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)

Hydrophobically modified dextrans, benzoyl dextran and valeryl dextran, have been used to study the interactions between tryptophan residues and benzoyl or valeryl groups by partitioning of tryptophan, tryptophan-tryptophan, (tryptophan)3, poly(lysine, tryptophan), beta-galactosidase and lysozyme in polymer aqueous two-phase systems. The two-phase systems used were polyethylene glycol (PEG)-benzoyl dextran, PEG-valeryl dextran, dextran-benzoyl dextran and dextran-valeryl dextran. Interaction between tryptophan residues and benzoyl or valeryl groups was observed by partitioning of tryptophan containing compounds to the phase containing hydrophobically modified dextran. At a certain phase composition the interactions were increased with increasing number of tryptophan per molecule. In a PEG-dextran system the partitioning of tryptophan peptides to the PEG phase was increased with increased number of tryptophan. In a PEG-benzoyl dextran system the opposite effect was obtained. At similar conditions benzoyl groups showed stronger interactions with tryptophans compared to valeryl groups. The partition coefficient of salts (sodium phosphate, NaCl, Nal and NaClO4) was determined in PEG-benzoyl dextran and PEG-valeryl dextran aqueous two-phase systems. The effect of addition of these salts on partitioning of poly(lysine, tryptophan), beta-galactosidase and lysozyme was studied. Salt effects on partitioning could be explained by the relative affinities of the ions for the polymers in the system. Charged molecules containing tryptophan were to an increasing degree partitioned to the phase for which the counterions had highest affinity. Strong effects on the partitioning of positively charged poly(lysine, tryptophan) and lysozyme were obtained with the ions I- and ClO4-.
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PMID:Interaction between tryptophan residues and hydrophobically modified dextran. Effect on partitioning of peptides and proteins in aqueous two-phase systems. 913 30

The mechanism of electrotransfer of DNA into Escherichia coli cells was investigated under conditions optimal for genetic transformation or transfection. Simple mixing in 10% polyethylene glycol 6000 did not cause binding of DNA to the recipient bacteria. When subjected to a high electric field, however, 90-98% of the input plasmid or phage DNAs were complexed with the cells. By application of the electric field, a significant amount of biotin-labeled DNA was bound onto the recipient surface, as detected by fluorescein isothiocyanate coupled avidin. When subjected to a high voltage pulse, DNA molecules were rapidly attracted toward the anode. Concurrently, the electric field induced the orientation of bacterial cells, along the field lines and their movement toward the anode. Since the bacterial movement was relatively slow, a substantial fraction of DNA molecules must strike the cathode-facing end or side of the recipient cells. Irrespective of the high efficiency of DNA transformation, the voltage pulse did not induce release of alkaline phosphate and beta-galactosidase. The electrotransferred DNA first remained sensitive to Tris-EDTA treatment, and became refractory to spheroplasting only after incubation at 37 degrees C. These results indicate that the infecting DNA is electrophoretically plugged to the outer membrane loosened by the voltage pulse.
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PMID:Initial stage of DNA-electrotransfer into E. coli cells. 927 94

Non-toxic concentrations ( 1%) of dimethyl sulfoxide (DMSO) enhance the liposomal delivery of DNA to both MCF-7 and MDA-MB-231 human breast tumor cells. Uptake of SV-40-luciferase was enhanced in MCF-7 cells by 14-fold while uptake of CMV-beta-galactosidase was increased 10-fold. In MDA-MB-231 cells, uptake of SV-40-luciferase was increased by approximately 70%. A mixture of ethanol and polyethylene glycol (45:55) at a concentration of 1% produced less pronounced improvements in transgene delivery to MCF-7 cells (a 70% increase in SV-40-luciferase uptake and a 4-fold increase in CMV-beta-galactosidase uptake) but no improvement in SV-40-luciferase gene delivery to MDA-MB-231 cells. These studies suggest that select pharmaceutical adjuvants which dissolve clinically useful drugs may have promise as non-toxic vehicles for improving transgene delivery. However, the relative effectiveness of these adjuvants is likely to vary depending on both the nature of the gene being delivered as well as the tumor cell which is the target for uptake of the exogenous gene.
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PMID:Enhancement of liposomal gene delivery in human breast cancer cells by dimethyl sulfoxide. 985 73

