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)

Recombinant adeno-associated virus (rAAV) has become a very popular gene therapy vector in the past several years. A cis-plasmid is used to generate the rAAV stocks. In this plasmid, the entire expression cassette is incorporated between two AAV inverted terminal repeats. The construction of cis-plasmid has been problematic because of the high-frequency recombination of the viral inverted terminal repeats. Here we describe the design and construction of several multiple cloning site cis-plasmids that are driven by five different promoters, including the ubiquitous cytomegalovirus enhancer/chicken beta-actin (CAG), cytomegalovirus (CMV), rous sarcoma virus (RSV), simian virus 40 (SV40), and a muscle-specific promoter (CK6). The application of these multiple cloning site cis-plasmids improves the cloning efficiency. As an example of the utilization of these multiple cloning site vectors, the prokaryotic beta-galactosidase cDNA was cloned in the multiple cloning site cis-plasmids. High-level rAAV-mediated beta-galactosidase expression was achieved in HeLa cells from CAG, CMV, RSV and SV40 promoters, respectively, but notfrom the CK6 promoter. In vivo application in the adult mdx mouse (mouse model for Duchenne muscular dystrophy) muscle revealed efficient transgene expression from CMV and CK6 promoters, followed by CAG and RSV promoters. The SV40 promoter was the least efficient.
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PMID:Development of multiple cloning site cis-vectors for recombinant adeno-associated virus production. 1223 77

A mutant herpes simplex virus 1, mtHSV, was constructed by inserting the E. coli beta-galactosidase gene into the loci of icp34.5, the apoptosis-inhibiting gene of HSV. The mtHSV replicated in and lysed U251 (human glioma cells), EJ (human bladder cells), and S-180 (mice sarcoma cells), but not Wish (human amnion cells) cells. With its intact tk (thymidine kinase) gene, mtHSV exhibited susceptibility to acyclovir (ACV), which provided an approach to control viral replication. An in vivo test with mtHSV was conducted in immune-competent mice bearing sarcoma S-180 tumors, which were treated with a single intratumoral injection of mtHSV or PBS. Tumor dimensions then were measured at serial time points, and the tumor volumes were calculated. Sarcoma growth was significantly inhibited with prolonged time and reduced tumor volume. There was microscopic evidence of necrosis of tumors in treated mice, whereas no damage was found in other organs. Immunohistochemical staining revealed that virus replication was exclusively confined to the treated tumor cells. HSV-1 DNA was detected in tumors, but not in the other organs by a polymerase chain reaction analysis. From these experiments, we concluded that mtHSV should be a safe and promising oncolytic agent for cancer treatment.
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PMID:Gene therapy for mice sarcoma with oncolytic herpes simplex virus-1 lacking the apoptosis-inhibiting gene, icp34.5. 1289 96

Further advantages in the treatment of soft-tissue sarcomas will only be achieved by tailoring the adjuvant therapy after surgery. The photochemically directed release of macro-molecules from endosomes and lysosomes into the cytosol is a novel technology, named photochemical internalization (PCI), that has been evaluated for treatment of sarcoma cells in vitro. Two human synovial sarcoma cell lines (SW 982 and CME-1) were treated with the photosensitizer meso-tetraphenylporphine with two sulfonate groups on adjacent phenyl rings (TPPS2a) and a plasmid encoding enhanced green fluorescent protein (EGFP) complexed to poly-L-lysine to investigate the influence of PCI on gene transfer and with 5 micrograms/mL gelonin to investigate PCI of a Type-I ribosome-inactivating protein toxin. In addition, both cell lines were transduced with an Adenovirus serotype 5 encoding the Escherichia coli lacZ gene (AdHCMV-lacZ, expressing beta-galactosidase) and treated with TPPS2a and light to evaluate the effect of PCI on the transduction rate. Photochemically induced transfection with the reporter gene EGFP in CME-1 cells increased from 0% of cells at no light to 40% of the cells after 60 s of light exposure. In contrast, the SW 982 cells showed no enhanced expression of the gene. The fraction of virally transduced cells was about doubled in both cell lines by means of PCI, although the transduction was more efficient in the CME-1 cells. Both cell lines became up to four-fold more sensitive to light when combining photochemical treatment with gelonin incubation. Our experiments showed that PCI induced the endocytic escape of therapeutic substances in cells derived from human soft-tissue sarcomas.
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PMID:Photochemical internalization enhances the cytotoxic effect of the protein toxin gelonin and transgene expression in sarcoma cells. 1455 16

