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)

In this report, reporter gene beta-galactosidase (LacZ) was chosen to compare two different intramuscular gene transfer methods, direct injection and gene suture. Evidence showed that gene suture can produce a higher foreign gene express efficiency in skeletal muscle compared with the direct injection method. The highly efficient eukaryotic expressing vectors of human atrial natriuretic factor (ANF) were constructed (pcD2/pAdVAntage/hANF and pcDNA3/hANF), and in vivo ANF gene delivery was performed by intramuscular gene suture. The effects of ANF gene transfer on blood pressure and renal sodium and water excretion were studied in three models of hypertensive animals. Results showed that a marked decrease of mean arterial pressure (MAP) and a significant increase of urine volume and urinary sodium excretion was produced in rats receiving the hANF construct due to the local expression of ANF and its secretion into plasma. Taken together, these results indicate that gene suture may represent a novel gene delivery modality in gene therapy.
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PMID:Gene suture--a novel method for intramuscular gene transfer and its application in hypertension therapy. 1057 91

Premature aging of the skin is a prominent side-effect of psoralen photoactivation, a therapy used for a variety of skin disorders. Recently, we demonstrated that treatment of human dermal fibroblasts with 8-methoxypsoralen and ultraviolet A irradiation resulted in a permanent growth arrest with a switch of mitotic to postmitotic fibroblasts. Furthermore, an upregulation of matrix-degrading metalloproteinases and a high level of de novo expression of the senescence-associated beta-galactosidase was detected in the PUVA-treated postmitotic fibroblasts. The molecular basis for this PUVA-induced change in the functional and morphologic phenotype of fibroblasts resembling or mimicking replicative senescence is, however, unknown. Herein after, we have used a polymerase chain reaction-based subtractive hybridization protocol to identify human genes that are induced by PUVA treatment. Application of polymerase chain reaction-Select resulted in the cloning of four PUVA genes. Sequence analysis and homology searches identified three cDNA clones of known genes related to cell cycle regulation (p21waf1/cip1), stress response (ferritin H) and connective tissue metabolism (tissue inhibitor of metalloproteinases-3), whereas one cDNA clone represented a novel gene (no. 478). Northern blot analyses were performed to confirm a PUVA-dependent increase in specific mRNA levels in human dermal fibroblasts in vitro. This report on the identification of growth arrest related genes in PUVA-treated fibroblasts may stimulate further research addressing the causal role of these known and novel genes in extrinsic and intrinsic aging processes on a molecular and cellular level.
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PMID:Isolation and identification of psoralen plus ultraviolet A (PUVA)-induced genes in human dermal fibroblasts by polymerase chain reaction-based subtractive hybridization. 1106 32

The use of the site-specific DNA recombinases FLP and Cre is well-established in a broad range of organisms. Here we investigate the applicability of both recombinases to the Xenopus system where they have not been analyzed yet. We show that injection of FLP mRNA triggers the excision of an FLP recombination target (FRT)-flanked green fluorescent protein (GFP) sequence in a coinjected reporter construct inducing the expression of a downstream beta-galactosidase gene (lacZ). The FLP-mediated gene activation can be controlled in Xenopus embryos by injecting a mRNA encoding a fusion of FLP to the mutant ligand binding domain of the human estrogen receptor whose activity is dependent on 4-hydroxytamoxifen. We also demonstrate that a Cre reporter injected into fertilized eggs is fully recombined by Cre recombinase before zygotic gene transcription initiates. Our results indicate that in Xenopus embryos Cre is more effective than FLP in recombining a given quantity of reporter molecules. Finally, we present FLP-inducible double reporter systems encoding two fluorescence proteins (EYFP, ECFP, DsRed or GFP). These novel gene expression systems enable the continuous analysis of two reporter activities within living embryos and are expected to allow cell-lineage studies based on recombinase-mediated DNA rearrangement in transgenic Xenopus lines.
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PMID:FLP and Cre recombinase function in Xenopus embryos. 1137 65

