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)

A series of anthracycline prodrugs containing an immolative spacer was synthesized for application in selective chemotherapy. The prodrugs having the general structure anthracycline-spacer-beta-glycoside were designed to be activated by beta-glucuronidase or beta-galactosidase. Prodrugs with -chloro, -bromo or -n-hexyl substituents on the spacer were synthesized as well as prodrugs containing a -beta-glucuronyl, -beta-glucosyl or -beta-galactosyl carbamate specifier. The key step in the synthesis of all prodrugs is the highly beta-diastereoselective addition reaction of the anomeric hydroxyl of a glycosyl donor to a spacer isocyanate resulting in the respective beta-glycosyl carbamate pro-moieties. The resulting protected pro-moieties were coupled to an anthracycline. Prodrugs were evaluated with respect to activation rate by the appropriate enzyme and additionally, their IC50 values were determined. Optimal prodrugs in this study were at least 100- to 200-fold less toxic than their corresponding drug in vitro and were activated to the parent drug in a half-life time of approximately 2 h.
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PMID:Novel anthracycline-spacer-beta-glucuronide,-beta-glucoside, and -beta-galactoside prodrugs for application in selective chemotherapy. 1048 52

Efficient pulmonary vascular gene transfer in neonates would aid in understanding the pathophysiology of, and ultimately allow the development of specific therapies for, pulmonary vascular diseases. The purpose of this study was to optimize efficiency, and evaluate the duration, of catheter-based adenoviral vector-mediated pulmonary artery gene transfer in newborn pigs. An adenovirus vector encoding LacZ was infused via percutaneously placed catheters that were advanced to segmental pulmonary arteries under fluoroscopic guidance. Optimal viral dose and duration of expression were determined by histochemical evaluation of gene transfer efficiency 72 hr, 2 weeks, and 1 month after gene delivery. The effect of protamine on the efficiency of gene transfer was studied by assaying transgene protein in lung at 72 hr. The optimal viral dose was 2 x 10(10) plaque-forming units (PFU). Seventy-two hours after infusion, expression predominated in medium-sized artery endothelial cells, 40% of which expressed beta-galactosidase. At 2 weeks, the distribution of expression had changed such that the majority of transduced cells were seen not in arteries but in gas exchange units of lung. No histochemical evidence of transgene expression was seen 1 month after virus infusion. The addition of protamine to virus infusate resulted in a fivefold increase in transgene protein product in lung tissue assayed 72 hr after gene transfer. Adenoviral vector-mediated gene transfer in neonatal swine results in high-efficiency transduction of arterial endothelial cells. However, the time course of gene transfer is not significantly prolonged compared with the adult. The addition of protamine results in a significant improvement in transduction efficiency, permitting lower doses of virus.
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PMID:Optimization of adenoviral vector-mediated gene transfer to pulmonary arteries in newborn swine. 1083 14

These studies were undertaken to determine the feasibility, safety, and efficacy of suicide gene therapy using adenoviral-mediated herpes simplex virus thymidine kinase (ADV/RSV-tk) and the prodrug ganciclovir (GCV) in an orthotopic murine bladder cancer model. We utilized a replication-defective adenoviral construct containing the beta-galactosidase gene as a control and the herpes simplex virus thymidine kinase gene as the therapeutic vector under the transcription control of the Rous sarcoma virus long terminal repeat promoter. Intravesically created, orthotopic bladder tumors were established in syngeneic C3H/He female mice. India ink injection and beta-galactosidase studies were performed to determine if transurethral administration, direct tumor injection, or the combination was the most efficient route of virus administration. Optimal dosing of ADV/RSV-tk was determined by direct tumor injection with increasing viral doses and treatment with GCV. Treatment efficacy, long-term survival, and toxicity were determined in separate but similar controlled experiments. Growth curve studies demonstrated reliable tumor formation by 14 days. Direct transvesical tumor injection resulted in the best distribution and intratumor gene expression as measured by X-gal staining. Dose-ranging experiments demonstrated an optimal viral dose of 5 x 10(8) plaque-forming units and a greater than twofold reduction in tumor growth for the animals treated with ADV/RSV-tk compared to controls. Efficacy studies demonstrated a greater than threefold reduction in tumor growth. No clinical or gross pathologic toxicity was detected. Long-term survival results suggested a survival benefit for the treatment animals compared to controls. We conclude that ADV/RSV-tk in combination with GCV provides effective therapy for orthotopic murine bladder cancer by significantly inhibiting tumor growth with limited toxicity to the host. These data provide further support for testing this suicide gene therapy strategy in human Phase I trials.
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PMID:In vivo adenovirus-mediated suicide gene therapy of orthotopic bladder cancer. 1098 51