Surgical resection coupled with adjuvant radiotherapy and/or doxorubicin based chemotherapy are the mainstays of synovial sarcoma (SS) treatment. Although effective as a SS adjuvant, the proposed mechanism of action of doxorubicin remains controversial. Current opinion supports DNA damage-induced apoptosis. This in vitro study used cDNA gene expression profiling to investigate whether apoptosis, alone or in combination with cell senescence, is induced by doxorubicin in SS cells. Cell cultures of the FU-SY-1 SS, the pleomorphic SW982 sarcoma, and a primary dermal fibroblast (NHDF), were exposed to 500 nM doxorubicin, and then processed for cDNA microarray analysis. The one class response option of SAM (Significance Analysis of Microarrays) was used to test for significant overexpression of 15 apoptosis-related genes and nine senescence-related genes. Drug-induced cell senescence was quantified by measuring beta-galactosidase activity. None of 15 apoptosis-related genes and only two of nine senescence-related genes were identified by SAM as significantly overexpressed in doxorubicin-treated cultures. Drug-induced senescence as reflected by beta-galactosidase activity was significantly increased (p < 0.05) only in FU-SY-1 SS cultures. Apoptosis does not appear to be a major determinant of doxorubicin-induced mortality in FU-SY-1 SS or NHDF cultures, but may impact SW982 cells via the overexpression of BAX relative to Bcl-2. Doxorubicin-induced cell senescence was prominent in FU-SY-1 SS cultures, but negligible in SW982 and NHDF cultures. Likely, both apoptosis and cell senescence contribute to doxorubicin-induced cell death in this synovial sarcoma cell line.
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PMID:Doxorubicin induces cell senescence preferentially over apoptosis in the FU-SY-1 synovial sarcoma cell line. 1670 98

Joint disease in mucopolysaccharidosis type VI (MPS VI) remains difficult to treat despite the success of enzyme replacement therapy in treating other symptoms. In this study, the efficacy of a lentiviral vector to transduce joint tissues and express N-acetylgalactosamine-4-sulphatase (4S), the enzyme deficient in MPS VI, was evaluated in vitro and the expression of beta-galactosidase was used to evaluate transduction in vivo. High viral copy number was achieved in MPS VI fibroblasts and 4-sulphatase activity reached 12 times the normal level. Storage of accumulated glycosaminoglycan was reduced in a dose dependent manner in both MPS VI skin fibroblasts and chondrocytes. Enzyme expression was maintained in skin fibroblasts for up to 41 days. Comparison of two promoters; the murine phosphoglycerate kinase gene promoter (pgk) and the myeloproliferative sarcoma virus long terminal repeat promoter (mpsv), demonstrated a higher level of marker gene expression driven by the mpsv promoter in both chondrocytes and synoviocytes in vitro. When injected into the rat knee, the expression of beta-galactosidase from the mpsv promoter was widespread across the synovial membrane and the fascia covering the cruciate ligaments and meniscus. No transduction of chondrocytes or ligament cells was observed. Transduction was maintained for at least 8 weeks after injection. These results indicate that the lentiviral vector can be used to deliver 4S to a range of joint tissues in vitro and efficiently transduce synovial cells and express beta-galactosidase in vivo.
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PMID:Lentiviral-mediated correction of MPS VI cells and gene transfer to joint tissues. 1930 42

Mutations in fused in sarcoma (FUS) have been reported to cause a subset of familial amyotrophic lateral sclerosis (ALS) cases. Wild-type FUS is mostly localized in the nuclei of neurons, but the ALS mutants are partly mislocalized in the cytoplasm and can form inclusions. We demonstrate that the C-terminal 32 amino acid residues of FUS constitute an effective nuclear localization sequence (NLS) as it targeted beta-galactosidase (LacZ, 116 kDa) to the nucleus. Deletion of or the ALS mutations within the NLS caused cytoplasmic mislocalization of FUS. Moreover, we identified the poly-A binding protein (PABP1), a stress granule marker, as an interacting partner of FUS. Large PABP1-positive cytoplasmic foci (i.e. stress granules) colocalized with the mutant FUS inclusions but were absent in wild-type FUS-expressing cells. Processing bodies, which are functionally related to stress granules, were adjacent to but not colocalized with the mutant FUS inclusions. Our results suggest that the ALS mutations in FUS NLS can impair FUS nuclear localization, induce cytoplasmic inclusions and stress granules, and potentially perturb RNA metabolism.
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PMID:Nuclear localization sequence of FUS and induction of stress granules by ALS mutants. 2067 93


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