p. route, MALP-2 (0.5 microg) was capable of increasing beta-gal-specific serum IgG titers by 675-3,560-fold (i.n.) and 64-128-fold (i.p.), respectively, as compared to immunization with beta-gal alone. Using MALP-2, almost maximal IgG responses were already stimulated following the first immunization, and the IgG titers were similar to those observed using 10 microg of cholera toxin B subunit (CTB) as adjuvant. The mucosal immune system was also effectively stimulated (p<0.05) when MALP-2 was administered by the i.n. route (36% and 23% of beta-gal-specific IgA in lung and vaginal lavages, respectively). The i.n. co-administration of MALP-2 stimulated a stronger cellular immune response than CTB, both in submandibular lymph nodes and spleen (p<0.05). The analysis of beta-gal-specific IgG isotypes and the profiles of cytokines secreted by in vitro re-stimulated cells showed that co-administration of MALP-2 triggered a dominant Th2-response pattern. A recruitment of B220(+) and MAC-1(+) cells with an up-regulated expression of MHC class I, CD80 (B7.1) and CD54 (ICAM-1) was observed in nasal associated lymphoid tissues from MALP-2 treated mice. Taken together, our results demonstrated that the synthetic lipopeptide MALP-2 represents a very promising adjuvant for the mucosal delivery of vaccine antigens.
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PMID:The Mycoplasma-derived lipopeptide MALP-2 is a potent mucosal adjuvant. 1235 38

Oral induction of a disseminated mucosal immune response with polyplex-based DNA vaccines requires the delivery of intact polyplexes (polyelectrolyte complexes formed by self-assembly of plasmid DNA with a cationic polymer) to subepithelial lymphoid tissue (e.g. Peyer's patches) within the gastrointestinal tract. This work describes the formulation of a microparticle polyplex carrier allowing the potential of this approach to be realised. PEGylated PEI/DNA polyplexes (DNA concentration 20 microg/ml) formed at N/P 5:0 (defined as the ratio of polycation amino groups to DNA phosphates) were stable to salt-induced aggregation and could be concentrated to a final DNA concentration of 1 mg/ml without polyplex size increase. Polyplexes containing 1:1 polyethylene glycol (PEG)/polyethylenimine (PEI) ratio (mass/mass) gave similar levels of luciferase gene expression in B16F10 cells compared to non-PEG complexes. Poly-(D,L-lactide-co-glycolide) (PLGA) microparticles containing PEGylated polyplexes (approximately 17% DNA encapsulation efficiency) were formulated using a modified double emulsion solvent evaporation method. The microencapsulation and release of intact polyplexes from the microparticle carrier was demonstrated using polyanion (heparin sulfate and poly(aspartic acid) (PAA)) displacement techniques and electron microscopy. Microparticles containing PEGylated polyplexes (24 microg beta-galactosidase DNA) were given orally to Wistar rats. Significant transgene expression (compared to background) was found in peripheral tissue (spleen) 72 h after administration. This work demonstrates the potential application of microparticle carriers for mucosal polyplex-based vaccination.
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PMID:Formulation of a microparticle carrier for oral polyplex-based DNA vaccines. 1537 19

The hyaluronan receptor LYVE-1 is expressed abundantly on the surfaces of lymphatic vessels and lymph node sinus endothelial cells from early development, where it has been suggested to function both in cell adhesion/transmigration and as a scavenger for hyaluronan turnover. To investigate the physiological role(s) of LYVE-1, we generated mice in which the gene for the receptor was inactivated by replacement with a beta-galactosidase reporter. LYVE-1(-/-) mice displayed an apparently normal phenotype, with no obvious alteration in lymphatic vessel ultrastructure or function and no apparent change in secondary lymphoid tissue structure or cellularity. In addition, the levels of hyaluronan in tissue and blood were unchanged. LYVE-1(-/-) mice also displayed normal trafficking of cutaneous CD11c(+) dendritic cells to draining lymph nodes via afferent lymphatics and normal resolution of oxazolone-induced skin inflammation. Finally, LYVE-1(-/-) mice supported normal growth of transplanted B16F10 melanomas and Lewis lung carcinomas. These results indicate that LYVE-1 is not obligatory for normal lymphatic development and function and suggest either the existence of compensatory receptors or a role more specific than that previously envisaged.
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PMID:Normal lymphatic development and function in mice deficient for the lymphatic hyaluronan receptor LYVE-1. 1710 72

Hec-6st is a highly specific high endothelial venule (HEV) gene that is crucial for regulating lymphocyte homing to lymph nodes (LN). The enzyme is also expressed in HEV-like vessels in tertiary lymphoid organs that form in chronic inflammation in autoimmunity, graft rejection, and microbial infection. Understanding the molecular nature of Hec-6st regulation is crucial for elucidating its function in development and disease. However, studies of HEV are limited because of the difficulties in isolating and maintaining the unique characteristics of these vessels in vitro. The novel pClasper yeast homologous recombination technique was used to isolate from a BAC clone a 60-kb DNA fragment that included the Hec-6st (Chst4) gene with flanking sequences. Transgenic mice were generated with the beta-galactosidase (LacZ) reporter gene inserted in-frame in the exon II of Hec-6st within the isolated BAC DNA fragment. LacZ was expressed specifically on HEV in LN, as indicated by its colocalization with peripheral node vascular addressin. LacZ was increased in nasal-associated lymphoid tissue during development and was reduced in LN and nasal-associated lymphoid tissue by LTbetaR-Ig (lymphotoxin-beta receptor human Ig fusion protein) treatment in a manner identical to the endogenous gene. The transgene was expressed at high levels in lymphoid accumulations with characteristics of tertiary lymphoid organs in the salivary glands of aged mice. Thus, the Hec-6s-LacZ construct faithfully reproduces Hec-6st tissue-specific expression and can be used in further studies to drive expression of reporter or effector genes, which could visualize or inhibit HEV in autoimmunity.
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PMID:Transgenic LacZ under control of Hec-6st regulatory sequences recapitulates endogenous gene expression on high endothelial venules. 1736 May 66

