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 describe a fast light microscopic procedure for the simultaneous enzyme cytochemical detection of three different DNA target sequences in contrasting colors in both interphase and metaphase cell preparations. Chromosome-specific DNA probes labeled with either biotin, digoxygenin, or fluorescein were hybridized as a mixture and detected clearly and accurately by precipitates of the horseradish peroxidase-diaminobenzidine (PO-DAB, brown color), alkaline phosphatase-Fast Red (APase-Fast Red, red color), or horseradish peroxidase-tetramethylbenzidine (PO-TMB, green color) reaction, respectively. The PO-TMB reaction product was stabilized effectively by the addition of sodium tungstate to the reaction mixture, thus making the PO-TMB reaction now generally applicable to in situ hybridization (ISH). To avoid mixing of the precipitates of the two PO reactions used in the triple-color ISH method, the first detected PO activity was always completely inactivated by a mild acid treatment before the second one was applied. Finally, the cell preparations were embedded in a thin protein layer cross-linked by formaldehyde to ensure permanent stabilization of the enzyme reaction products and optimal visualization of color contrast. The triple-color ISH detection procedure could be combined with beta-galactosidase-5-bromo-4-chloro-3-indolyl-beta- D-galactoside (beta-Gal-BCIG) immunocytochemistry (ICC), leading to the simultaneous localization of multiple DNA targets and a protein target in the same cell. The described procedure may therefore be a valuable tool in the areas of cytogenetics, cell biology, and molecular pathology.
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PMID:A novel triple-color detection procedure for brightfield microscopy, combining in situ hybridization with immunocytochemistry. 793 May 13

The Tat protein of human immunodeficiency virus 1 (HIV-1) can enter cells efficiently when added exogenously in tissue culture. To assess if Tat can carry other molecules into cells, we chemically cross-linked Tat peptides (residues 1-72 or 37-72) to beta-galactosidase, horseradish peroxidase, RNase A, and domain III of Pseudomonas exotoxin A (PE) and monitored uptake colorimetrically or by cytotoxicity. The Tat chimeras were effective on all cell types tested, with staining showing uptake into all cells in each experiment. In mice, treatment with Tat-beta-galactosidase chimeras resulted in delivery to several tissues, with high levels in heart, liver, and spleen, low-to-moderate levels in lung and skeletal muscle, and little or no activity in kidney and brain. The primary target within these tissues was the cells surrounding the blood vessels, suggesting endothelial cells, Kupffer cells, and/or splenic macrophages. Tat-mediated uptake may allow the therapeutic delivery of macromolecules previously thought to be impermeable to living cells.
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PMID:Tat-mediated delivery of heterologous proteins into cells. 829 May 79

Bio- and chemiluminescence have proved sensitive enough to compete with chromogenic and radioisotopic tracers for in situ detection. However, they must also provide a discriminant morphological analysis of the specific signal. We have tested seven bio- or chemiluminescent reagents for tissue antigen and nucleic acid detection by immunocytochemistry (ICC) or in situ hybridization (ISH). They were based on luminescent detection of peroxidase, alkaline phosphatase, beta-galactosidase or xanthine oxidase. We also explored whether high molecular weight polymers could increase the spatial definition of the photon emission. An ICCD camera was used to collect the light signal provided by immunolabelling of endothelial cells and by ISH of human papilloma virus on cell smears. Among the enzyme-luminescent substrate combinations tested, the enhanced luminol chemiluminescence (ECL) gave the best resolution of the specific signal. The other systems were mainly hampered by a high diffusion of the reaction product over the tissue section. Unfortunately, in this case, the high molecular weight polymers tested were inefficient. However, the addition of polyvinylalcohol (PVA) or polyvinylpyrrolidone (PVP) significantly improved respectively the definition and intensity of ECL photon emission. We demonstrate that chemiluminescence gives a morphological resolution allowing histological examination. The extension of this new application, now depends on physicochemical adaptation of chemiluminescent reagents to the constraints of tissue detection.
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PMID:Comparison of seven bio- and chemiluminescent reagents for in situ detection of antigens and nucleic acids. 853 6

