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 constructed a shuttle plasmid for Bacillus megaterium and Escherichia coli that contains the promoter and repressor gene of the B. megaterium-borne operon for xylose utilization. A polylinker downstream of the promoter allows versatile cloning of genes under its transcriptional control. We have placed gdhA (encoding glucose dehydrogenase) from B. megaterium, lacZ (encoding beta-galactosidase) from E. coli, mro (encoding mutarotase) from Acinetobacter calcoaceticus, and human puk (encoding single-chain urokinase-like plasminogen activator, rscuPA) under xylose control in this vector. All four genes were between 130-fold and 350-fold inducible by 0.5% xylose in the growth medium in B. megaterium. Enzymatically active glucose dehydrogenase and mutarotase accumulated to 20% and 30% of the total soluble protein, respectively. beta-Galactosidase and rscuPA were also expressed at a high level. A gel analysis of the products demonstrated their proteolytic stability in the cytoplasm, even up to 5 h after induction. The expression properties of this new host-vector system are discussed in comparison to the ones available for B. subtilis and E. coli.
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PMID:Inducible high-level expression of heterologous genes in Bacillus megaterium using the regulatory elements of the xylose-utilization operon. 136 76

The use of intravascular stents may be limited by both local thrombosis and restenosis due to intimal proliferation. In an effort to provide solutions to these problems, we seeded stents with genetically engineered endothelial cells in vitro. Using retroviral-mediated gene transfer, we inserted the gene for either bacterial beta-galactosidase or human tissue-type plasminogen activator (t-PA) into cultured sheep endothelial cells. The endothelial cells were seeded onto stainless steel stents and grown until the stents were covered. Expression of intracellular beta-galactosidase and high level secretion of t-PA were demonstrated both before and after the transduced cells were seeded onto the stents. Eight stents were expanded by in vitro balloon inflation, with observation of the seeded endothelial layer both prior to and after expansion. Most of the endothelial cells remained on the stents after balloon inflation. We conclude that intravascular stents can be coated with a layer of genetically engineered endothelial cells that can be either specifically labeled or made to secrete high levels of a therapeutic protein. Much of the layer of genetically engineered cells remains after the expansion of the stent in vitro. In vivo implantation of stents coated with genetically engineered endothelial cells may allow 1) introduction of genetically engineered endothelial cells directly into the vascular wall and 2) improvement of stent function through localized delivery of anticoagulant, thrombolytic, or antiproliferative molecules.
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PMID:Seeding of intravascular stents with genetically engineered endothelial cells. 280 83

When messenger RNA (mRNA) from both untreated and phorbol ester-treated melanoma cells is translated in simple reticulocyte lysates, tissue-type plasminogen activator can be immunoprecipitated by an affinity-purified antibody as a approximately 52,000 mol wt protein, with no detectable biological (plasminogen activating) activity. When the reticulocyte lysate system is supplemented with a preparation of microsomal membranes, biological activity becomes detectable and a 63,000 mol wt protein can be immunoprecipitated with the same antibody. Furthermore, when natural tissue-type plasminogen activator (mol wt approximately equal to 70,000) is incubated with different glycosidases, distinct alterations in the electrophoretic mobility of the molecules are observed, together with alterations in the level of biological activity. While treatment with neuraminidase and beta-galactosidase caused decreases in activity, alpha-mannosidase caused an increase. These results suggest that the carbohydrate part of the molecule can influence its biological behavior.
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PMID:Influence of carbohydrate side chains on activity of tissue-type plasminogen activator. 308 8

