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)

To examine the role of reactive oxygen species on the invasive phenotype of cancer cells, we overexpressed manganese- and copper-zinc-containing superoxide dismutases (MnSOD, CuZnSOD) and catalase (Cat) in hamster cheek pouch carcinoma (HCPC-1) cells in vitro using adenoviral vector-mediated gene transfer. Hamster cheek pouch carcinoma cells were transduced with these adenoviral vector constructs alone, or in combination, at concentrations [i.e., multiplicity of infectivity (MOI)] of 100 MOI each. The Escherichia coli beta-galactosidase reporter construct was used as a control virus. Protein expression was examined by Western blot analysis and enzymatic activities were measured using spectrophotometry. To observe the effects of transgene overexpression on in vitro tumor cell invasion, we used the membrane invasion culture system, an accurate and reliable method for examining tumor cell invasion, in vitro. This assay measures the ability of tumor cells to invade a basement membrane matrix consisting of type IV collagen, laminin, and gelatin. MnSOD overexpression resulted in a 50% increase in HCPC-1 cell invasiveness (p < .001); co-overexpression of MnSOD with Cat partially inhibited this effect (p < .05). Moreover, co-overexpression of both SODs resulted in a significant increase in invasiveness compared with the parental HCPC-1 cells (p < .05). These changes could not be correlated with the 72 kDa collagenase IV or stromolysin activities using zymography, or the downregulation of the adhesion molecules E-cadherin or the alpha4 subunit of the alpha4beta1 integrin. These results suggest that hydrogen peroxide may play a role in the process of tumor cell invasion, but that the process does not rely on changes in matrix metalloproteinase activity in the cells, or the expression of cell adhesion molecules.
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PMID:Effects of antioxidant enzyme overexpression on the invasive phenotype of hamster cheek pouch carcinoma cells. 1049 Feb 77

The present study was aimed at investigating whether the expression of Fas ligand (FasL) by CHO cells transfected with IL-4 (CHO/IL-4) or IL-10 (CHO/IL-10) genes would improve the effect of the cytokine. DBA/ 1 mice immunized with type II collagen were treated with suboptimal doses of transfected CHO cells (a single s. c. injection of 2 x 10(5) cells) around onset of arthritis. Severe collagen-induced arthritis (CIA) developed in the control groups injected with PBS, CHO /beta-galactosidase/FasL, CHO/IL-4 or CHO/IL-10 cells. In contrast, administration of CHO/IL-4/FasL, but not CHO/IL-10/FasL, cells significantly reduced the clinical severity and resulted in rapid and sustained suppressive effect. Amelioration of CIA was not due to a prolonged in vivo secretion of IL-4 since expression of FasL by CHO cells shortened the in vivo survival of the xenogeneic cells. In fact, administration of FasL(+) cells was associated with a decreased proportion of Mac1(+) neutrophils in the blood and an increased expression of myeloperoxidase at the site of engineered cell engraftment. These findings suggest that the mechanism underlying the beneficial effect of IL-4 delivered by cells expressing FasL involves the combination of the anti-inflammatory properties of IL-4 and the apoptosis of Fas(+) Mac1(+) granulocytes participating in the pathogenic process.
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PMID:Expression of Fas ligand improves the effect of IL-4 in collagen-induced arthritis. 1060 54

The effects of steroids on a cloned pluripotential cell from bone marrow stroma were examined in vitro in culture and in vivo after the cells were transfected with a traceable gene and transplanted into host mice. Bipedal chickens were treated with steroids to establish a model for osteonecrosis. The effects of a lipid lowering agent, lovastatin, on the prevention of steroid induced adipogenesis in vitro in cell culture, and on adipogenesis and osteonecrosis in vivo in chickens, were evaluated. On treatment with dexamethasone, cloned pluripotential cells began to differentiate into adipocytes and expressed a fat specific gene, whereas the expression of Type I collagen and osteocalcin messenger ribonucleic acid decreased. Addition of lovastatin in culture inhibited steroid induced fat gene expression and counteracted the inhibitory effect of steroids on osteoblastic gene expression. Cloned pluripotential cells were transduced with a traceable retrovirus vector encoding the beta-galactosidase and neomycin resistance genes. The transfected cells were administered to mice either by tail vein or by direct intramedullary injection. Half of the animals in each group were treated with steroids. Histologic sections showed the appearance of transplanted cells in the marrow. Analysis of marrow blowouts by flow cytometry revealed that steroid treatment produced adipogenesis in transplanted cells. Evidence of osteonecrosis was observed in steroid treated chickens, whereas sections from animals treated with steroids and lovastatin showed less adipogenesis and no bone death. The results indicate that steroid induced adipogenesis in the marrow may contribute to osteonecrosis and that lovastatin may be helpful in preventing the development of steroid induced osteonecrosis.
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PMID:The Nicolas Andry award. The pathogenesis and prevention of steroid-induced osteonecrosis. 1066 Jul 25

