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)

We have characterized the epitopes for ten murine monoclonal antibodies (Mabs) to human low density lipoprotein (LDL) and studied their ability to interfere with the LDL-receptor interaction. The epitopes for the antibodies were defined by using the following approaches: 1) interaction with apoB-48; 2) interaction with apoB-100 thrombolytic fragments; and 3) interaction with beta-galactosidase-apoB fusion proteins spanning different areas of the apoB-100 sequence. The results obtained are consistent with the following map of epitopes: Mab 6E, amino acids (aa) 1-1297, Mabs 5A and 6B, aa 1480-1693, Mabs 2A, 7A, 3B, and 4B, aa 2152-2377, Mabs 8A and 9A, aa 2657-3248 and 3H, aa 4082-4306. Four Mabs (2A, 5A, 7A, and 9A) whose epitopes are located in three different areas of apoB, dramatically reduced (up to 95%) the LDL-receptor interaction on cultured human fibroblasts; Fab fragments were as effective as the whole antibodies. Mab 3H, on the other hand, increased LDL binding up to threefold. These findings are consistent with the hypothesis that several areas of apoB-100 are involved independently or in concert in modulating the apoprotein B conformation required for interaction with the LDL receptor.
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PMID:Monoclonal antibodies to human low density lipoprotein identify distinct areas on apolipoprotein B-100 relevant to the low density lipoprotein-receptor interaction. 127 88

Liver-directed gene therapy is being considered in the treatment of inherited metabolic diseases. One approach we are considering is the transplantation of autologous hepatocytes that have been genetically modified with recombinant retroviruses ex vivo. We describe, in this report, techniques for isolating human hepatocytes and efficiently transducing recombinant genes into primary cultures. Hepatocytes were isolated from tissue of four different donors, plated in primary culture, and exposed to recombinant retroviruses expressing either the LacZ reporter gene or the cDNA for rabbit LDL receptor. The efficiency of gene transfer under optimal conditions, as determined by Southern blot analysis, varied from a maximum of one proviral copy per cell to a minimum of 0.1 proviral copy per cell. Cytochemical assays were used to detect expression of the recombinant derived proteins, E. coli beta-galactosidase and rabbit LDL receptor. Hepatocytes transduced with the LDL receptor gene expressed levels of receptor protein that exceeded the normal endogenous levels. The ability to isolate and genetically modify human hepatocytes, as described in this report, is an important step towards the development of liver-directed gene therapies in humans.
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PMID:Towards liver-directed gene therapy: retrovirus-mediated gene transfer into human hepatocytes. 176 37

We have reported the use of a retroviral vector to introduce the low density lipoprotein (LDL) receptor gene into receptor-deficient Watanabe heritable hyperlipidemic (WHHL) rabbit fibroblasts (Miyanohara, A., M. F. Sharkey, D. Steinberg, J. L. Witztum, and T. Friedmann. 1988. Proc. Natl. Acad. Sci. USA. 85: 6538-6542). Because the cDNA for the LDL receptor did not contain the 5' sterol regulatory element that confers sterol-mediated inhibition of LDL receptor transcription, we did not anticipate that LDL receptor activity transduced by this vector would be sterol-responsive. However, we now demonstrate sterol-mediated down-regulation of receptor protein in the infected cells by a mechanism that appears to be mediated at a post-transcriptional level. Down-regulation of LDL receptor activity occurred when infected WHHL cells were preincubated with either LDL or cholesterol plus 25-hydroxycholesterol. Identically organized vectors bearing cDNAs encoding irrelevant genes such as firefly luciferase or bacterial beta-galactosidase exhibited no sterol regulation of reporter gene activity. Insulin receptor activity in WHHL fibroblasts and in WHHL fibroblasts infected with the LDL receptor retroviral vector was also unchanged by sterol. Transfection of LDL receptor-deficient Chinese hamster ovary (CHO) cells with a nonretroviral vector containing the same LDL receptor cDNA also resulted in sterol responsiveness of the transduced LDL receptor. These experiments suggest that the effect of sterol was specific for the LDL receptor transcript. Transgene LDL receptor mRNA levels from the infected cells were unaffected by sterol, indicating that the sterol-mediated reduction in LDL receptor activity did not result from alterations in steady state mRNA levels. These data suggest the existence of post-transcriptional level mechanisms that are responsible for sterol regulation of expression of the transduced LDL receptor gene.
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PMID:Post-transcriptional regulation of retroviral vector-transduced low density lipoprotein receptor activity. 209 Jul 10