Enzymatic activity was investigated in metal-binding proteins from rat epidermal cells. Tris-HCl buffer soluble and KSCN solubilized proteins were extracted stepwise from granular and cornified cells of 2-day old rat epidermis. Each extract was separately applied to a Cu2+ or Zn2+ chelate Sepharose 6B column and the proteins were eluted with buffers of different pHs and finally with EDTA solution. Metal chelate-binding proteins were found in both soluble and solubilized proteins but there was a larger amount in the latter. Affinity of the proteins to bind with Cu2+ chelate was greater than that with Zn2+ chelate. In Tris-HCl buffer extract, histidase activity was detected in Cu2+ chelate-binding proteins, but not in Zn2+ chelate-binding proteins. Acid phosphatase, cysteine proteinase, dipeptidase, cathepsin D, beta-galactosidase, gelatin hydrolase, and superoxide dismutase did not bind to metal chelates although these enzymes, except acid phosphatase, were inhibited by Cu2+, but not by Zn2+. In contrast, KSCN solubilized metal chelate-binding proteins showed plasminogen activator, acid phosphatase, and gelatin and casein hydrolases while histone hydrolase did not bind to either chelate column. Since metal-binding proteins in rat epidermal cells have been shown previously to be histidine- and cysteine-rich proteins concentrated in keratohyalin granules, interaction of metals and the structural proteins with certain enzymes may be involved in the regulation of epidermal cell functions.
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PMID:Enzymatic activity of metal-binding proteins in epidermal cells. 653 44

The present study describes a novel method for the histochemical demonstration of beta-galactosidase activity on tissue sections. We have replaced 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal) with 5-bromo-indolyl-beta-o-galactopyranoside (Bluo-Gal) as a chromogenic substrate for the bacterial beta-galactosidase (lacZ). After beta-galactosidic cleavage, Bluo-Gal precipitates in form of fine birefringent crystals, whereas X-gal gives rise to an amorphous precipitate. Upon microscopic examination under polarized light, the crystals emit a strong signal consisting of yellow reflected light. This property of Bluo-Gal results in greatly enhanced sensitivity of the staining method for beta-galactosidase and allows for optimal morphological resolution. To exemplify the applications of this technique, the expression is demonstrated in transgenic mice of beta-galactosidase driven by a fragment of the human tissue-type plasminogen activator promoter.
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PMID:Improved in situ beta-galactosidase staining for histological analysis of transgenic mice. 753 99

Levels of hydrolytic enzymes increase in skeletal muscle after denervation and their activities in the extracellular matrix appear to be important for interaction between muscle and nerve. Using enzymatic assays for beta-glucuronidase, beta-galactosidase, and plasminogen activator, we show that secretion of these enzymes from mouse skeletal muscle increases after denervation and that drugs interfering with the secretory pathway or the reuptake of enzymes modulate this release. Thus, brefeldin A inhibited secretion of plasminogen activator activity and mannan increased secreted amounts of beta-glucuronidase, but not of beta-galactosidase, in denervated muscle. In innervated muscle, brefeldin A decreased secreted activity of plasminogen activator, but mannan had no effect on secretion of either beta-glucuronidase or beta-galactosidase. Furthermore, secretion of plasminogen activator was temperature dependent. These observations, together with previous studies, suggest that secretion of hydrolytic enzymes from adult skeletal muscle may be of physiological significance in nerve-muscle communication.
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PMID:Secretion of plasminogen activator and lysosomal enzymes from mouse skeletal muscle: effect of denervation. 765 63

To examine whether the epidermal growth factor (EGF)-like domain Pro47-Asp87 is involved in the interaction of tissue plasminogen activator (t-PA) with platelets, we have expressed this domain in E. coli. The peptide fragment was produced from a plasmid expression vector as a fusion protein with beta-galactosidase Met1-Val444 at high yield in eight clones of E. coli. The fusion protein was purified and subjected to mild acid hydrolysis with formic acid, then the peptide Pro47-Asp87, identified by immunoblotting using specific antibodies to t-PA, was isolated by HPLC. After incubation with blood platelets spin labelled with 16-doxylstearic acid or 5-doxylstearic acid, the Pro47-Asp87 peptide fragment reduced fluidity of the membrane lipid bilayer to the same extent as did intact t-PA as indicated by ESR measurements. Our data suggest that the EGF-like domain of t-PA can directly interact with blood platelets and thus it seems to contain those sites of the t-PA molecule that bind the platelet membrane components.
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PMID:The epidermal growth factor-like domain from tissue plasminogen activator. Cloning in E. coli, purification and ESR studies of its interaction with human blood platelets. 803 Mar 71