CRACM1 (also called Orai1) constitutes the pore subunit of store-operated calcium release-activated calcium channels. A point mutation in the gene encoding CRACM1 is associated with severe combined immunodeficiency disease in humans. Here we generated CRACM1-deficient mice in which beta-galactosidase activity 'reported' CRACM1 expression. CRACM1-deficient mice were smaller in size. Mast cells derived from CRACM1-deficient mice showed grossly defective degranulation and cytokine secretion, and the allergic reactions elicited in vivo were inhibited in CRACM1-deficient mice. We detected robust CRACM1 expression in skeletal muscles and some regions of the brain, heart and kidney but not in the lymphoid regions of thymus and spleen. In contrast, we found CRACM2 expression to be much higher in mouse T cells. In agreement with those findings, the store-operated calcium influx and development and proliferation of CRACM1-deficient T cells was unaffected. Thus, CRACM1 is crucial in mouse mast cell effector function, but mouse T cell calcium release-activated calcium channels are functional in the absence of CRACM1.
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PMID:Defective mast cell effector functions in mice lacking the CRACM1 pore subunit of store-operated calcium release-activated calcium channels. 1805 70

Intratracheal instillation of L-selectin-deficient (L-Sel(-/-)) mice with an adenovirus 2 (Ad2) vector resulted in the lack of respiratory Ad2- or beta-galactosidase-specific CTLs with concomitant long-lived beta-galactosidase transgene expression in the lungs. The absence of Ag-specific CTLs was attributed to a deficiency in lymphoid CD11c(+)CD8(+) dendritic cells (DCs) in the lower respiratory lymph nodes (LRLNs). To enable L-Sel(-/-) CTL activity, cell-sorted L-Sel(-/-)CD8(+) T cells were cocultured with cell-sorted L-Sel(+/+)CD8(+) or CD8(-) DCs or L-Sel(-/-)CD8(-) DCs. Only the CD8(+) DCs restored CTL activity; L-Sel(-/-)CD8(-) DCs failed to support L-Sel(+/+) CTLs because these remained immature, lacking the ability to express costimulatory molecules CD40, CD80, or CD86. Although no lung CD8(+) DCs were detected, the DC environment remained suppressive in L-Sel(-/-) mice evident by the lack of CTL responses following adenoviral challenge with OVA in recipient L-Sel(-/-) adoptively transferred with OT-1 CD8(+) T cells. To assess whether the L-Sel(-/-)CD8(-) DCs could be induced into maturity, microbial stimulation studies were performed showing the failure of L-Sel(-/-) LRLN to make matured DCs. When L-Sel(-/-) mice were subjected in vivo to microbial activation before Ad2 vector dosing, CTL activity was restored stimulating the renewed presence of LRLN CD8(+) DCs in L-Sel(-/-) mice. These studies show that impairment of L-Sel(-/-) DC maturation results in insufficient mature DCs that require microbial activation to restore increases in respiratory CD8(+) DCs to support CTL responses.
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PMID:The absence of lymphoid CD8+ dendritic cell maturation in L-selectin-/- respiratory compartment attenuates antiviral immunity. 1860 89

A new langerin(+) DC subset has recently been identified in murine dermis (langerin(+) dDC), but the lineage and functional relationships between these cells and langerin(+) epidermal Langerhans cells (LC) are incompletely characterized. Selective expression of the cell adhesion molecule EpCAM by LC allowed viable LC to be easily distinguished from langerin(+) dDC in skin and lymphoid tissue and ex vivo as well. Differential expression of EpCAM and langerin revealed the presence of at least 3 distinct skin DC subsets. We determined that LC and langerin(+) dDC exhibit different migratory capabilities in vitro and repopulate distinct anatomic compartments in skin at different rates after conditional depletion in vivo. Langerin(+) dDC, in contrast to LC, did not require TGFbeta1 for development. Carefully timed gene gun immunization studies designed to take advantage of the distinct repopulation kinetics of langerin(+) dDC and LC revealed that langerin(+) dDC were required for optimal production of beta-galactosidase-specific IgG2a/c and IgG2b in the acute phase. In contrast, immunization via LC-deficient skin resulted in persistent and strikingly reduced IgG1 and enhanced IgG2a Ab production. Our data support the concepts that LC and langerin(+) dDC represent distinct DC subsets that have specialized functions and that LC are important immunoregulatory cells. The presence of at least 3 functionally distinct skin DC subsets may have particular relevance for vaccines that are administered epicutaneously.
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PMID:Murine epidermal Langerhans cells and langerin-expressing dermal dendritic cells are unrelated and exhibit distinct functions. 1921 33


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