Efficient gene delivery to the skin is important for gene therapy of skin diseases and in-depth biologic studies of epidermis. In this report, we investigated three nonviral transfection systems for gene transfer in cultured human keratinocytes and organotypic cultures. SuperFect is a highly branched polycationic transfection reagent, PrimeFector a polycationic liposome compound, and the AVET (adenovirus-enhanced transferrin-mediated) system consists of a ternary complex of biotinylated chemically inactivated adenovirus noncovalently complexed with plasmid DNA and polylysine-transferrin. After AVET transfection of cultured keratinocytes with pCIbetagal, a CMV/beta-galactosidase reporter plasmid, 28.8% +/- 1.4% of the cells were stained blue. SuperFect was about 2-fold less efficient, whereas Primefector did not transfect keratinocytes. Similar results were obtained when transfection efficiencies were measured by enzyme assays. Addition of holotransferrin to the culture medium or replacement of polylysine-transferrin by polylysine in the ternary complex did not affect the transfection efficiency. Using AVET complexes without adenovirus, however, strongly diminished gene delivery. This indicates that the AVET complex is taken up by an adenovirus receptor. Separation of AVET/pCIbetagal transfected keratinocytes by adhesion to collagen IV into two fractions (rapidly and slowly adhering cells) showed that the latter were transfected at a 3-fold higher level. Therefore, it seems that putative stem cells adhering rapidly to collagen IV are not efficiently transfected by AVET. AVET-transfected keratinocytes derived from keratinocyte trans- glutaminase negative lamellar ichthyosis patients with a CMV-TGK expression plasmid showed that it is possible to reach a level of total enzyme activity similar to that found in cultured keratinocytes from normal individuals. In organotypic cultures from outer root sheath cells AVET transfection was not successful, which might be due to the presence of the cornified layer or inaccessibility of the adenovirus receptor. In summary, the AVET system provides a powerful tool for transient in vitro transfection of keratinocytes.
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PMID:Efficient in vitro transfection of human keratinocytes with an adenovirus-enhanced receptor-mediated system. 1073 70

Type XI collagen, a heterotrimer composed of alpha1(XI), alpha2(XI) and alpha3(XI), is primarily synthesized by chondrocytes in cartilage and is also present in some other tissues. Type XI collagen plays a critical role in collagen fibril formation and skeletal morphogenesis. We investigated a tissue-specific transcriptional enhancer in the first intron of the alpha2(XI) collagen gene (Col11a2). Transient transfection assays using reporter gene constructs revealed that a 60-base pair (bp) segment within intron 1 increased promoter activity of Col11a2 in rat chondrosarcoma cells but not in either BalB/3T3 cells or undifferentiated ATDC5 cells, suggesting that it contained cell type-specific enhancer activity. In transgenic mice, this 60-bp fragment was also able to target beta-galactosidase expression to cartilage including the limbs and axial skeleton, with similar localization specificity as the full-length intron 1 fragment. Competition experiments in gel shift assays using mutated oligonucleotides showed that recombinant Sox9 bound to a 7-bp sequence, CTCAAAG, within the 60-bp segment. Anti-Sox9 antibodies supershifted the complex of the 60-bp segment with recombinant Sox9 or with rat chondrosarcoma cell extracts, confirming the binding of Sox9 to the enhancer. Moreover, a site-specific mutation within the 7-bp segment resulted in essentially complete loss of the enhancer activity in chondrosarcoma cells and transgenic mice. These results suggest that the 7-bp sequence within intron 1 plays a critical role in the cartilage-specific enhancer activity of Col11a2 through Sox9-mediated transcriptional activation.
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PMID:Identification of an enhancer sequence within the first intron required for cartilage-specific transcription of the alpha2(XI) collagen gene. 1923 80