A partial rat apo E-beta-galactosidase fusion protein was produced in Escherichia coli Y1089 infected with recombinant lambda GT11 obtained by immunoscreening of a rat liver cDNA library with an anti-rat LDL antiserum. Partial cDNA overlapped the apo E mRNA sequence coding for apo E binding domain towards the LDL(B/E) receptor up to codon for Arg-139. Fusion protein specifically bound to human fibroblasts. The high-affinity component exhibited a Kd of 5 x 10(-8) M and 4.1 x 10(5) sites per cell. Fusion protein binding to fibroblasts was mediated by their apo E moiety and not by beta-galactosidase since: (1) specific binding of fusion protein was competed out by human LDL; (2) beta-galactosidase did not compete with fusion protein binding; and (3) human fibroblasts from a patient with familial hypercholesterolemia, deficient in LDL(B/E) receptor, bound fusion protein 10-times lower than control fibroblasts. It was demonstrated that partial fusion protein retained the functional activity of the native apo E. However, compared to full-length native or engineered apo E, fusion protein was able to bind fibroblasts without being complexed with phospholipids. Fusion proteins might be a useful tool for studying the functional efficiency of the LDL(B/E) receptor and for mapping residues and domains involved in the binding process.
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PMID:Partial apolipoprotein E-beta-galactosidase fusion protein expressed in Escherichia coli retains binding activity to the LDL(B/E) receptor. 222 83

The ex vivo approach to hepatic gene therapy involves several steps, which include the isolation and culture of hepatocytes, followed by their transduction with a retrovirus. Subsequently, autologous hepatocytes are transplanted. The number of hepatocytes that can be transduced by retroviruses bearing the therapeutic gene is one of the limiting steps that can impair the success of this strategy. We presently describe an experimental approach that leads to improved transduction efficiency in mouse and human hepatocytes in vitro. By using a recombinant retrovirus bearing the Escherichia coli beta-galactosidase gene, we show that addition of growth factors to the cells, namely human hepatocyte growth factor (HGF), allows marked increase in the transduction efficiency in mouse (up to 80%) and human (40%) hepatocytes. Familial hypercholesterolemia (FH) is due to mutation in the low-density lipoprotein (LDL) receptor gene and results in a deficiency in LDL receptors. Transduction of the human LDL receptor cDNA under the transcriptional control of the L-type pyruvate kinase promoter-activator into mouse hepatocytes led to an elevated tissue-specific expression of the human protein. These results suggest that the ex vivo approach remains a promising alternative for hepatic gene therapy.
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PMID:Efficient retroviral-mediated gene transfer into primary culture of murine and human hepatocytes: expression of the LDL receptor. 753 67