Directed evolution of Escherichia coli beta-galactosidase into variants featuring beta-glucosidase activity was challenged. To this end, mutagenesis of lacZ was performed by replication in E. coli CC954, a mutator strain containing a DNA polymerase III defective in 3'-->5' exonuclease activity. beta-Galactosidase variants can be isolated upon mutagenesis of lacZ hosted into the self-transmissible episome F'128. Optimal evolution of lacZ can be achieved by propagation of E. coli CC954/F'128 cultures for 15 generations; further growth of mutator cultures for 37 or 55 generations imposes a high mutational load on lacZ and hinders the selection of efficiently evolved clones.
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PMID:Directed evolution of beta-galactosidase from Escherichia coli by mutator strains defective in the 3'-->5' exonuclease activity of DNA polymerase III. 1128 11

During freezing in sodium and potassium phosphate (NaP and KP) buffer solutions, changes in pH may impact the stability of proteins. Since the degradation pathways for the model proteins, monomeric and tetrameric beta-galactosidase (beta-gal), chosen for this study are governed by conformational changes (i.e., physical instability) as opposed to chemical transformations, we explored how the stresses of freezing and thawing alter the protein's native structure and if preservation of the native conformation during freeze-thawing is a requisite for optimal recovery of activity. During freezing in NaP buffer, a significant pH decrease from 7.0 to as low as 3.8 was observed due to the selective precipitation of the disodium phosphate; however, the pH during freezing in KP buffer only increased by at most 0.3 pH units. pH-induced inactivation was evident as seen by the lower recovery of activity when freeze-thawing in NaP buffer as compared to KP buffer for both sources of beta-gal. In addition, we investigated the effects of cooling rate and warming rate on the recovery of activity for monomeric and tetrameric beta-gal. Optimal recovery of activity for the NaP samples was obtained when the processing protocol involved a fast cool/fast warm combination, which minimizes exposure to acidic conditions and concentrated solutes. Alterations in the native secondary structure of monomeric beta-gal as measured by infrared spectroscopy were more significant when freezing and thawing in NaP buffer as opposed to KP buffer. Conformational and activity analyses indicate that pH changes during freezing in NaP buffer contribute to denaturation of beta-gal. These results suggest that proteins formulated in NaP buffer should be frozen and thawed rapidly to minimize exposure to low pH and high buffer salts.
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PMID:Protein denaturation during freezing and thawing in phosphate buffer systems: monomeric and tetrameric beta-galactosidase. 1136 30

The nasal mucosa may provide a simple, non-invasive route to deliver DNA encoding genes that stimulate a specific immune response. Based on this, a new approach using pCMVbeta-gal plasmid DNA complexed to the Opc meningococcal outer membrane protein was assayed for. Optimal conditions of interaction were established between recombinant Opc protein and pCMVbeta-gal plasmid DNA. Complexes were fully characterized by electrophoresis analysis, DNAse resistance assay and transmission electron microscopy. DNA-protein complexes were also evaluated in in vitro transfection experiments. After the characterisation of complexes, Balb/c mice were intranasal (i.n.) and intramuscularly (i.m.) immunized. The humoral immune response against beta-galactosidase was measured by ELISA. The proliferative response in the spleen lymph nodes was also measured. Complexes administered by i.n. route induced both systemic and mucosal antibody responses. This behavior was not observed with the naked DNA. Finally, a lymphoproliferative response specific to beta-galactosidase induced by DNA-protein complexes was also detected.
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PMID:Plasmid DNA-recombinant Opc protein complexes for nasal DNA immunization. 1139 3

The feasibility of non-viral gene transfer using liposomes is described for human fetal nigral tissue. Ventral mesencephalic explants from 6 to 12 week old fetuses were grown as free-floating roller tube cultures. For the transfection, a vector coding for beta-galactosidase driven by the Rous Sarcoma Virus promoter was used. The developmental stage of the human tissue, time in vitro and the amount of vector DNA used significantly influenced the transfection efficiency. Optimal transfection results were obtained with tissue from a 10 week old fetus, cultured for 4 days and transfected with mixtures containing 4 microg vector DNA. Histological analysis suggested that a specific population of ventral mesencephalic precursor cells were the target for the gene transfer. This finding might have implications for gene delivery and cell replacement strategies in Parkinson's disease.
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PMID:Liposome-mediated gene transfer to fetal human ventral mesencephalic explant cultures. 1147 15