Peroxisome proliferators cause a rapid and coordinated transcriptional activation of genes encoding the enzymes of the peroxisomal beta-oxidation pathway in rats and mice. Cis-acting peroxisome proliferator responsive elements (PPREs) have been identified in the 5'-flanking region of H202-producing rat acyl-CoA oxidase (ACOX) gene and in other genes inducible by peroxisome proliferators. To gain more insight into the purported nonresponsiveness of human liver cells to peroxisome volume density and in the activity of the beta-oxidation enzyme system, we have previously cloned the human ACOX gene, the first and rate-limiting enzyme of the peroxisomal beta-oxidation system. We now present information on a regulatory element for the peroxidase proliferator-activated receptor (PPAR)/retinoid X receptor (RXR) heterodimers. The PPRE, consists of AGGTCA C TGGTCA, which is a direct repeat of hexamer half-sites interspaced by a single nucleotide (DR1 motif). It is located at -1918 to -1906 base pairs upstream of the transcription initiation site of this human ACOX gene. This PPRE specifically binds to baculovirus-expressed recombinant rat PPAR alpha/RXR alpha heterodimers. In transient transfection experiments, the maximum induction of luciferase expression by ciprofibrate and/or 9-cis-retinoic acid is dependent upon cotransfection of expression plasmids for PPAR alpha and RXR alpha. The functionally of this human ACOX promoter was further demonstrated by linking it to a beta-galactosidase reporter gene or to a rat urate oxidase cDNA and establishing stably transfected African green monkey kidney (CV1) cell lines expressing reporter protein. The human ACOX promoter has been found to be responsive to peroxisome proliferators in CV1 cells stably expressing PPAR alpha, whereas only a basal level of promoter activity is detected in stably transfected cells lacking PPAR alpha. The presence of a PPRE in the promoter of this human peroxisomal ACOX gene and its responsiveness to peroxisome proliferators suggests that factors other than the PPRE in the 5'-flanking sequence of the human ACOX gene may account for differences, if any, in the pleiotropic responses of humans to peroxisome proliferators.
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PMID:Identification of a peroxisome proliferator-responsive element upstream of the human peroxisomal fatty acyl coenzyme A oxidase gene. 856 72

Protein renaturation is of particular interest not only for the basic mechanisms of protein folding but also as a practical problem for proteins overexpressed in microorganisms, since recombinant proteins may accumulate as misfolded aggregates in "inclusion bodies" that are inactive after purification. We have established a systematic screening method to identify conditions which promote protein renaturation. A matrix of 50 different buffers, which were originally developed for protein crystallization, were found to facilitate the renaturation for eight of nine different proteins examined. The proteins tested include the adhesive protein bindin, recombinant bindin, and a variety of enzymes, including bacterial alkaline phophatase, horseradish peroxidase, lysozyme, trypsin, beta-galactosidase, rabbit carboxylesterase, and acetylcholinesterase. The total amount of activity recovered varied from 9 to 333% depending on the protein. The conditions that were found to promote renaturation are very different from the optimal conditions for enzyme activity. The finding that most of the proteins tested renatured to a significant extent in one or more of the buffers in the matrix suggests that the sparse matrix screen may be of general utility for establishing initial renaturation conditions for a wide variety of proteins. One initial renaturation conditions have been identified, the conditions may be optimized by systematically altering other parameters of the renaturation process.
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PMID:A sparse matrix screen to establish initial conditions for protein renaturation. 858 34