A carboxylesterase that is responsible for conversion of 1,4-butanediol diacrylate (BDA) to 4-hydroxybutyl acrylate (4HBA) was found in Brevibacterium lines IFO 12171, and purified to homogeneity. The purified enzyme was active toward a variety of diesters of ethylene glycol, 1,4-butanediol, and 1,6-hexanediol. The K(m) and kcat of the enzyme for BDA were 3.04 mM and 203,000 s-1, respectively. The reaction with the purified enzyme gave 98 mM 4HBA from 100 mM BDA for 60 min. The enzyme gene was cloned from the chromosomal DNA of the bacterium. The open reading frame encoding the enzyme was 1176 bp long, corresponding to a protein of 393 amino acid residues (molecular mass = 42,569 Da). The deduced amino acid sequence contained the tetra peptide motif sequence, STTK, and the serine residue was confirmed to be the catalytic center of BDA esterase by site-directed mutagenesis for several amino acid residues. The gene was expressed in Escherichia coli under the control of the lac promoter, and the gene product (a fusion protein with 6 amino acid residues from beta-galactosidase) showed the same catalytic properties as the enzyme from the parent strain.
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PMID:A new carboxylesterase from Brevibacterium linens IFO 12171 responsible for the conversion of 1,4-butanediol diacrylate to 4-hydroxybutyl acrylate: purification, characterization, gene cloning, and gene expression in Escherichia coli. 1036 81

Crystals of an extracellular beta-galactosidase from Penicillium sp. (MW = 120 +/- 5 kDa) have been obtained from a sodium phosphate buffer using PEG as precipitant. The crystals belong to the tetragonal space group P4(1)or P4(3), with unit-cell parameters a = b = 110.82, c = 161.28 A, and diffract to 1.85 A resolution at a synchrotron source.
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PMID:Purification, crystallization and preliminary diffraction study of beta-galactosidase from Penicillium sp. 1105 67

During freezing in phosphate buffers, selective precipitation of a less soluble buffer component and subsequent pH shifts may induce protein denaturation. Previous reports indicate significantly more inactivation and secondary structural perturbation of monomeric and tetrameric beta-galactosidase (beta-gal) during freeze-thawing in sodium phosphate (NaP) buffer as compared with potassium phosphate (KP) buffer. This observation was attributed to the significant pH shifts (from 7.0 to as low as 3.8) observed during freezing in the NaP buffer (1). In the current study, we investigated the impact of the additional stress of dehydration after freezing on the recovery of active protein on reconstitution and the retention of the native structure in the dried state. Freeze-drying monomeric and tetrameric beta-gal in either NaP or KP buffer resulted in significant secondary structural perturbations, which were greatest for the NaP samples. However, similar recoveries of active monomeric protein were observed after freeze-thawing and freeze-drying, indicating that most dehydration-induced unfolding was reversible on reconstitution of the freeze-dried protein. In contrast, the tetrameric protein was more susceptible to dehydration-induced denaturation as seen by the greater loss in activity after reconstitution of the freeze-dried samples relative to that measured after freeze-thawing. To ensure optimal protein stability during freeze-drying, the protein must be protected from both freezing and dehydration stresses. Although poly(ethylene glycol) and dextran are preferentially excluded solutes and should confer protection during freezing, they were unable to prevent lyophilization-induced denaturation. In addition, Tween did not foster maintenance of native protein during freeze-drying. However, sucrose, which hydrogen bonds to dried protein in the place of lost water, greatly reduced freezing- and drying-induced denaturation, as observed by the high retention of native protein in the dried state as well as the complete recovery of active beta-gal on reconstitution. These results indicate that addition of an effective stabilizer, such as sucrose, may minimize protein denaturation during freeze-drying in phosphate buffers, even if there are large-scale changes in solution pH during freezing.
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PMID:Lyophilization-induced protein denaturation in phosphate buffer systems: monomeric and tetrameric beta-galactosidase. 1174 78

The therapeutic effects of the Sonic hedgehog (Shh) have been difficult to evaluate because of its relatively short serum half-life. To address this issue polyethylene glycol modification (PEGylation) was investigated as an approach to improve systemic exposure. Shh was PEGylated by a targeted approach using cysteines that were engineered into the protein by site-directed mutagenesis as the sites of attachment. Sixteen different versions of the protein containing one, two, three, or four sites of attachment were characterized. Two forms were selected for extensive testing in animals, Shh A192C, which provided a single site for PEGylation, and Shh A192C/N91C, which provided two sites. The PEGylated proteins were evaluated for reaction specificity by SDS-PAGE and peptide mapping, in vitro potency, pharmacokinetic and pharmacodynamic properties, and efficacy in a sciatic nerve injury model. Targeted PEGylation was highly selective for the engineered cysteines and had no deleterious effect on Shh function in vitro. Systemic clearance values in rats decreased from 117.4 mL/h/kg for unmodified Shh to 29.4 mL/h/kg for mono-PEGylated Shh A192C that was modified with 20 kDa PEG-maleimide and to 2.5 mL/h/kg for di-PEGylated Shh A192C/N91C modified with 2, 20 kDa PEG vinylsulfone adducts. Serum half-life increased from 1 h for unmodified Shh to 7.0 and 12.6 h for the mono- and di-PEGylated products. These changes in clearance and half-life resulted in higher serum levels of Shh in the PEG-Shh-treated animals. In Ptc-LacZ knock-in mice expressing lacZ under regulation of the Shh receptor Patched, about a 10-fold lower dose of PEG-Shh was needed to induce beta-galactosidase than for the unmodified protein. Therapeutic treatment of mice with PEG-Shh enhanced the regeneration of injured sciatic nerves. These studies demonstrate that targeted PEGylation greatly alters the pharmacokinetic and pharmacodynamic properties of Shh, resulting in a form with improved pharmaceutical properties.
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PMID:Long-acting forms of Sonic hedgehog with improved pharmacokinetic and pharmacodynamic properties are efficacious in a nerve injury model. 1183 97