Studies of N-ethyl-N-nitrosourea (ENU)-induced mutagenesis with a tyrosine auxotroph of Escherichia coli revealed a new type of revertant. This mutant strain was interesting because: (i) it was not a true revertant of the nonsense (ochre) defect nor a tRNA suppressor mutation; and (ii) it was induced by ENU to greater extent in a UmuC-defective host. Genetic mapping located the probable mutation to a region of the E. coli chromosome containing a newly described gene called tas. To investigate this mutation, the upstream region of the tas gene from both wild-type and mutant cells was cloned into a promoterless lacZ expression vector and recombined onto a lambda bacteriophage. Recombinant bacteriophage were inserted into the bacterial chromosome and beta-galactosidase (betaGal) assays were performed. These assays revealed an almost three-fold greater expression of betaGal from the mutant DNA than from the wild-type DNA. Sequence analysis of the region directly upstream of the tas gene revealed a G:C to A:T transition at base number 2263 (numbering based on GenBank Accession #AE000367), located within a potential promoter site. Further sequencing indicated no other mutations within the 1454bp region analyzed; however, there were several nucleotide differences seen in our B/r strain of E. coli, when compared with the published E. coli K-12 sequence. A total of 10 base differences were discovered; one in mutH, six within a potential open reading frame (ORF-o237) and three in non-coding regions. Yet, none of the changes altered the predicted amino acid sequences. These results provide evidence of a mechanism for increased expression of the novel gene tas and support the neutral drift hypothesis for the evolution of DNA sequences.
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PMID:Identification of a mutation causing increased expression of the tas gene in Escherichia coli FX-11. 1147 Apr 87

Previous attempts to establish 293cell-based stable and high-titer adeno-associated virus (AAV) packaging cell lines were unsuccessful, primarily due to adenovirus E1-activated Rep gene expression, which exerts cytostatic and cytotoxic effects on the host cells. Control of the two large AAV Rep proteins (Rep78/68) was insufficient to eliminate the adverse effects, because of the leaky expression of the two small Rep proteins (Rep52/40). However, it was unsuccessful to control Rep52/40 gene expression since its promoter is located within the coding sequence of Rep78/68. To tightly regulate all four Rep proteins by using their own promoters, we have developed a novel gene control paradigm termed "dual splicing switch," which disrupts all four Rep genes by inserting into their shared coding region an intron that harbors transcription termination sequences flanked the LoxP sites. As a result, the structure and activities of the Rep gene promoters, both p5 and p19, are not affected; however, all of the Rep transcripts are prematurely terminated and the genes were inactivated. Removal of the terminator by Cre protein reactivates the transcription of all four Rep proteins derived from their own promoters. This switch system was initially tested in the lacZ gene and a 600-fold induction of beta-galactosidase activity was observed. Using the dual splicing switch strategy, we have subsequently established a number of AAV packaging cell lines from 293 cells, which showed a normal growth rate, high stability, and more importantly, high yields of AAV vectors. Such a gene control paradigm is also useful for other viruses, e.g., autonomous parvoviruses. Finally, the high-titer 293-based AAV packaging cell lines should greatly reduce the risk of wild-type adenovirus contamination and provide a scalable AAV vector production method for both preclinical and clinical studies.
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PMID:A novel gene expression control system and its use in stable, high-titer 293 cell-based adeno-associated virus packaging cell lines. 1243 27

The limited efficacy of non-viral gene delivery systems currently hampers their wider therapeutic use. In order to further develop novel gene delivery systems, it is important to quantify their efficacy. Many reporter gene assays have limitations when being used to quantify expression in vivo. We have developed a simple assay which allows the quantification of beta-galactosidase transgene activity in vivo. The assay is based on beta-galactosidase cleavage of the DDAO-galactopyranoside substrate to DDAO, which shifts the fluorescence towards longer wavelengths. Reaction conditions were optimised to minimise degradation, activity of endogenous beta-galactosidase, and non-specific background fluorescence. The spectrofluorimetric quantification of the reaction product DDAO in the red part of the spectrum avoided interference from haemoglobin or other bio-molecules which hamper many in vivo assays. Routinely, amounts of less than 1 ng of beta-galactosidase (1 mU) per gram tissue could be detected and quantified. After intravenous administration of beta-galactosidase complexed with linear polyethylenimine (PEI, 22 kD) in mice, 134 mU g(-1) beta-galactosidase were detected in the lung, but only 2.9 mU g(-1) were found in the liver.
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PMID:Quantification of beta-galactosidase activity after non-viral transfection in vivo. 1293 52

