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 manometric sensor previously developed to measure urea was modified to measure glucose and lactose through enzymatic oxidation. Change in pressure in an enclosed cavity was correlated to the depletion of oxygen resulting from the enzymatic oxidation of glucose or lactose. The response of the sensor was linear and could be made adjustable over a large range by adjusting the amount of sample loaded into the fixed volume reactor. Because of the slow mutarotation of glucose, the oxidation of glucose was not allowed to proceed to completion. Therefore, the precision of the sensor (approximately 0.2 mM in a range from 0 to 5 mM) was limited by variations in the oxidation rate of glucose by glucose oxidase. Because the assay for lactose measured glucose subsequent to the hydrolysis of lactose by beta-galactosidase, the same degree of precision was observed in lactose. Milk lactose, typically at concentrations of about 150 mM, was estimated using the lactose assay after first diluting the samples. For many fluids such as milk, the use of manometric sensors for oxidizable substrates may be preferable to optical and electrochemical methods because they are robust and suffer a low degree of optical and chemical interferences. Glucose and lactose are representative of many important oxidizable substrates, which may be determined in this manner, many of which do not suffer from limitations caused by mutarotation. In theory, detection limits less than 1 microM may be achieved using these methods.
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PMID:Adaptation of a manometric biosensor to measure glucose and lactose. 1244 50

The gas vesicle formation in Haloferax mediterranei occurs in the stationary growth phase and involves the 14 genes mc-gvpACNO and mc-gvpDEFGHIJKLM. The appearance of the two regulatory proteins GvpD and GvpE, and also of GvpF, was investigated during the growth of H. mediterranei. GvpD was only found during the stationary growth phase, GvpE was present from the late exponential to stationary growth phase, and GvpF was present only during the exponential growth, although the three genes were co-transcribed. The impact of GvpD and GvpE on the activity of the promoter of the mc-gvpACNO gene cluster encoding the gas vesicle structural proteins was analysed in H. volcanii transformants containing the mc-gvpA gene or a fusion of the mcA promoter with the bgaH reading frame encoding a halobacterial beta-galactosidase as reporter. The experiments proved that GvpE is a transcriptional activator, whereas GvpD is involved in the repression. Protein-protein affinity chromatography was used to search for putative binding partners of GvpD and GvpE. Both proteins were synthesized in Escherichia coli as his-tagged proteins, isolated under denaturing conditions and refolded by dialysis against buffers containing decreasing urea and increasing KCl concentrations up to 2.5 M. The Ni-NTA matrix tagged with GvpD-his or GvpE-his was incubated with soluble proteins of gas vesicle producing H. mediterranei cells. A 21 kDa protein was purified using the matrix tagged with GvpD-his which proved to be GvpE by Western analysis. Vice versa, GvpD was purified using the GvpE-his-Ni-NTA matrix. These results strongly suggested that GvpD and GvpE were able to interact and might constitute a regulatory system.
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PMID:Regulation of the expression of gas vesicle genes in Haloferax mediterranei: interaction of the two regulatory proteins GvpD and GvpE. 1286 59

Disulfide reduction of Kluyveromyces lactis and Aspergillus oryzae beta-galactosidases and beta-lactoglobulin was assessed. Reduction was performed using one of two thiol-containing agents: dithiothreitol (DTT) or thiopropyl-agarose with a high degree of substitution (1000 micromol of SH groups/g of dried gel). Both reductants allowed an increase of three- (for K. lactis beta-galactosidase) and fourfold (for A. oryzae beta-galactosidase) in the initial content of SH groups in the lactases. Nearly sevenfold fewer micromoles of SH groups per milligram of protein were needed to perform the reduction of K. lactis beta-galactosidase with thiopropyl-agarose than for the same reduction with DTT. However, for A. oryzae beta-galactosidase, nearly twice as many micromoles of SH groups per milligram of protein were needed with thiopropylagarose than with DTT. Disulfide bonds in beta-lactoglobulin were not accessible to thiopropyl-agarose, since this reduction was only possible in the presence of 6 M urea. These results proved that highly substituted thiopropyl-agarose is as good a reducing agent as DTT, for the reduction of disulfide bonds in proteins. Moreover, excess reducing agent was very simply separated from the reduced protein by filtration, making it easier to control the reaction and providing reduced protein solutions free of reductant. All these advantages substantially cut down the time required and therefore the cost of the overall process.
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PMID:Solid-phase reducing agents as alternative for reducing disulfide bonds in proteins. 1290 29

