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 study, an in vitro model has been developed to examine the interactions of macrophages with ultrahigh molecular-weight polyethylene (UHMWPE) and high-density polyethylene (HDPE) particles. Polyethylene particles are the major constituent of the material debris formed as a result of orthopedic implant wear. However, the study of polyethylene particle interactions with cells has been limited. UHMWPE (18-20 microns) and HDPE (4-10 microns) were suspended in soluble collagen type I and subsequently solidified on glass coverslips. The particle chemistry was characterized by Fourier transform infra-red spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS). Mouse cell line macrophages (IC-21) were established on the collagen-particle substrata and maintained for up to 24 h. The response of the cells to the particles was examined by light and transmission electron microscopy (LM and TEM), as well as by scanning electron microscopy (SEM), and compared to cells on control collagen surfaces without particles. Histological analysis of the samples revealed that the macrophages surrounded larger particles (18-20 microns) and the cells appeared to be attached to the surface of the particles, and the smaller particles (4-10 microns) had been phagocytosed within 2 h. Inflammatory cytokines (TNF-alpha, IL-1 alpha, IL-1 beta, and IL-6), lysosomal enzymes (beta-galactosidase and hexosaminidase), and prostaglandin E2 were released into the medium, and IL-1 alpha, IL-1 beta, PGE2, beta-galactosidase, and hexosaminidase levels were significantly increased over collagen control values. The results demonstrate active phagochemotaxis by macrophages for wear particulates and validate this model as a means of studying the specific in vitro interactions of polyethylene with cells.
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PMID:Macrophage phagocytosis of polyethylene particulate in vitro. 942 95

Prolonged culture of mesangial cells forms multifocal nodular structures, termed "hillocks," composed of cells and extracellular matrix (ECM), which may mimic the situation in the glomerular mesangium. Mesangial cells incorporated in hillocks show repressed expression of alpha-smooth muscle actin, a marker of mesangial cell activation/dedifferentiation. The aim of this study is to elucidate molecular mechanisms involved in this phenomenon, focusing on the activity of CArG box elements located in 5'-flanking region of the alpha-smooth muscle actin gene. Reporter mesangial cells were created to monitor the activity of CArG elements. These clones expressed beta-galactosidase gene (lacZ) under the control of CArG boxes. Within the hillocks, reporter cells showed repressed expression of lacZ as well as alpha-smooth muscle actin compared to the cells in two-dimensional cultures. Consistent with this result, the reporter cells embedded in collagen gel exhibited down-regulation of lacZ and alpha-smooth muscle actin transcripts. Deactivation of CArG box elements by transfection with either a dominant negative mutant of serum response factor or a dominant negative form of ternary complex factor Elk-1 led to depressed expression of alpha-smooth muscle actin gene. These data suggested that three-dimensional ECM primes mesangial cells to down-regulation of alpha-smooth muscle actin via deactivation of CArG box elements.
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PMID:Three-dimensional matrix primes mesangial cells to down-regulation of alpha-smooth muscle actin via deactivation of CArG box elements. 950 15

The proliferative capacity and cellular and biochemical characteristics of human trabecular bone osteoblasts were analysed throughout their replicative lifespan in vitro. Like several other cell types, human osteoblasts demonstrated a typical Hayflick phenomenon of cellular aging comprising a period of rapid proliferation until cumulative population doubling level (CPDL) 22 to 24, followed by a phase of slow growth and the final cessation of cell division at CPDL 32 to 34. Comparing young cells (less than 20% lifespan completed) and old cells (more than 90% lifespan completed) revealed a progressive increase in population doubling (PD) time, a decrease in attachment frequency, a decrease in the number of S-phase positive cells, a decrease in the rates of DNA, RNA and protein synthesis, an increase in the protein content per cell and an increased proportion of senescence-specific beta-galactosidase positive cells. While osteoblastic production of collagen type I decreased progressively during aging, alkaline phosphatase activity dropped rapidly after the first few passages and then remained constant during the rest of the proliferative lifespan, Significant morphological changes from thin and spindle-shaped early passage young cells to large, flattened and irregularly shaped late passage old cells full of intracellular debris were observed. In comparison, osteoblasts established from an osteoporotic bone sample showed a maximum CPDL of less than 5, had a longer PD time and exhibited abnormal senescent morphology. Thus, we have demonstrated for the first time that human osteoblasts, like several other diploid cell types, have a limited proliferative capacity in vitro and undergo aging and senescence as measured by various cellular and biochemical markers. In addition, preliminary studies show that cells from osteoporotic bone have a severely reduced proliferative capacity. This model of bone cell aging facilitates study of the molecular mechanisms of osteoblast senescence as well as factors related to osteoblast dysfunction in patients with osteoporosis.
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PMID:Demonstration of cellular aging and senescence in serially passaged long-term cultures of human trabecular osteoblasts. 960 46