A novel gene therapy approach for treating damaged cartilage is proposed that involves placing endotoxin-free cDNA containing the gene for bone morphogenetic protein-2 (BMP-2) in type I collagen sponges and then transferring the naked plasmid DNA construct to the injury site. A full-thickness cartilaginous defect in rabbits implanted with plasmid containing a marker gene (beta-galactosidase) showed expressed protein as detected by immunostaining. At 1 week postimplantation, mesenchymal cells subjacent to the defect had incorporated the implanted naked plasmid DNA and, once transfected, served as local bioreactors, transiently producing the gene product. Plasmids containing the gene for BMP-2 implanted in collagen sponges in cartilage lesions stimulated hyalinelike articular cartilage repair at 12 weeks postimplantation, nearly equivalent in quality to that induced by collagen sponges with recombinant BMP-2 protein. Our approach circumvents the risks of inflammation and immunogenic response associated with the use of viral vectors. Naked plasmid DNA as a vehicle for transferring therapeutic genes has been shown to be effective in a therapeutic model within rabbit articular cartilage and appears to be safe and cost effective.
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PMID:Regional gene therapy for full-thickness articular cartilage lesions using naked DNA with a collagen matrix. 1660 67

The aim of this investigation was to develop and evaluate a novel nanoparticles-in-microsphere oral system (NiMOS) for gene delivery and transfection in specific regions of the gastrointestinal (GI) tract. Plasmid DNA, encoding either for beta-galactosidase (CMV-betagal) or enhanced green fluorescent protein (EFGP-N1), was encapsulated in type B gelatin nanoparticles. NiMOS were prepared by further protecting the DNA-loaded nanoparticles in a poly(epsilon-caprolactone) (PCL) matrix to form microspheres of less than 5.0 microm in diameter. In order to evaluate the biodistribution following oral administration, radiolabeled ((111)In-labeled) gelatin nanoparticles and NiMOS were administered orally to fasted Wistar rats. The results of biodistribution studies showed that, while gelatin nanoparticles traversed through the GI tract fairly quickly with more than 85% of the administered dose per gram localizing in the large intestine within the first hour, NiMOS resided in the stomach and small intestine for relatively longer duration. Following oral administration of CMV-betagal or EFGP-N1 plasmid DNA at 100 microg dose in the control and test formulations, the qualitative results presented in this study provide the proof-of-concept for the transfection capability of NiMOS upon oral administration. After 5 days post-administration, we observed transgene expression in the small and large intestine of rats. Based on these preliminary results, NiMOS show significant potential as novel gene delivery vehicle for therapeutic and vaccination purposes.
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PMID:Gastrointestinal distribution and in vivo gene transfection studies with nanoparticles-in-microsphere oral system (NiMOS). 1747 58

A novel gene encoding transglycosylating beta-galactosidase (BGase) was cloned from Penicillium expansum F3. The sequence contained a 3,036-bp open reading frame encoding a 1,011-amino-acid protein. This gene was subsequently expressed on the cell surface of Saccharomyces cerevisiae EBY-100 by galactose induction. The BGase-anchored yeast could directly utilize lactose to produce galactooligosaccharide (GOS), as well as the by-products glucose and a small quantity of galactose. The glucose was consumed by the yeast, and the galactose was used for BGase expression, thus greatly facilitating GOS synthesis. The GOS yield reached 43.64% when the recombinant yeast was cultivated in yeast nitrogen base-Casamino Acids medium containing 100 g/liter initial lactose at 25 degrees C for 5 days. The yeast cells were harvested and recycled for the next batch of GOS synthesis. During sequential operations, both oligosaccharide synthesis and BGase expression were maintained at high levels with GOS yields of over 40%, and approximately 8 U/ml of BGase was detected in each batch.
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PMID:Cell surface engineering of a beta-galactosidase for galactooligosaccharide synthesis. 1961 84


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