It has been suggested that Purkinje cells (PC) play a role in organizing topographic relationships of several cerebellar afferent systems, including olivocerebellar fibers. This hypothesis is based on the observation that PC in the rat express biochemical heterogeneities during the presumptive period of olivocerebellar fiber ingrowth to the cerebellum. Previous studies designed to investigate the organization of murine olivocerebellar fibers during embryogenesis have suggested that interactions with PC may play a role in segregating olivocerebellar fibers after they enter the cerebellum. To determine whether PC heterogeneities are related to olivocerebellar fiber organization, transgenic mice carrying a beta-galactosidase (beta-gal) reporter gene linked to the promoter from the PC-specific gene L7/pcp-2 were used in neuroanatomical tracing experiments. Expression of the transgene mirrors endogenous L7/pcp-2 expression, which is upregulated earliest in parasagittal strips of the vermal cortex. Studies were conducted in vitro by using brainstem-cerebellar explants from embryonic day 17/18 (E17/18) and 18/19 mice. Applications of neuroanatomical tracer (horseradish peroxidase or neurobiotin) were made in either the caudal medial accessory olive (cMAO) or the rostral olive. These studies indicate that groups of olivocerebellar fibers and clusters of L7/lacZ+ and L7/lacZ-Purkinje cells respect common distribution boundaries during late embryogenesis. The strong correspondence between the distribution patterns generated by these two markers suggests that expression of L7/pcp-2 and the topographic organization of olivocerebellar (OC) fibers are not interdependent, but may be regulated by a common event or interaction, of a presently unknown nature, which occurs earlier during cerebellar development.
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PMID:Correspondence between L7-lacZ-expressing Purkinje cells and labeled olivocerebellar fibers during late embryogenesis in the mouse. 890 10

The newly developed peroxidase-labelled Enhanced Polymer One-Step (EPOS) reagents were applied, together with an unlabelled primary mouse antibody, in a multistep double-labelling protocol. Enzyme label reporter combinations consisted of either peroxidase and alkaline phosphatase in red and blue, respectively, or beta-galactosidase and alkaline phosphatase in turquoise and red, respectively. The latter enzyme combination was introduced using a rabbit antiperoxidase antibody and an enzyme-labelled anti-rabbit immunoglobulin antibody. The multistep procedure was tested using five different antibody combinations on cryostat and Carnoy- or formalin-fixed, paraffin-embedded sections. In each instance, clear and distinct labelling was obtained, either with the two antigens at separate sites, or with an overlap in distribution. In the latter situation, the sites of co-localization were marked by mixed colours, which were distinct and readily discriminated from the two basic colours.
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PMID:The use of enhanced polymer one-step staining reagents for immunoenzyme double-labelling. 895 Jun

The estrogenic activity of dieldrin, toxaphene, and an equimolar mixture of both compounds (dieldrin/toxaphene) was investigated in the 21-day-old B6C3F1 mouse uterus, MCF-7 human breast cancer cells, and in yeast-based reporter gene assays. Treatment of the animals with 17beta-estradiol (E2) (0.0053 kg/day x3) resulted in a 3.1-, 4.8-, and 7.8-fold increase in uterine wet weight, peroxidase activity, and progesterone receptor binding, respectively. In contrast, treatment with 2.5, 15 and 60 micromol/kg (x3) doses of toxaphene, dieldrin, or dieldrin/toxaphene (equimolar) did not significantly induce a dose-dependent increase in any of the E2-induced responses. The organochlorine pesticides alone and the binary mixture did not bind to the mouse uterine estrogen receptor (ER) in a competitive binding assay using [3H]E2 as the radioligand. In parallel studies, estrogenic activities were determined in MCF-7 cells by using a cell proliferation assay and by determining induction of chloramphenicol acetyl transferase (CAT) activity in MCF-7 cells transiently transfected with plasmids containing estrogen-responsive 5'-promoter regions from the rat creatine kinase B and human cathepsin D genes. E2 caused a 24-fold increase in CAT activity in MCF-7 cells transiently transfected with creatine kinase B and a 3.8-fold increase in cells transiently transfected with the human cathepsin D construct. Treatment of MCF-7 cells with dieldrin, toxaphene, or an equimolar mixture of dieldrin plus toxaphene (10(-8)-10(-5) M) did not significantly induce cell proliferation or CAT activity in the transient transfection experiment with both plasmids. The relative competitive binding of the organochlorine pesticides was determined by incubating MCF-7 cells with 10(-9) M [3H]E2 in the presence or absence of 2 x 10(-7) M unlabeled E2 (to determine nonspecific binding), toxaphene (10(-5) M), dieldrin (10(-5) M), and equimolar concentrations of the dieldrin plus toxaphene mixture (10(-5) M). The binding observed for [3H]E2 in the whole cell extracts was displaced by unlabeled E2, whereas the organochlorine pesticides and binary mixture exhibited minimal to nondetectable competitive binding activity. E2 caused a 5000-fold induction of beta-galactosidase (beta-gal) activity in yeast transformed with the human ER and a double estrogen responsive element upstream of the beta-gal reporter gene. Treatment with 10(-6)-10(-4) M chlordane, dieldrin, toxaphene, or an equimolar mixture of dieldrin/toxaphene did not induce activity, whereas 10(-4) M endosulfan caused a 2000-fold increase in beta-gal activity. Diethylstilbestrol caused a 20-fold increase in activity in yeast transformed with the mouse ER and a single estrogen responsive element upstream of the beta-gal reporter gene. Dieldrin, chlordane, toxaphene, and endosulfan induced a 1.5- to 4-fold increase in activity at a concentration of 2.5 x 10(-5) M. Synergistic transactivation was not observed for any equimolar binary mixture of the pesticides at concentrations of either 2.5 x 10(-5) M or 2.5 x 10(-4) M. The results of this study demonstrate that for several estrogen-responsive assays in the mouse uterus, MCF-7 human breast cancer cells, and yeast-based reporter gene assays, the activities of both dieldrin and toxaphene were minimal, and no synergistic interactions were observed with a binary mixture of the two compounds.
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PMID:Estrogenic activity of a dieldrin/toxaphene mixture in the mouse uterus, MCF-7 human breast cancer cells, and yeast-based estrogen receptor assays: no apparent synergism. 907 11