Apolipoprotein (apo) B-100 is the major protein component in low density lipoprotein (LDL); it contains the binding domain for the LDL receptor and the attachment site for apolipoprotein(a) in lipoprotein(a). ApoB-48 is colinear with the amino-terminal half of apoB-100 and misses the part of the molecule required for LDL receptor interaction and lipoprotein(a) formation. ApoB-48 mRNA is produced by the editing of apoB-100 mRNA, a process by which the codon CAA for Gln-2153 is changed to UAA, an in-frame stop codon. We used the cloned catalytic component of the rat apoB mRNA-editing enzyme (REPR) to construct a replication-defective recombinant adenoviral vector containing REPR cDNA (AvREPR) and a control vector (Av1LacZ4) containing a beta-galactosidase cDNA to investigate the effect of REPR gene delivery in C57BL/6 mice. Intravenous injection of AvREPR in mice resulted in efficient transduction of liver cells, where REPR mRNA and protein were overexpressed, reaching a peak at 7 and 12 days, returning toward control levels at 39 days after AvREPR administration. ApoB mRNA editing activity in liver extracts showed changes parallel to those of REPR mRNA expression; the proportion of edited apoB mRNA in the total hepatic apoB mRNA increased from approximately 60% to more than 90% at the peak of REPR expression. The proportion of plasma apoB-100 in AvREPR-transduced animals decreased from approximately 50% to < 10% of total plasma apoB concentration. Plasma very low density lipoproteins were polydisperse in control animals with an average diameter of 54.9 +/- 20.6 nm (uninjected control) and 54.7 +/- 16.8 nm (Av1LacZ4-treated), respectively. They became much smaller (average diameter 39.3 +/- 12.7 nm) and more uniform in size at day 12 following AvREPR administration. On the same day, the normal plasma LDL (26.2-25.5 nm) was almost completely eliminated in treated animals. Adenovirus-mediated transfer of the REPR cDNA is an efficient method to reduce plasma apoB-100 and normal LDL production.
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PMID:Adenovirus-mediated gene transfer of rat apolipoprotein B mRNA-editing protein in mice virtually eliminates apolipoprotein B-100 and normal low density lipoprotein production. 796 18

We have explored the use of adenovirus-mediated gene transfer to transiently elicit production of low density lipoprotein (LDL) receptors in mice. A recombinant adenovirus carrying the human LDL receptor cDNA restored LDL receptor function in receptor-deficient cultured cells. Intravenous injection of recombinant virus acutely lowered plasma cholesterol levels and increased the rate of 125I-labeled LDL clearance from the circulation in normal mice. At 4 days after virus injection, the t1/2 of plasma LDL was reduced up to 10-fold. An estimated 90% of the parenchymal cells in liver expressed the adenovirus-transferred genes as judged by immunofluorescence of LDL receptors or by beta-galactosidase staining. These results demonstrate that adenovirus-mediated transfer of the LDL receptor gene provides an efficient way of augmenting LDL receptor gene function in the liver over the short term.
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PMID:Adenovirus-mediated transfer of low density lipoprotein receptor gene acutely accelerates cholesterol clearance in normal mice. 846 93

Several lines of evidence suggest that the cellular enzyme 15 lipoxygenase (15-LO) may be important in promoting the oxidation of lipoproteins in vivo. In previous studies we have shown that fibroblasts transfected with 15-LO "seed" LDL with lipoperoxides such that subsequent oxidation readily generates an LDL that is taken up by macrophages through scavenger receptors. We now demonstrate that LDL incubated with 15-LO cells is "minimally modified" and has bioactive properties. Characterization of LDL incubated with 15-LO cells reveals that lipid peroxidation is modest, with low levels of TBARS generated (12.6 +/- 4.7 nmole MDA per mg protein) and small amounts of 18:2 lost as a result of oxidation (7%, compared with extensive loss [82%] with copper oxidation). The 15-LO-conditioned LDL showed mildly increased electrophoretic mobility on agarose gels, and on polyacrylamide gels it showed only mild protein degradation compared with copper-oxidized LDL. Additionally 15-LO-conditioned LDL competed very well for the LDL receptor of fibroblasts but did not compete for macrophage uptake of 125I-acetylated LDL. Importantly, compared with LDL incubated on beta-galactosidase (lac Z)-transfected control cells, LDL incubated on 15-LO cells stimulated monocyte chemotaxis (15-LO-LDL, 6.9 +/- 1.2 monocytes per field versus lac Z-LDL, 0 +/- 0.9 monocytes per field) and when added to endothelial cells enhanced adhesion (15-LO-LDL, 31.1 +/- 5.0 monocytes per field versus lac Z-LDL, 0 +/- 2.0 monocytes per field). Preincubation of 15-LO cells with 15-LO inhibitors significantly inhibited the generation of bioactive LDL. Lipid extracts of LDL conditioned on 15-LO cells showed chemotactic activity not related to lysophosphatidylcholine levels. Preincubation of target endothelial cells with several different platelet-activating factor receptor antagonists prevented stimulation of monocyte adhesion by 15-LO-conditioned LDL. When probucol- or vitamin E-enriched LDL was incubated with 15-LO cells it was less oxidized and less bioactive, which suggests that these cells seed LDL with LOOH, which then requires further propagation of lipid peroxidation to yield bioactivity. These studies demonstrate that fibroblasts expressing 15-LO reliably produce a bioactive "minimally modified" LDL, which may explain in part how cellular 15-LO activity may generate atherogenic LDL in vivo.
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PMID:Fibroblasts that overexpress 15-lipoxygenase generate bioactive and minimally modified LDL. 943 16