Systemic tumor-targeted gene delivery is attracting increasing attention as a promising alternative to conventional therapeutical strategies. To be considered as a viable option, however, the respective transgene has to be administered with high tumor specificity. Here, we describe novel polyethylenimine (PEI)-based DNA complexes, shielded by covalent attachment of polyethylene glycol (PEG), that make use of epidermal growth factor (EGF) as a ligand for targeting gene delivery to EGF receptor-expressing human hepatocellular carcinoma (HCC) cells. In vitro transfection of luciferase reporter DNA resulted in high levels of gene expression in the human HCC cell lines Huh-7 and HepG2. An excess of free EGF during transfection clearly reduced expression levels, indicating a specific EGF receptor-mediated uptake of the DNA particles. Following intravenous injection into human HCC xenograft-bearing SCID mice, luciferase expression was predominantly found in the tumor, with levels up to 2 logs higher than in the liver, which was the highest expressing major organ. Histologic investigation showed reporter gene expression (beta-galactosidase) localized to tumor cells. Assessing DNA distribution within the tumor by immunofluorescence microscopy, rhodamine-labelled transgene DNA was found to be mainly associated with HCC cells. In the liver, DNA was taken up almost exclusively by Kupffer cells and, as indicated by the low expression, subsequently degraded. In conclusion, we have shown that intravenous injection of PEGylated EGF-containing DNA/PEI complexes allows for highly specific expression of a transgene in human HCC tumors.
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PMID:Specific systemic nonviral gene delivery to human hepatocellular carcinoma xenografts in SCID mice. 1239 20

A gene transfer vector has been developed utilising anionic liposomes as a carrier of plasmid DNA (pEGlacZ, 7.6 kb) to transfect CD3+ T lymphocytes (Jurkat cells). The plasmid DNA that contained the Escherichia coli beta-galactosidase reporter gene was condensed using poly-l-lysine of molecular mass 20,700 (PLK99) to form a polyplex which was interacted with several anionic liposome formulations to form lipopolyplexes. The liposome formulations where based on dioleoylphosphatidylethanolamine (DOPE) in combination with cholesterol and dioleoylphosphatidylcholine (DOPC) and oleic acid, or dimyristoylphosphatidylethanolamine (DMPE). For targeting to the Jurkat cells distearoylphosphatidylethanolamine (DSPE) linked to poly (ethylene glycol) molecular mass 2,000 and coupled to anti-CD3 antibody was incorporated. The polyplexes and lipopolyplexes were characterised in terms of size, zeta potential, agarose gel electrophoresis and electron microscopy and the permeability of the lipopolyplexes to liposome-encapsulated glucose was determined. The polyplexes consisted of a mixed population of rod-like structures (53-160 nm long and 23-31 nm diameter) and spheres (18-30 nm diameter). The lipopolyplexes retained a permeability barrier although were more permeable to glucose than their component liposomes. The poly-l-lysine condensing agent was still susceptible to pronase digestion suggesting that the polyplex was associated with the outer surface of the liposome. The lipopolyplexes with lipid composition DOPE/cholesterol/OA/DSPE-PEG2000 anti-CD3+ PLK99-plasmid DNA had significant gene transfer activity, as monitored by beta-galactosidase expression, that depended on the charge ratio of the component polyplex and the lipid/DNA weight ratio. The anti-CD3 antibody, the liposomal lipid and pH sensitivity were essential for transfection activity.
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PMID:The transfection of Jurkat T-leukemic cells by use of pH sensitive immunoliposomes. 1251 28

An effective pH-sensitive gene transfer vector has been developed utilising anionic liposomes with various formulations as a carrier of plasmid DNA (pEGlacZ, 7.6 kb) to transfect CD3 T+ lymphocytes (Jurkat cells). The plasmid DNA was condensed using poly-l-lysines with a range of molecular masses to form polyplexes that were interacted with several anionic liposome formulations to form lipopolyplexes. For targeting to the Jurkat cells, distearoylphosphatidylethanolamine (DSPE) linked to poly (ethylene glycol) molecular mass 2000 and coupled to anti-CD3 antibody was incorporated in the liposomes. The polyplexes and lipopolyplexes were characterised in terms of size, zeta potential, gel electrophoresis and electron microscopy. The gene transfer activity of the lipopolyplexes, assessed from beta-galactosidase expression, depended on the charge ratio (NH(3)+/PO(4)-) of the component polyplex and the lipid/DNA weight ratio of the lipopolyplex.
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PMID:The transfection of Jurkat T-leukemic cells by use of pH-sensitive immunoliposomes. 1260 37


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