Affinity-based reversed micellar extraction and separation (ARMES) is an effective method for purifying both low and high molecular weight glycoproteins via liquid-liquid extraction. A range of extraction conditions were examined to gain insight into the mechanism of ARMES. Concanavalin A (Con A) was used as the model affinity ligand to bind soybean peroxidase (SBP) and beta-galactosidase as model glycoproteins. Factorial design was used to investigate the effect of various system variables on the extraction of SBP via ARMES. A quadratic model described the systems well, resulting in a standard deviation of 7% between calculated and experimental extraction efficiencies. Sensitivity analysis suggested that the key criteria in ARMES were the NaCl concentration and pH of the aqueous feed phase. Extraction of both glycoproteins decreased above pH 7 but fell to zero only at pH values significantly above the pI of the model glycoproteins and the Con A affinity ligand. It is proposed that the complex of the affinity lectin with the glycoprotein results in a sufficiently hydrophobic species that can be extracted into a reversed micellar organic phase even at pH's far above the pI's of the individual proteins that comprise the complex. This finding has practical considerations for the use of ARMES in the resolution and purification of protein glycoforms.
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PMID:Parameters affecting the efficiency of affinity-based reversed micellar extraction and separation (ARMES) in glycoprotein purification. 926 79

An in situ PCR protocol by which we can monitor the presence or absence of lac mRNA in individual cells of a Salmonella typhimurium F' lac+ strain has been developed. In this protocol, fixed cells are permeabilized with lysozyme and subjected to a seminested reverse transcriptase PCR using reporter molecule-labeled primers, and subsequently, intracellular reporter molecules are detected microscopically at the individual-cell level by use of a horseradish peroxidase-conjugated antifluorescein antibody assay. In order to determine the sensitivity of the in situ PCR assay, the ability to detect lac mRNA in suboptimally isopropyl-beta-D-thiogalactopyranoside-induced cells was investigated. By use of a single-cell beta-galactosidase assay, it was confirmed that homogeneous suboptimally induced cultures of S. typhimurium F' lacY cells could be established, and the number of functional lac mRNAs in individual cells was estimated from standard population level beta-galactosidase assays. Cells estimated to contain a single lac mRNA were detected as containing lac mRNA by the in situ PCR method. Conclusively, we demonstrate the potential of in situ PCR for detection of even poorly expressed mRNA in individual bacterial cells.
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PMID:Visualization of specific gene expression in individual Salmonella typhimurium cells by in situ PCR. 936 4


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