Treatments for rheumatoid arthritis and other inflammatory arthropathies are often ineffective at preventing joint destruction. Long-term genetic modification of the cells lining the joint space (synoviocytes) in vivo represents a potential method for the treatment of these chronic conditions. However, a vector capable of efficiently transducing synoviocytes in vivo for a persistent period has not been available. The present report describes the genetic modification of synoviocytes in vivo using recombinant adeno-associated virus. High-titer adeno-associated virus encoding the gene for Escherichia coli beta-galactosidase was injected into the knee joints of mice. Synovial tissues were then examined for beta-galactosidase transgene expression by in situ staining and by fluorometry. High-efficiency, persistent transgene expression was observed in the synovium with no evidence of vector-induced inflammation. Expression was observed for at least 7 months and was higher in arthritic than nonarthritic mice. Gene transfer of murine IL-4 to the joints of mice with collagen-induced arthritis led to detectable levels of IL-4 in the joint and protection from articular cartilage destruction. These data suggest that adeno-associated virus may be a useful vector for gene delivery to the synovium for the treatment of inflammatory arthropathies.
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PMID:Adeno-associated virus mediates long-term gene transfer and delivery of chondroprotective IL-4 to murine synovium. 1094 42

Vascular endothelial growth factor (VEGF) is required for endothelial cell differentiation, vasculogenesis, and normal glomerular vascularization. To examine whether VEGF plays a role as a chemoattractant for the developing kidney vasculature, avascular metanephric kidneys from rat embryos (E14) were cocultured with endothelial cells. To determine whether VEGF directly provides chemoattractive guidance for migration, we examined migration of endothelial cells toward VEGF-coated beads. Mouse glomerular endothelial cells expressing beta-galactosidase (MGEC) were isolated from Flk-1(+/-) heterozygous mice and passaged 4-12 times. Upon 24 h culture on collagen I gels MGEC formed a lattice or capillary-like network. Embryonic metanephroi were cocultured with MGEC on collagen I gels for 1-6 days in defined media, stained for beta-galactosidase, and examined by light microscopy. Metanephric organs induced a rearrangement of the endothelial cell lattice and attracted MGEC. MGEC invaded the metanephric organs forming capillary-like structures within and surrounding the forming nephrons. This process was accelerated and amplified by low oxygen (3% O(2)) and was prevented by anti-VEGF neutralizing antibodies. MGECs migrated toward VEGF-coated beads, whereas PBS-coated beads did not alter MGEC networks. We conclude that VEGF produced by the differentiating nephrons acts as a chemoattractant providing spatial direction to developing capillaries toward forming nephrons during metanephric development in vitro.
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PMID:VEGF spatially directs angiogenesis during metanephric development in vitro. 1107 74

A key impediment to the development of effective virus-mediated gene therapy for cancer is the low level of gene transfer that occurs after the administration of recombinant viral vectors. Improving in vivo infection and transduction efficiency is an important goal for gene therapy. The limited distribution of gene delivery is particularly problematic when large vectors such as recombinant adenoviruses and retroviruses are used to mediate transgene delivery to solid tumors. To facilitate the spread of virus, we have investigated the potential of administering proteases prior to the intratumoral inoculation of recombinant replication deficient adenovirus. For these studies, we chose proteases that are active against collagen and the other extracellular matrix proteins found in primary brain tumor tissue, but are not widely expressed in normal brain. Various concentrations of a mixture of collagenase/dispase or trypsin were inoculated into xenografts of human glioblastoma multiforme-derived brain tumor cell lines U87, U251, and SF767. Subsequently, recombinant adenovirus encoding the beta-galactosidase gene was administered and tumor tissue was examined for evidence of virus infection. Both collagenase/dispase and trypsin enhanced virus infection, indicating that protease pretreatment may be a useful strategy for enhancing virus-mediated gene transduction for many in vivo applications.
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PMID:Pretreatment with protease is a useful experimental strategy for enhancing adenovirus-mediated cancer gene therapy. 1108 79