The aim of this study was to investigate the role of carbohydrate moieties attached to IgA1 hinge region in IgA1 self-aggregation and adhesion to extracellular matrix (ECM) proteins previously reported in IgA nephropathy. Serum IgA1 samples isolated from healthy individuals were digested with neuraminidase (NA), NA + beta-galactosidase, and NA + beta-galactosidase + alpha-N-acetylgalactosaminidase to remove the carbohydrates from the hinge region and were named asialo, agalacto, and naked IgA1, respectively. First, polyacrylamide gel electrophoresis was performed under the native condition, and consequently, a broad band indicating IgA1 self-aggregation was clearly observed in asialo, agalacto, and naked IgA1, but not in native IgA1. However, the broad band disappeared in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under the nonreducing condition. Second, it was shown that IgA1 adhesion activities to type IV collagen, fibronectin, and laminin were significantly higher in asialo, agalacto, and naked IgA1 than in native IgA1, using enzyme-linked immunosorbent assay (asialo, agalacto, and naked versus native: P < 0.01). In addition, agalacto IgA1 had the highest affinity for all of the ECM proteins among the deglycosylated IgA1 (agalacto versus asialo and naked, P < 0.05). These results indicated that the removal of carbohydrates from the IgA1 molecule resulted in noncovalent self-aggregation and a significant increase in adhesion to the ECM proteins. It was therefore suggested that the IgA1 glycans played a protective role against aggregation and adhesion and that the underglycosylation of the IgA1 molecule found in IgA nephropathy could be involved in the nonimmunologic glomerular accumulation of IgA1.
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PMID:Protective role of IgA1 glycans against IgA1 self-aggregation and adhesion to extracellular matrix proteins. 980 90

The organotypic (raft) culture system has been shown to be a useful model for examining the effects of biochemical manipulations on various epithelial cell types, using in vitro conditions that simulate the in vivo environment of the tissue of origin. To investigate this method as a model for topical gene therapy, we cultured the oral head and neck squamous cell carcinoma cell line TR146 on fibroblast-containing collagen gels at the air-medium interface and assessed the efficiency of transduction of a topically applied adenoviral vector containing beta-galactosidase cDNA. Diffuse expression of -galactosidase activity in multiple cell layers demonstrated effective penetration of the vector. Transduction efficiency and therapeutic activity of a replication-defective recombinant adenovirus containing wild-type p53 cDNA linked to a FLAG marker (AdCMV-p53-FLAG) were then assessed in TR146 organotypic cultures transduced by topical application. Twenty-four, 48, and 72 h after transduction, the cultures were harvested, and residual cell number and FLAG peptide expression were determined. The number of cells in p53 transduced cultures was significantly reduced in comparison to controls at all three time points (P < 0.001), which resulted from the induction of apoptosis as determined by in situ DNA end labeling. In addition, the FLAG peptide was expressed diffusely in the residual cells, further confirming effective transduction and expression of the exogenous gene products throughout multiple layers. We conclude that the organotypic culture is an effective in vitro model for assessing the efficacy of topically applied gene therapy on head and neck squamous carcinomas and premalignancies.
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PMID:Evaluation of topical gene therapy for head and neck squamous cell carcinoma in an organotypic model. 981 13

