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)

To obtain silica supports for high-performance affinity chromatography, a method of preparing CNBr-activated diol-silica under anhydrous conditions was developed. Activation of the silane-derived hydroxyls with cyanogen bromide and triethylamine was optimized and demonstrated to efficiently couple several amino ligands (tryptophan, 6-aminohexyl-Cibacron Blue, and DNA). Sonication and vacuum degassing, a procedure used to remove air from the silica beads, increased activation. Coupling of an amino ligand under slightly basic conditions (pH 8.0) gave the highest yield. The linkage between the immobilized ligands and silica was stable from pH 2-10. Anhydrous acetone was the most effective solvent for activation but dimethylformamide and 2-propanol were also good choices. The high-performance affinity chromatography columns obtained by coupling sequence-specific DNA binding sequences for Lac repressor-beta-galactosidase fusion protein were compared to affinity columns obtained by coupling the same DNA element to Sepharose beads; 5 microm silica gave the best performance.
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PMID:Cyanogen bromide activation and coupling of ligands to diol-containing silica for high-performance affinity chromatography optimization of conditions. 1235 Jan 29

Chitosan has the potential for DNA complexation and is useful as a non-viral vector for gene delivery. Highly purified low molecular weight chitosan (LMWC) was prepared. Lactobionic acid (LA) bearing galactose group was coupled with LMWC for liver-specificity. A series of galactosylated-LMWC (gal-LMWC) samples covering a range of galactose group contents were prepared. The chitosan/DNA complexes were obtained using a complex coacervation process. Gal-LMWCs were used to transfer pSV-beta-galactosidase reporter gene into human hepatocellular carcinoma cell (HepG2), L-02, SMMC-7721, and human cervix adenocarcinoma cell line (HeLa) cell lines in vitro. Transfection efficiency of gal-LMWCs was evaluated by beta-galactosidase assay and compared with those of lipofectin, calcium phosphate (CaP), high molecular weigh chitosan (HMWC) and LMWC. Gal-LMWC/DNA complex shows a very efficient cell selective transfection to hepatocyte. The transfection efficiency of gal-LMWCs increased with the improvement of the galactosylation degree. Cytotoxicity of gal-LMWC was determined by 3-(4,5-dimethylthiazd-2-yl)-2,5-diphenyltentrazolium bromide (MTT) assay and the results show that the modified chitosan has relatively low cytotoxicity, giving the evidence that the modified chitosan vector has the potential to be used as a safe gene-delivery system.
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PMID:Galactosylated low molecular weight chitosan as DNA carrier for hepatocyte-targeting. 1267 2

With the aim to improve the specificity and to reduce the cytotoxicity of polyethylenimine (PEI), we have synthesized the conjugates of the branched PEI (25 kDa) with transferrin. The transferrin-PEI (TP) conjugates with five compositions were synthesized using periodate oxidation method and confirmed by FT-IR spectroscopy and gel permeation chromatography. The free amine contents of TP conjugates, which were able to condense and deliver DNA, increased as the amount of PEI increased. TP/DNA polyplexes were characterized by measuring gel electrophoresis, ethidium bromide fluorescence quenching, particle size and zeta potential of complexes. Complete complexation of the polyplexes was observed above the N/P ratio of 5 in TP/ DNA, and above 3 in PEI/DNA, respectively. The zeta potential of the complexes decreased as the amount of transferrin in TP conjugates increased. Transfection efficiency of TP conjugates was evaluated in HeLa cell and Jurkat cell systems. Among the five compositions of TP conjugates, TP-2 system mediated a higher beta-galactosidase gene expression than PEI system in Jurkat cell which was known to express elevated numbers of transferrin receptors. From the results of the cell viability based on MTT assay, TP conjugates showed lower cytotoxicity compared with the PEI system. We expect that the TP conjugate can be used efficiently as a nonviral gene delivery vector.
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PMID:Evaluation of transferrin-polyethylenimine conjugate for targeted gene delivery. 1604 83

Immobilization of insect cells using porous biomass support particles (BSPs) and production of a recombinant protein by the immobilized cells after infection with a baculovirus were investigated in a shake-flask culture. Sf9 cells were passively immobilized in reticulated polyvinyl formal (PVF) resin BSPs (2 x 2 x 2 mm cubes) with matrices of 60 mum mean pore diameter in situ in shake-flasks. The cell density in the BSPs was over 5 x 10(7) cells/cm3-BSP in cultures with regular replacement of the culture medium, as estimated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After infection with a recombinant baculovirus carrying the beta-galactosidase gene, immobilized cells within the BSPs showed a high specific productivity, comparable to the maximum productivity in shake-flask cultures of non-immobilized cells, as long as nutrients in the medium were not depleted. Even when immobilized cells at a high density of 5 x 10(7) cells/cm3-BSP were infected with the baculovirus, efficient beta-galactosidase production with a high specific productivity was possible by replacing the medium at appropriate intervals to avoid nutrient depletion.
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PMID:Production of recombinant protein by baculovirus-infected insect cells in immobilized culture using porous biomass support particles. 1623 92