High-performance anion-exchange chromatography (HPAEC) with pulsed-amperometric detection was used to monitor the consistency of the oligosaccharides released from several partially purified preparations of a tissue-type plasminogen activator mutant (TNK-tPA). Differences in the oligosaccharide map were observed, primarily in the neutral carbohydrate region of the separation. Subsequent investigations using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF/MS) identified the neutral oligosaccharides to be primarily asialo-diantennary complex-type glycans with 2, 1, or 0 galactose residues. Additional asialo-triantennary and asialo-tetrantennary structures were also observed with varying amounts of galactose. Hydrolysis of the chromogenic substrate 4-methyl umbelliferyl-beta-galactoside confirmed that host-cell lysosomal beta-galactosidase is present at the first step in the purification process and is the cause of the observed glycan degradation. Subsequent steps in the purification process quantitatively remove this enzyme. Comparison of HPAEC and MALDI/TOF/MS analysis of time-course samples revealed quite similar rates of degradation and demonstrates the quantitative utility of these methods. HPAEC did not reveal significant changes in the sialylated structures as evidenced by nearly identical profiles for the sialic acid-containing structures.
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PMID:The use of high-performance anion-exchange chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to monitor and identify oligosaccharide degradation. 866 Jun 30

Midkine (MK) is a growth factor that promotes neurite outgrowth and survival of neurons, and enhances the plasminogen activator in endothelial cells. A highly sensitive enzyme-linked immunoassay for MK was developed, involving affinity-purified anti-MK antibodies, their biotinylated form, and avidin-beta-galactosidase. The amount of bound avidin-beta-galactosidase was determined using a fluorogenic substrate, 4-methylumbelliferyl-beta-D-galactoside. This method allowed the detection of human and mouse MK in the range of 50 pg-10 ng. Pleiotrophin, which is related to MK in its amino acid sequence, did not show any cross reactivity. Employing this method, the MK levels in the developing mouse brain were determined. The MK level was 2 micrograms/g of wet tissue on the 12th day of gestation, and then steadily decreased during embryogenesis and postnatal development to 30 ng/g two months after birth. The assay method can also be applied to serum samples. Although the MK levels in the sera of normal human subjects were low or undetectable, 0.6-8 ng/ml of MK was detected in samples in the majority of cases of hepatocellular carcinomas.
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PMID:Enzyme-linked immunoassay for midkine, and its application to evaluation of midkine levels in developing mouse brain and sera from patients with hepatocellular carcinomas. 882 54

Recent research in various areas has appreciably expanded our knowledge of streptokinase, a plasminogen activator produced by all human group A (GAS), group C (GCS), and group G (GGS) streptococci. Several molecular genetic approaches are described here to study the expression of the streptokinase gene, skn. Southern hybridization analysis demonstrated homology of synteny of ska, skc, and skg in the genomes of the above serogroups. S1 nuclease mapping, the use of transcriptional fusions to beta-galactosidase and luciferase reporter genes, in conjunction with site-directed mutagenesis, led to the localization of the core promoter region of skc and the identification of a cis-active upstream region required for full promoter activity. Circular permutation analysis of the promoter upstream region identified an intrinsic DNA bending locus as the pivotal DNA element stimulating the activity of the core promoter. The detection of skn allele-specific expression phenotypes, which proved not to be due to different skn mRNA half-lives, prompted allele swap experiments, showing that promoter activity is dictated by the host genetic background, rather than the sequence of the regulatory region. These findings suggest the involvement in skn expression of an as yet unidentified transcriptional activator that contacts the bent DNA region. Transcription termination of skc is directed by a bidirectional terminator whose structural requirements for termination efficiency were determined with base substitution mutants fused to a chloramphenicol acetyl transferase reporter. Finally, mutagenic plasmids are described for insertion-duplication and allele replacement mutagenesis of the skn locus.
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PMID:Expression and regulation of the streptokinase gene. 1081 72


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