Continuously renewing tissues, such as the epidermis, are populated by a hierarchy of dividing transient amplifying cells, which are maintained by stem cells. Transient amplifying cells divide to maintain the tissue, but they are limited to a finite number of cell divisions before they differentiate and are sloughed. Only the stem cells remain for the life of the tissue. Thus, it is critical to target stem cells when designing gene therapy regimes for genetically inherited diseases, such as epidermolysis bullosa simplex (EBS). Unfortunately, isolating pure epithelial stem cells has been problematic. In this study, we used rapid adherence to collagen type IV to successfully enrich for epidermal stem cells from adult human skin. These preselected stem cells were slow to proliferate, but they ultimately formed large colonies. When recombined with the dermal substrate AlloDerm, the stem cells re-formed a stratified squamous epidermis within 1 week after raising the AlloDerm to the air-liquid interface. These organotypic cultures grew continuously and, even after 6 weeks in culture, they maintained a proliferative basal layer. When transduced with a retroviral LacZ vector, preselected stem cells formed beta-galactosidase-positive clones in submerged and organotypic cultures. Transduced cells showed persistent expression through 12 weeks in organotypic culture, demonstrating the feasibility of using preselected stem cells for gene therapy. Currently, we are developing two models of EBS to test a gene therapy approach, which is based on the premise that EBS stem cells with a mutant keratin (K)14 gene corrected to wild type will have a growth advantage over noncorrected EBS stem cells.
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PMID:Transduction of a preselected population of human epidermal stem cells: consequences for gene therapy. 1035 57

The main cause of acute coronary syndrome may be recurrent thrombosis, which is initiated by the activation of the extrinsic coagulation pathway. Tissue factor (TF) pathway inhibitor (TFPI) efficiently inhibits an early step in this pathway by the formation of a complex with factor VIIa, TF, and factor Xa. We determined whether local TFPI gene transfer can inhibit thrombosis in an injured artery without inducing systemic side effects. Balloon-injured rabbit carotid arteries were infected with an adenoviral vector that expressed either human TFPI (AdCATFPI) or bacterial beta-galactosidase (AdCALacZ). Two to 6 days after gene transfer, thrombosis was induced by the production of constant stenosis of the artery, and blood flow was measured continuously with an electromagnetic flow probe. A cyclic flow variation, which is thought to reflect the recurrent formation and dislodgment of mural thrombi, was observed in all AdCALacZ-infected arteries as well as in saline-infused arteries. In contrast, no cyclic flow variation was detectable in AdCATFPI-transfected arteries, even in the presence of epinephrine (1 microg. kg-1. min-1 infusion). Prothrombin time, activated partial thromboplastin time, and the ex vivo platelet aggregation induced by either adenosine diphosphate or collagen were unaltered in AdCATFPI-infected rabbits. We found that in vivo TFPI gene transfer into an injured artery completely inhibits the recurrent thrombosis induced by shear stress even in the presence of catecholamine, without affecting systemic coagulation status. Adenovirus-mediated local expression of TFPI may have the potential for the treatment of human thrombosis.
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PMID:Adenovirus-mediated local expression of human tissue factor pathway inhibitor eliminates shear stress-induced recurrent thrombosis in the injured carotid artery of the rabbit. 1038 97

Collagen-induced arthritis (CIA) is an experimental model of arthritis widely used to dissect the pathogenesis of human rheumatoid arthritis and to identify potential therapeutic targets. Among these, TNF-alpha has been recognized to play an important role. Here we investigate the feasibility and therapeutic efficacy of prolonged blockade of TNF-alpha activity through the adenovirus-mediated gene delivery of a dimeric chimeric human p55 TNFR-IgG fusion protein and compare it to protein therapy in established CIA. A single i.v. administration of the replication-deficient adenovirus yielded microgram serum levels of the chimeric fusion protein and ameliorated CIA for 10 days. Subsequently, benefit was lost and a rebound to greater inflammatory activity was observed despite the continual presence of bioactive TNFR fusion protein. A similar trend was also observed in mice injected directly with comparable amounts of a human TNFR-IgG fusion protein, whereas the administration of a control adenovirus-encoding beta-galactosidase or of a control human IgG1 protein did not significantly affect the disease course. The mechanisms of the rebound of CIA were investigated, and augmented Ab response to collagen type II and TNFR were identified as potential causes. Our results confirm the feasibility of adenovirus-mediated gene delivery of cytokine inhibitors in animal models of autoimmune diseases for investigational purposes and highlight the importance of prolonged studies. Further investigations are needed to optimize ways of exploiting the potential of adenoviral gene therapy in RA.
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PMID:Paradoxical effects of adenovirus-mediated blockade of TNF activity in murine collagen-induced arthritis. 1039 98