The purpose of this research was to evaluate chitosan lactate (CL) of different molecular weights (MWs) as a DNA complexing agent for its efficiency in transfecting COS-1 cells (green monkey fibroblasts) and its effect on cell viability compared with polyethylenimine (PEI), a commercially available cationic polymer. CL and chitosan base dissolved in dilute acetic acid (chitosan acetate [CA]) of different MWs (20, 45, 200, 460 kDa) and N/P ratios (2:1, 4:1, 8:1, 12:1, 24:1) formed complexes with pSV beta-galactosidase plasmid DNA. The complexes were characterized by agarose gel electrophoresis and investigated for their ability to transfect COS-1 cells compared with PEI. Additionally, the effect of CL on the viability of COS-1 cells was investigated using 3-(4,5-dimethyliazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The binding of CL/DNA and CA/DNA was dependent on chitosan MWs. The N/P ratio of CL to completely form the complex with the DNA was higher than that of CA. Both CL and CA were comparable in transfection efficiencies at an N/P ratio of 12:1, but less efficient than PEI (P < .05). The cell viability in the presence of CL and CA at all MWs was over 90%, whereas that of PEI-treated cells was approximately 50%. These results suggest the advantage of CL for in vitro gene transfection, with the ease of preparation of polymer/DNA complexes and low cytotoxicity.
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PMID:Chitosan lactate as a nonviral gene delivery vector in COS-1 cells. 1702 47

We have reported previously that a basic peptide, arginine peptide, can be used as an efficient system for delivery of foreign genes. In this work, to better understand the mechanism of arginine peptide-mediated gene delivery, we further evaluated the process of cellular uptake and nuclear localization of the peptide/DNA complex. To investigate the effect of cellular proteoglycans on arginine peptide/DNA complexes, interactions between polyanionic glycosaminoglycans (GAGs) and peptide/DNA complexes were examined by the ethidium bromide interaction assay. Sulfated GAGs were found to relax the complexed DNA at low peptide/DNA charge ratios. Condensed peptide/DNA complexes facilitate cellular uptake, but their mechanism of uptake is poorly understood. Studies of various endocytosis inhibitors suggested that the peptide/DNA complex internalization involved the caveolar-related endocytosis pathway. A critical step in the gene delivery is the cytosol-to-nucleus transport of exogenous DNA following initial complex uptake. Nuclear localization of peptide/DNA complex was confirmed by confocal laser scanning microscopic observation. Further, we show that transfections with peptides result in an early accumulation of plasmid DNA in the nucleus of growth-arrested cells, which suggest nuclear transport. To assess the potential for arginine peptide as an agent for therapeutic gene delivery, in vivo complexed DNA transduction studies were performed. Mice were injected subcutaneously with the reporter gene beta-galactosidase, resulting in high levels of gene expression in dermal tissue.
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PMID:Characterization of gene delivery in vitro and in vivo by the arginine peptide system. 1715 Mar 15

The enzyme beta-galactosidase, encoded by the bacterial gene lac-Z, is commonly used as a histochemical reporter to track transplanted cells in vivo or to analyze temporospatial gene expression patterns by coupling expression of specific target genes to beta-galactosidase activity. Previously, endogenous beta-galactosidase activity has been recognized as a confounding factor in the study of different soft tissues, but there is no description of the typical background on bone marrow sections when using the chromogenic substrate 5-Bromo-4-chloro-3-indolyl beta-D-Galactoside (X-Gal). In this report, we show that osteoclasts in bone marrow sections specifically and robustly stain blue with X-Gal. This leads to a typical background when bone marrow is examined that is present from the first day post partum throughout the adult life of experimental mice and can be confused with transgenic, bacterial beta-galactosidase expressing hematopoietic or stromal cells. Experimental variations in the X-Gal staining procedure, such as pH and time of exposure to substrate, were not sufficient to avoid this background. Therefore, these data demonstrate the need for strenuous controls when evaluating beta-galactosidase positive bone marrow cells. Verifiable bacterial beta-galactosidase positive bone marrow cells should be further identified using immunohistological or other approaches. Specifically, beta-galactosidase positive hematopoietic or stromal cells should be proven specifically not to be osteoclasts by co-staining or staining adjacent sections for specific markers of hematopoietic and stromal cells.
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PMID:Beta-galactosidase staining on bone marrow. The osteoclast pitfall. 1752 74