We have investigated the interaction of apolipoprotein E2(Arg158-Cys) (apoE2) and apolipoprotein E3-Leiden (apoE3-Leiden) with the very low density lipoprotein (VLDL) receptor in vivo and in vitro to define the possible role of this receptor in lipoprotein metabolism and atherosclerosis. The in vivo binding specificity of the VLDL receptor for apoE2 and apoE3-Leiden was investigated by adenovirus-mediated gene transfer of the VLDL receptor in apoE2 and apoE3-Leiden transgenic mice lacking endogenous mouse apoE (Apoe-/-). Ectopic overexpression of the VLDL receptor gene in the liver resulted in a >50% decrease of plasma cholesterol levels in both apoE2 and apoE3-Leiden transgenic mice compared with liver expression of the beta-galactosidase gene. This reduction in plasma cholesterol was mainly due to a reduction in the VLDL level. Overexpression of the VLDL receptor did not affect the hepatic VLDL triglyceride production, indicating that the hypocholesterolemic effect is due to an increased level of plasma clearance mediated by the VLDL receptor. In vitro binding analysis showed that both apoE2 and apoE3-Leiden VLDL compete efficiently with rabbit beta-VLDL for binding to the VLDL receptor expressed on LDL receptor-deficient Chinese hamster ovary cells. We conclude from these data that both apoE2 and apoE3-Leiden function as proper ligands for the VLDL receptor in vitro and in vivo. This finding substantiates a possible role for the VLDL receptor in atherosclerosis in hyperlipidemic subjects homozygous for apoE2 or carrying apoE3-Leiden and indicates that the VLDL receptor expressed on the liver has therapeutic potential as an alternative route for clearance of binding-defective lipoproteins.
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PMID:Reversal of hypercholesterolemia in apolipoprotein E2 and apolipoprotein E3-Leiden transgenic mice by adenovirus-mediated gene transfer of the VLDL receptor. 944 49

1. Recent studies have revealed an association between coronary risk factors and both the number and function of bone marrow-derived endothelial progenitor cells (EPC). We investigated the effect of oxidized low-density lipoprotein (ox-LDL) on the senescence of EPC, leading to cellular dysfunction. 2. Endothelial progenitor cells were isolated from human peripheral blood and characterized. The exposure of cultured EPC to ox-LDL (10 microg/mL) significantly accelerated the rate of senescence compared with control during 20 days in culture as determined by acidic beta-galactosidase staining. Oxidized LDL-induced EPC senescence was significantly inhibited by pretreatment with either lectin-like ox-LDL receptor-1 (LOX-1) antibody (Ab) or atorvastatin (P < 0.01). 3. Because cellular senescence is critically influenced by telomerase, which elongates telomeres, we measured telomerase activity using a polymerase chain reaction-ELISA-based assay. Oxidized LDL significantly diminished telomerase activity to approximately 50%, an effect that was significantly abolished by pretreatment with either LOX-1 Ab or atorvastatin (P < 0.01). 4. We examined whether ox-LDL-induced EPC senescence translates into EPC dysfunction. An MTS assay disclosed an inhibitory effect of ox-LDL on EPC proliferation. In a Matrigel assay, EPC treated with ox-LDL were less likely to participate in network formation compared with controls. 5. In conclusions, ox-LDL accelerates the onset of EPC senescence, which may be related to telomerase inactivation. Oxidized LDL-induced EPC senescence leads to the impairment of proliferative capacity and network formation.
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PMID:Oxidized low-density lipoprotein induces endothelial progenitor cell senescence, leading to cellular dysfunction. 1523 25


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