It has been demonstrated that BMPs, IGFs, and TGFbetas improve the process of bone healing in vivo. We have suggested the use of gene therapy as a possible way to deliver growth factors to fracture sites in order to improve repair. The aim of this study was to develop a minimally invasive gene therapy approach to treat bone injuries locally without damaging the local blood circulation. A segmental defect of 1.3 cm was created in the diaphysis of the femur in mature NZW rabbits. Internal fixation with 7-hole DCP plates and 2.7 mm screws was used to stabilize the bone. After building a chamber by tightly closing the muscles around the segmental defect, 0.5 ml of either saline solution or a collagen gel containing 1 x 10(10) particles of adenovirus carrying cDNA encoding either the bacterial beta-galactosidase gene (LacZ), or the firefly luciferase gene were injected into the gap. The control side received 0.5 ml of saline solution without virus particles. Bone marrow, cortical and trabecular bone and surrounding muscle were harvested from the injected femur and were analyzed for local gene expression through X-gal staining or measurement of local luciferase activity. To determine whether distant sites were transduced, tissue from the spleen, liver, and lung were harvested as well as bone, bone marrow and muscle from the contralateral diaphysis of the femur. The delivery of the adenoviral vector suspended in saline solution led to local transduction of the bone, bone marrow and the muscle surrounding the gap. No luciferase activity was found in the contralateral femur, lung, or spleen, and only transient luciferase activity was seen in the liver. While marker gene expression persisted within the surrounding soft tissues for at least 2 weeks, the expression in bone lasted up to 6 weeks. This study has shown that it is possible to use adenoviral vectors to transfer and express genes locally within a segmental defect. Gene expression persisted for several weeks, which may be already sufficient to accelerate repair.
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PMID:A gene therapy approach to accelerating bone healing. Evaluation of gene expression in a New Zealand white rabbit model. 1040 58

In previous studies, RGD-CAP (collagen-associated protein containing the RGD sequence) isolated from a collagen fiber-rich fraction of pig cartilage was found to be orthologous to human (beta)ig-h3, which is synthesized by lung adenocarcinoma cells in response to transforming growth factor-beta. In the present study, we examined the effect of recombinant chick RGD-CAP on the spreading of chondrocytes and fibroblasts using RGD-CAP-coated dishes. When rabbit articular chondrocytes, chick embryonic sternal chondrocytes, rabbit peritoneal fibroblasts or human MRC5 fibroblasts were seeded on plastic dishes coated with RGD-CAP, cell spreading was enhanced compared with that on control dishes (bovine serum albumin- or beta-galactosidase-coated dishes). The effect of RGD-CAP on the cell spreading required divalent cations (Mg(2+) or Mn(2+)), and was reduced by EDTA. Monoclonal antibodies (mAbs) to the human integrin alpha(1) or beta(1) subunit, but not to the alpha(2), alpha(3), alpha(5) or beta(2) subunits, suppressed the RGD-CAP-induced spreading of human MRC5 fibroblasts. In a parallel experiment, the mAb to the alpha(5) subunit, but not the mAb to the alpha(1) subunit, suppressed fibronectin-induced spreading of these cells. These findings suggest that RGD-CAP is a novel ligand for integrin alpha(1)beta(1) that dose not bind to the RGD motif. Accordingly, an RGD-CAP fragment, which carries a deletion in the C-terminal region containing the RGD motif, was still capable of stimulating cell spreading.
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PMID:RGD-CAP ((beta)ig-h3) enhances the spreading of chondrocytes and fibroblasts via integrin alpha(1)beta(1). 1044 1


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