Oncogenic Ras induces premature senescence in primary cells. Such an oncogene-induced senescence involves activation of tumor suppressor genes that provide a checkpoint mechanism against malignant transformation. In mouse, the ARF-p53 pathway mediates Ha-Ras(G12V)-induced senescence, and p19(ARF-/-) and p53(-/-) cells undergo transformation upon Ras activation. In addition, mouse cells, unlike human cells, express constitutively active telomerase and have long telomeres. However, it is unclear how Ras activation affects human cells of epithelial origin with p53 mutation and/or telomerase activation. In order to address this question, Ha-Ras(G12V) was expressed ectopically in primary as well as hTERT-immortalized human esophageal keratinocytes stably expressing dominant-negative p53 mutants. In human esophageal keratinocytes, we found that Ha-Ras(G12V) induced senescence regardless of p53 status and telomerase activation. Ras activation resulted in changes of cellular morphology, activation of senescence-associated beta-galactosidase, and suppression of cell proliferation, all coupled with reduction in the hyperphosphorylated form of the retinoblastoma protein (pRb). Furthermore, Ha-Ras(G12V) upregulated p16(INK4a) and downregulated cyclin-dependent kinase Cdk4 in human esophageal keratinocytes. Thus, Ras-mediated senescence may involve distinct mechanisms between human and mouse cells. Inactivation of the pRb pathway may be necessary for Ras to overcome senescence and transform human esophageal epithelial cells.
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PMID:Ha-Ras(G12V) induces senescence in primary and immortalized human esophageal keratinocytes with p53 dysfunction. 1527 25

The urea transporters, UT-A1 and UT-A3, two members of the UT-A gene family, are localized to the terminal portion of the inner medullary collecting duct (IMCD). In this manuscript, we demonstrate that 4.2 kb of the 5'-flanking region of the UT-A gene (UT-Aalpha promoter) is sufficient to drive the IMCD-specific expression of a heterologous reporter gene, beta-galactosidase (beta-Gal), in transgenic mice. RT-PCR, immunoblotting, and immunohistochemistry demonstrate that within the kidney, transgene expression is confined to the terminal portion of the IMCD. Colocalization studies with aquaporin-2 show that expression is localized to the principal cells of the IMCD2 and IMCD3 regions. Utilizing beta-Gal activity assays, we further show that within the kidney, the beta-Gal transgene can be regulated by both water restriction and glucocorticoids, similar to the regulation of the endogenous UT-A gene. These results demonstrate that 4.2 kb of the UT-Aalpha promoter is sufficient to drive expression of a heterologous reporter gene in a tissue-specific and cell-specific fashion in transgenic mice.
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PMID:UT-A urea transporter promoter, UT-Aalpha, targets principal cells of the renal inner medullary collecting duct. 1609 80

To enhance the transduction efficiency (TE) of a recombinant adeno-associated virus 2 (rAAV2) in human cancer cells, we examined the combined effects of various chemicals known to influence the rAAV2 transduction process at distinct steps. Among the agents tested were trichostatin A, a histone deacetylase inhibitor, MG-132, a proteosome inhibitor, the genotoxic agents hydroxyurea, aphidicolin, etoposide and camptothecin, and tyrphostin-1, an epidermal growth factor receptor inhibitor. During or after chemical treatment, various human cancer cells were infected with rAAV2 expressing beta-galactosidase. Treatment with hydroxy-urea or etoposide plus tyrphostin-1 dramatically increased the TE in most cell lines. The combination of hydroxyurea plus tyrphostin-1 increased TE to 37.7+/-7.9%, 32.8+/-2.0% and 31.8+/-2.1% in SK-Hep1, HeLa, and HCT116 cells, respectively. In addition, following rAAV2 infection and treatment with hydroxyurea plus tyrphostin-1, long-term transgene expression was observed for up to 6 months, with no damage to the transduced cells. These results indicate that rAAV2 transgene expression can be significantly enhanced by a combination of chemical agents with distinct activity and prolonged gene expression can occur following rAAV2 gene transfer into human cancer cells.
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PMID:Treatment with hydroxyurea and tyrphostin-1 significantly improves the transduction efficiency of recombinant adeno-associated viruses in human cancer cells. 1627 41

The Forkhead box m1 (Foxm1) gene is critical for G(1)/S transition and essential for mitotic progression. However, the transcriptional mechanisms downstream of FoxM1 that control these cell cycle events remain to be determined. Here, we show that both early-passage Foxm1(-)(/)(-) mouse embryonic fibroblasts (MEFs) and human osteosarcoma U2OS cells depleted of FoxM1 protein by small interfering RNA fail to grow in culture due to a mitotic block and accumulate nuclear levels of cyclin-dependent kinase inhibitor (CDKI) proteins p21(Cip1) and p27(Kip1). Using quantitative chromatin immunoprecipitation and expression assays, we show that FoxM1 is essential for transcription of the mitotic regulatory genes Cdc25B, Aurora B kinase, survivin, centromere protein A (CENPA), and CENPB. We also identify the mechanism by which FoxM1 deficiency causes elevated nuclear levels of the CDKI proteins p21(Cip1) and p27(Kip1). We provide evidence that FoxM1 is essential for transcription of Skp2 and Cks1, which are specificity subunits of the Skp1-Cullin 1-F-box (SCF) ubiquitin ligase complex that targets these CDKI proteins for degradation during the G(1)/S transition. Moreover, early-passage Foxm1(-)(/)(-) MEFs display premature senescence as evidenced by high expression of the senescence-associated beta-galactosidase, p19(ARF), and p16(INK4A) proteins. Taken together, these results demonstrate that FoxM1 regulates transcription of cell cycle genes critical for progression into S-phase and mitosis.
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PMID:Forkhead box M1 regulates the transcriptional network of genes essential for mitotic progression and genes encoding the SCF (Skp2-Cks1) ubiquitin ligase. 1631 12