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a potent immune stimulant when administered with different vaccines. Optimal use of GM-CSF resides in its ability to act locally to stimulate the proliferation and maturation of professional antigen-presenting cells (APCs) (i.e., Langerhans' cells) at the injection site. GM-CSF was engineered into a replication-incompetent recombinant avian (fowlpox) virus (rF-GM-CSF) and a single subcutaneous injection resulted in a sustained enrichment of activated dendritic cells within the regional draining lymph nodes. Those changes were attributed to local GM-CSF production at the injection site by rF-GM-CSF-infected cells. Studies were carried out in which mice were administered different types of beta-galactosidase (beta-gal)-based vaccines--whole protein, peptide, recombinant poxviruses--and GM-CSF was administered either as a single injection of rF-GM-CSF or four daily bolus injections of the recombinant protein. The use of rF-GM-CSF either improved the immune adjuvant effect, as observed for poxvirus-based vaccines, or was equivalent to rGM-CSF, as observed with the beta-gal protein vaccine. It is important to note that with either the replication-competent (vaccinia) or replication-incompetent (fowlpox) vaccines expressing LacZ, strong CTL responses directed against beta-gal were induced only when rF-GM-CSF was used as the immune adjuvant. Engineering GM-CSF into a recombinant fowlpox virus offers an excellent vehicle for the delivery of this cytokine as an immune adjuvant with specific vaccine platforms. In particular, delivery of GM-CSF via the rF-GM-CSF construct would be preferred over bolus injections of rGM-CSF when used as an immune adjuvant with whole protein or recombinant poxvirus-based vaccines. The study underscores the importance of defining the appropriate delivery form of an immune adjuvant, such as GM-CSF, relative to the immunization strategy to maximize the host immune responses against a specific antigen.
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PMID:Comparative studies of Avipox-GM-CSF versus recombinant GM-CSF protein as immune adjuvants with different vaccine platforms. 1578 Jul 40

Beta-Galactosidase production by Bifidobacterium longum CCRC 15708, Bifidobacterium longum B6 and Bifidobacterium infantis CCRC 14633 was first examined with B. longum CCRC 15708 showing the highest production of beta-galactosidase and the highest specific activity. Further study with B. longum CCRC 15708 revealed that the highest level of beta-galactosidase was produced with lactose and yeast extract as carbon and nitrogen sources, respectively. Optimal enzyme production occurred at an initial pH of 6.5 and at 37 degrees C. Under these optimum culture conditions, a maximumbeta-galactosidase activity of 18.6 U/ml could be obtained after 16 h of fermentation in a medium contain 4% lactose, 3.5% yeast extract, 0.3% K2HPO4, 0.1% KH2PO4, 0.05% MgSO4.7H2O and 0.03% L-cysteine. The highest transgalactosylation activity was also detected in this culture after 14-16 h of fermentation.
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PMID:Production of beta-galactosidase by Bifidobacteria as influenced by various culture conditions. 1598 5

A glycoside hydrolase characterized by beta-fucosidase (EC 3.2.1.38) and beta-glucosidase (EC 3.2.1.21) activities was purified from the culture medium of the anaerobic ruminal phycomycete Neocallimastix frontalis grown on 0.5% Avicel. The enzyme had a molecular mass of 120 kilodaltons and a pI of 3.85. Optimal activity against p-nitrophenyl-beta-d-fucoside and p-nitrophenyl-beta-D-glucoside occurred at pH 6.0 and 50 degrees C. The beta-fucosidase and beta-glucosidase activities were stable from pH 6.0 to pH 7.8 and up to 40 degrees C. They were both inhibited by gluconolactone, sodium dodecyl sulfate, p-chloromercuribenzoate, and Hg cation. The enzyme had K(m)s of 0.26 mg/ml for p-nitrophenyl-beta-d-fucoside and 0.08 mg/ml for p-nitrophenyl-beta-d-glucoside. The purified protein also had low beta-galactosidase activity.
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PMID:Purification and Characterization of an Aspecific Glycoside Hydrolase from the Anaerobic Ruminal Fungus Neocallimastix frontalis. 1634 24


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