Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.31 (beta-glucuronidase)
 7,680 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Methoxypoly(ethylene glycol) (PEG) modification of Escherichia coli beta-glucuronidase (betaG) was examined as a method to improve the stability and pharmacokinetics of antibody-betaG conjugates for the targeted activation of glucuronide prodrugs at tumor cells. Introduction of 3 PEG molecules did not affect betaG activity whereas higher degrees of PEG modification produced progressively greater loss of enzymatic activity. The enzyme was found to be stable in serum regardless of PEG modification. PEG-modified betaG was coupled via a thioether bond to mAb RH1, an IgG2a antibody that binds to the surface of AS-30D hepatoma cells, to produce conjugates with 3 (RH1-betaG-3PEG), 5.2 (RH1-betaG-5PEG) or 9.8 (RH1-betaG-10PEG) PEG molecules per betaG with retention of 75%, 45% and 40% of the combined antigen-binding and enzymatic activity of the unmodified conjugate RH1-betaG. In contrast to the rapid serum clearance of RH1-betaG observed in mice, the PEG-modified conjugates displayed extended serum half-lives. RH1-betaG-3PEG and RH1-betaG-5PEG also exhibited reduced spleen uptake and greater tumor accumulation than RH1-betaG. BHAMG, the glucuronide prodrug of p-hydroxyaniline mustard (pHAM), was relatively nontoxic in vivo. Injection of 6 mg/kg or 12 mg/kg pHAM i.v. depressed white blood cell numbers by 46% and 71% whereas 80 mg/kg BHAMG reduced these levels by 22%. Although the tumor/blood ratio of RH1-betaG-5PEG was adversely affected by slow clearance from serum, combined therapy of small solid hepatoma tumors with this conjugate, followed 4 and 5 days later with i.v. injections of BHAMG, cured all of seven mice with severe combined immunodeficiency. Combined treatment with a control antibody-betaG conjugate and BHAMG delayed tumor growth and cured two of six mice while treatment with pHAM or BHAMG alone was ineffective.
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PMID:Poly(ethylene glycol) modification of beta-glucuronidase-antibody conjugates for solid-tumor therapy by targeted activation of glucuronide prodrugs. 929 32

A novel murine system was developed to study the in vivo localization of xenotransplanted human cells and assess their therapeutic effect in an authentic model of disease. The beta-glucuronidase (GUSB) mutation of the mucopolysaccharidosis type VII (MPSVII) mouse was backcrossed onto the nonobese diabetic/severe combined immunodeficient (NOD/SCID) xenotransplantation strain. The resulting NOD/SCID/MPSVII mice displayed the characteristic features of lysosomal storage disease because of GUSB deficiency and were also capable of engrafting human cells. Human CD34+ hematopoietic progenitor cells from healthy, GUSB+ donors engrafted NOD/SCID/MPSVII mice in a manner similar to that of standard NOD/SCID mice. Six to 12 weeks following transplantation, 1% to 86% of the host bone marrow was positive for human CD45. By using a GUSB-specific histochemical assay, human engraftment was detected with single-cell sensitivity not only in well-characterized hematopoietic tissues like bone marrow, spleen, lymph node, and thymus, but also in other nonhematopoietic organs like liver, kidney, lung, heart, brain, and eye. Quantitative measurements of GUSB activity confirmed this expansive tissue distribution. The GUSB-specific assays were validated for their accuracy in identifying human cells through colocalization of human CD45 expression with GUSB activity in tissues of mice receiving transplants. An analysis of the therapeutic effects of engrafted human cells revealed a reduction of pathologic storage material in host organs, including the bone, spleen, and liver. Such xenotransplantation experiments in the NOD/SCID/MPSVII mouse represent a powerful approach to both study the in vivo biology of human cells and gather preclinical data regarding treatment approaches for a human disease.
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PMID:Engraftment of human CD34+ cells leads to widespread distribution of donor-derived cells and correction of tissue pathology in a novel murine xenotransplantation model of lysosomal storage disease. 1240 86

The 230-kbp murine cytomegalovirus (MCMV) genome is predicted to encode 182 open reading frames (orfs). One gene whose functional role is not known is encoded by the 762-bp m136 orf. Sequence analysis of rat cytomegalovirus (RCMV) strains Maastricht and English revealed homologous orfs, pr136, and ORF HJ4, respectively. Conservation of these orfs suggested that m136 and the RCMV homologs might play a role during virus replication. Expression of an epitope tagged form of m136 (m136-V5) yielded a polypeptide of 34 kDa that localized to the perinuclear region of transfected mouse 3T3 fibroblasts. Three independently generated MCMV m136 mutants were isolated and characterized. Mutations were introduced into the m136 orf by inserting either a beta-glucuronidase (m136-beta-gluc) or a guanosine phosphoribosyl transferase (m136-gpt) expression cassette into a unique BglII site, or by inserting a gpt cassette into a deleted region (Deltam136) of m136. No differences were observed in viral yield, plaque size, and plaque morphology between the parental strain and any of the m136 mutant viruses. In vivo analysis using a SCID mouse virulence model showed a consistently measurable attenuated phenotype for all three m136 mutants. The results showed that although the m136 gene was not essential for replication in vitro or in vivo, an intact m136 gene was necessary to yield wild type virulence during infection of the host.
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PMID:Characterization of the murine cytomegalovirus m136 gene. 1714 24