A method is described for generating and screening variants of the beta-galactosidase from Lactobacillus delbrueckii subsp. bulgaricus sensitive to several environmental stresses, with potential application in the food industry. Chemical mutagenesis with hydroxylamine or methoxylamine was performed on the beta-galactosidase gene carried on an Escherichia coli expression vector. Mutants sensitive to cold, heat, low pH, low magnesium concentration, and the presence of urea were isolated by screening for reduced color development on beta-galactosidase indicator plates. The mutations responsible for three variant beta-galactosidases were localized, and the base substitutions were determined by DNA sequencing. The amino acid alterations associated with one low-pH-sensitive (pHs) and two urea-sensitive (Us) variants correspond to P584L (pHs1), G400S/R479Q (Us26), and G167E/E168K/E363K/V492M (Us17), respectively. Mutant pHs1 is also heat, cold, low magnesium, and urea sensitive; Us26 is also cold sensitive; and Us17 is also low-pH sensitive.
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PMID:Generation and Characterization of Environmentally Sensitive Variants of the beta-Galactosidase from Lactobacillus delbrueckii subsp. bulgaricus. 1634 30

The glycoprotein B (gB) of human cytomegalovirus represents a dominant antigen for the humoral immune response. The immunodominant region on gB is the antigenic domain 1 (AD-1), a complex structure that requires a minimal continuous sequence of more than 75 amino acids for antibody binding. In this study, this domain was expressed in Escherichia coli as a fusion protein with beta-galactosidase but yielded insoluble protein aggregates as inclusion bodies. To recover the fusion protein, inclusion bodies were solubilized by two extractions with urea 8 M: and the fusion protein then isolated using gel filtration chromatography. After confirmation of fusion protein antigenicity by Western blotting, the purified product was used as the capturing antigen in an enzyme-linked immunosorbent assay (ELISA) to determine the presence of viral antibodies in serum samples of pregnant women. A cut-off point of approximately 0.2 absorbance units could discriminate the results of seropositive from seronegative pregnant women. The data indicates the potential usefulness of the fusion protein for the development of immunoassay for detection of the HCMV antibodies.
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PMID:Isolation of a fusion protein containing the antigenic domain 1 of human cytomegalovirus glycoprotein B and its application in ELISA tests. 1636 88

This study demonstrated that engineered polyhydroxyalkanoate (PHA) synthases can be employed as molecular tools to covalently immobilize enzymes at the PHA granule surface. The beta-galactosidase was fused to the N terminus of the class II PHA synthase from Pseudomonas aeruginosa. The open reading frame was confirmed to encode the complete fusion protein by T7 promoter-dependent overexpression. Restoration of PHA biosynthesis in the PHA-negative mutant of P. aeruginosa PAO1 showed a PHA synthase function of the fusion protein. PHA granules were isolated and showed beta-galactosidase activity. PHA granule attached proteins were analyzed and confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Surprisingly, the beta-galactosidase-PHA synthase fusion protein was detectable at a high copy number at the PHA granule, compared with PHA synthase alone, which was barely detectable at PHA granules. Localization of the beta-galactosidase at the PHA granule surface was confirmed by enzyme-linked immunosorbent assay using anti-beta-galactosidase antibodies. Treatment of these beta-galactosidase-PHA granules with urea suggested a covalent binding of the beta-galactosidase-PHA synthase to the PHA granule. The immobilized beta-galactosidase was enzymologically characterized, suggesting a Michaelis-Menten reaction kinetics. A Km of 630 microM and a Vmax of 17.6 nmol/min for orthonitrophenyl-beta-D-galactopyranoside as a substrate was obtained. The immobilized beta-galactosidase was stable for at least several months under various storage conditions. This study demonstrated that protein engineering of PHA synthase enables the manufacture of PHA granules with covalently attached enzymes, suggesting an application in recycling of biocatalysts, such as in fine-chemical production.
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PMID:In vivo enzyme immobilization by use of engineered polyhydroxyalkanoate synthase. 1651 22


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