The objective of this study was to investigate the effect of formulation parameters (i.e. polymer molecular weight and homogenization speed) on various physicochemical and biological properties of cationic nanoparticles. Cationic nanoparticles were prepared using different molecular weights of poly(DL-lactide-co-glycolide) (PLGA) and poly(DL-lactic acid) (PLA) by double emulsion solvent evaporation at two different homogenization speeds, and were characterized in terms of size, surface charge, morphology, loading efficiency, plasmid release, plasmid integrity, cytotoxicity, and transfection efficiency. Cationic surfactant, cetyltrimethylammonium bromide (CTAB), was used to provide positive charge on the surface of nanoparticles. Reporter plasmid gWIZ Beta-gal was loaded on the surface of nanoparticles by incubation. Use of higher homogenization speed and lower molecular weight polymer led to a decrease in mean particle size, increase in zeta potential, increase in plasmid loading efficiency, and a decrease in burst release. The nanoparticles displayed good morphology as evident from scanning electron micrographs. In vitro cytotoxicity study by MTT assay showed a low toxicity. Structural integrity of the pDNA released from nanoparticles was maintained. Transfecting human embryonic kidney (HEK293) cells with nanoparticles prepared from low molecular weight PLGA and PLA resulted in an increased expression of beta-galactosidase as compared to those prepared from high molecular weight polymer. Our results demonstrate that the PLGA and PLA cationic nanoparticles can be used to achieve prolonged release of pDNA, and the plasmid release rate and transfection efficiency are dependent on the formulation variables.
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PMID:Preparation, characterization, cytotoxicity and transfection efficiency of poly(DL-lactide-co-glycolide) and poly(DL-lactic acid) cationic nanoparticles for controlled delivery of plasmid DNA. 1761 Oct 54

5-Bromo-2-deoxyuridine (BrdU) is a thymidine analogue that is incorporated into replicating DNA. Although originally designed as a chemotherapeutic agent, sublethal concentrations of BrdU have long been known to alter the growth and phenotype of a wide range of cell types. Mechanisms underlying these BrdU-mediated effects remain unknown, however. We have characterized the effects of BrdU on A549 lung cancer cells by examining DNA damage responses, cell cycle effects and phenotypic changes. A549 cells express wild-type p53, but are p16-null. Sublethal concentrations of BrdU evoke a DNA damage response in these cells that involves the activation of Chk1, Chk2 and p53. Increased numbers of enlarged nuclei and multinucleated cells are evident in the treated populations. Cell cycle inhibition occurs, resulting in reduced proliferation and accumulation of cells in the S, G2/M and G0 phases. BrdU induces an early inhibition of p21 expression that coincides with nuclear localization of proliferating cell nuclear antigen. Subsequently, p21 levels increase, whereas proliferating cell nuclear antigen levels decrease compared with control cells. Upregulation of p27 and p57 expression also occurs. By day 7 of exposure to BrdU, treated cells acquire a senescent-like phenotype with an increase in cell size, granularity and beta-galactosidase activity. We conclude that BrdU induces a DNA damage response in A549 cells, which results in reduced proliferation mitotic exit and phenotypic changes that resemble senescence.
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PMID:5-Bromo-2-deoxyuridine activates DNA damage signalling responses and induces a senescence-like phenotype in p16-null lung cancer cells. 1770 56

Disposal of lactose in whey and whey permeates is one of the most significant problems with regard to economics and environmental impact faced by the dairy industries. The enzymatic hydrolysis of whey lactose to glucose and galactose by beta-galactosidase constitutes the basis of the most biotechnological processes currently developed to exploit the sugar content of whey. Keeping this in view, lactose hydrolysis in whey was performed using CTAB permeabilized Kluyveromyces marxianus cells. Permeabilization of K. marxianus cells in relation to beta-galactosidase activity was carried out using cetyltrimethyl ammonium bromide (CTAB) to avoid the problem of enzyme extraction. Different process parameters (biomass load, pH, temperature, and incubation time) were optimized to enhance the lactose hydrolysis in whey. Maximum hydrolysis (90.5%) of whey lactose was observed with 200 mg DW yeast biomass after 90 min of incubation period at optimum pH of 6.5 and temperature of 40 degrees C.
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PMID:Hydrolysis of whey lactose using CTAB-permeabilized yeast cells. 1843 1


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