Amplification of multipotential stem cells, with or without ex vivo gene transfer, offers the potential for their use for beneficial repopulation of a host in which there is specific cellular deficiency or functional impairment. The aims of the current study were to immunoselect, genetically mark, and determine the fate of fibroblastic progenitor cells in vivo. A monoclonal antibody, HOP-26, which has high reactivity with a cell surface antigen present on human osteoprogenitors in bone marrow fibroblast populations, was used to select these cells by immunopanning. Following culture in 10% FCS in alphaMEM containing ascorbate-2-phosphate and dexamethasone the amplified cells expressed the osteoblast phenotype as determined by expression of osteocalcin protein determined immunohistochemically, and Type I collagen and osteocalcin mRNA expressions determined by RT-PCR analysis. The selected cells were genetically labeled using a murine leukemia virus (MuLV) encoding a reporter gene (lacZ) with a selective marker gene (neo(r)) using a triple transient transfection protocol. Transfected cells were implanted in CB17 scid/scid mice by local subcutaneous injection over the calvariae. Localization of the genetically marked cells within the calvarial tissues was detected by beta-galactosidase histochemistry and immunocytochemistry. Genetically marked cells were observed within the periosteal layer in close association with the osteoblast layer, covering mineralized bone surfaces and within bone osteoid at 5 and 7 days after injection. This study demonstrates the successful selection, expansion, and retroviral-marking of human osteoprogenitors and their migration and localization within calvariae of SCID mice following in vivo implantation. These basic studies indicate the migration of these cells to skeletal sites and support possibilities for future uses of human osteoprogenitors in therapy of bone deficiency diseases and the potential for development of gene therapy procedures in these conditions.
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PMID:Retroviral marking of human bone marrow fibroblasts: in vitro expansion and localization in calvarial sites after subcutaneous transplantation in vivo. 1116 57

Regeneration of damaged periodontal tissues is mediated by periodontal cells, but a major sub-population comprises highly differentiated cells that do not renew. To overcome the loss of specialized cell types caused by disease, various therapeutic approaches including cell transplants have been developed to promote cell re-population in periodontal tissues. As previous transplantation studies used unlabeled cells, that are indistinguishable from host cells, it has been difficult to assess the contributions of transplanted cells to the healing processes. To track the fate and differentiation of rat periodontal cells transplanted into periodontal wounds, we used collagen-coated fluorescent beads as a permanent endocytosed marker, or cells constitutively expressing beta-galactosidase. We assessed osteogenic cell differentiation with immunohistochemical staining for osteopontin and bone sialoprotein. Cells were transplanted into periodontal wounds created in Sprague--Dawley male rats that are null for beta-galactosidase. Defects were allowed to heal spontaneously (controls), or were closed with collagen implants mixed with beta-galactosidase-positive (Lac-Z) periodontal cells, or closed with collagen implants mixed with periodontal cells loaded with fluorescent beads. Animals were killed at 1 and 2 weeks after surgery and tissues were prepared for morphometric assessment and immunostaining for osteopontin (OPN) and bone sialoprotein (BSP). Transplanted cells were easily distinguished by fluorescent beads or by beta-galactosidase-positive expression and were distributed throughout the regenerating periodontal ligament (PL) and alveolar bone. At 1 week after wounding, animals treated with beta-galactosidase-positive cells exhibited a slightly higher percentage of labeled cells in the PL compared with the fluorescent bead-labeled cell implant group (2% vs. 1% respectively; P > 0.2). At Week 2 percentages of labeled cells were slightly increased in the regenerating PL (approximately 3% for both groups, P > 0.2). In regenerating alveolar bone at 1 week, animals that were treated with beta-galactosidase-positive cells and fluorescent bead-loaded cells exhibited approximately 30% and 25% of labeled cells respectively. At 2 weeks after wounding there was an increase in the percentage of transplanted beta-galactosidase-positive cells (approximately 39% at week 2; P < 0.05), but not of transplanted cells with fluorescent beads (approximately 25% at week 2). In sites with transplanted cells there were higher percentages of OPN positive and BSP positive cells in nascent bone and more newly formed bone than in controls (>40%; P < 0.05). Transplantation of beta-galactosidase-positive cells or cells loaded with fluorescent beads is a useful method for assessing the fate and differentiation of periodontal cells in vivo. Fluorescent beads, however, are diluted at mitosis and this method underestimates the percentage of transplanted cells. As transplanted periodontal cells in both groups promoted regeneration of alveolar bone, cell transplantation could improve the restoration of periodontium destroyed by periodontitis.
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PMID:Transplantation of labeled periodontal ligament cells promotes regeneration of alveolar bone. 1116 14


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