Transplanted adult progenitor cells distribute to peripheral organs and can promote endogenous cellular repair in damaged tissues. However, development of cell-based regenerative therapies has been hindered by the lack of preclinical models to efficiently assess multiple organ distribution and difficulty defining human cells with regenerative function. After transplantation into beta-glucuronidase (GUSB)-deficient NOD/SCID/mucopolysaccharidosis type VII mice, we characterized the distribution of lineage-depleted human umbilical cord blood-derived cells purified by selection using high aldehyde dehydrogenase (ALDH) activity with CD133 coexpression. ALDH(hi) or ALDH(hi)CD133+ cells produced robust hematopoietic reconstitution and variable levels of tissue distribution in multiple organs. GUSB+ donor cells that coexpressed human leukocyte antigen (HLA-A,B,C) and hematopoietic (CD45+) cell surface markers were the primary cell phenotype found adjacent to the vascular beds of several tissues, including islet and ductal regions of mouse pancreata. In contrast, variable phenotypes were detected in the chimeric liver, with HLA+/CD45+ cells demonstrating robust GUSB expression adjacent to blood vessels and CD45-/HLA- cells with diluted GUSB expression predominant in the liver parenchyma. However, true nonhematopoietic human (HLA+/CD45-) cells were rarely detected in other peripheral tissues, suggesting that these GUSB+/HLA-/CD45- cells in the liver were a result of downregulated human surface marker expression in vivo, not widespread seeding of nonhematopoietic cells. However, relying solely on continued expression of cell surface markers, as used in traditional xenotransplantation models, may underestimate true tissue distribution. ALDH-expressing progenitor cells demonstrated widespread and tissue-specific distribution of variable cellular phenotypes, indicating that these adult progenitor cells should be explored in transplantation models of tissue damage.
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PMID:Widespread nonhematopoietic tissue distribution by transplanted human progenitor cells with high aldehyde dehydrogenase activity. 1805 47

Bone marrow-derived mesenchymal stem cells (MSCs) are a promising platform for cell- and gene-based treatment of inherited and acquired disorders. We recently showed that human MSCs distribute widely in a murine xenotransplantation model. In the current study, we have determined the distribution, persistence, and ability of lentivirally transduced human MSCs to express therapeutic levels of enzyme in a xenotransplantation model of human disease (nonobese diabetic severe combined immunodeficient mucopolysaccharidosis type VII [NOD-SCID MPSVII]). Primary human bone marrow-derived MSCs were transduced ex vivo with a lentiviral vector expressing either enhanced green fluorescent protein or the lysosomal enzyme beta-glucuronidase (MSCs-GUSB). Lentiviral transduction did not affect any in vitro parameters of MSC function or potency. One million cells from each population were transplanted intraperitoneally into separate groups of neonatal NOD-SCID MPSVII mice. Transduced MSCs persisted in the animals that underwent transplantation, and comparable numbers of donor MSCs were detected at 2 and 4 months after transplantation in multiple organs. MSCs-GUSB expressed therapeutic levels of protein in the recipients, raising circulating serum levels of GUSB to nearly 40% of normal. This level of circulating enzyme was sufficient to normalize the secondary elevation of other lysosomal enzymes and reduce lysosomal distention in several tissues. In addition, at least one physiologic marker of disease, retinal function, was normalized following transplantation of MSCs-GUSB. These data provide evidence that transduced human MSCs retain their normal trafficking ability in vivo and persist for at least 4 months, delivering therapeutic levels of protein in an authentic xenotransplantation model of human disease.
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PMID:Lentiviral-transduced human mesenchymal stem cells persistently express therapeutic levels of enzyme in a xenotransplantation model of human disease. 1843 61

Breast cancers expressing human embryonic stem cell (hESC)-associated genes are more likely to progress than well-differentiated cancers and are thus associated with poor patient prognosis. Elevated proliferation and evasion of growth control are similarly associated with disease progression, and are classical hallmarks of cancer. In the current study we demonstrate that the hESC-associated factor Nodal promotes breast cancer growth. Specifically, we show that Nodal is elevated in aggressive MDA-MB-231, MDA-MB-468 and Hs578t human breast cancer cell lines, compared to poorly aggressive MCF-7 and T47D breast cancer cell lines. Nodal knockdown in aggressive breast cancer cells via shRNA reduces tumour incidence and significantly blunts tumour growth at primary sites. In vitro, using Trypan Blue exclusion assays, Western blot analysis of phosphorylated histone H3 and cleaved caspase-9, and real time RT-PCR analysis of BAX and BCL2 gene expression, we demonstrate that Nodal promotes expansion of breast cancer cells, likely via a combinatorial mechanism involving increased proliferation and decreased apopotosis. In an experimental model of metastasis using beta-glucuronidase (GUSB)-deficient NOD/SCID/mucopolysaccharidosis type VII (MPSVII) mice, we show that although Nodal is not required for the formation of small (<100 cells) micrometastases at secondary sites, it supports an elevated proliferation:apoptosis ratio (Ki67:TUNEL) in micrometastatic lesions. Indeed, at longer time points (8 weeks), we determined that Nodal is necessary for the subsequent development of macrometastatic lesions. Our findings demonstrate that Nodal supports tumour growth at primary and secondary sites by increasing the ratio of proliferation:apoptosis in breast cancer cells. As Nodal expression is relatively limited to embryonic systems and cancer, this study establishes Nodal as a potential tumour-specific target for the treatment of breast cancer.
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PMID:Embryonic morphogen nodal promotes breast cancer growth and progression. 2314 58