SEARCH · Search PubMed

Results for “GALACTOSIDASE”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,405 records · Page 78Linked to original sources

PubMed1968

Effect of infection with ribonucleic acid bacteriophage R23 on the inducible synthesis of beta-galactosidase in Escherichia coli.

Infection by ribonucleic acid (RNA) bacteriophage R23 inhibited the synthesis of beta-galactosidase in Escherichia coli. The inhibition, although not complete, was apparent shortly after infection and was maximal after the first 20 min of infection. R23 diminished the beta-galactosidase-synthesizing capacity when inducer was added after phage infection, but not when infection followed inducer removal. These findings suggested that the primary effect of R23 on enzyme-forming capacity was limitation of synthesis of enzyme-specific messenger RNA. Studies with ultraviolet irradiated phage and amber mutants of R23 indicated that the inhibitory process could be separated into two phases. Early inhibition did not require the expression of the viral genome, whereas late inhibition required the expression of the viral RNA synthetase cistron.

Bacterial Proteins↗

PubMed1992

Detection of replication-competent and pseudotyped human immunodeficiency virus with a sensitive cell line on the basis of activation of an integrated beta-galactosidase gene.

We have constructed a HeLa cell line that both expresses high levels of CD4 and contains a single integrated copy of a beta-galactosidase gene that is under the control of a truncated human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). This cell line, called CD4-LTR/beta-gal, can be used to determine quantitatively the titer of laboratory-adapted HIV strains, and the method used to do so is as sensitive as the determination of viral titers in a T-cell line by end point dilution. Using this cell line as a titer system, we calculated that HIV-1 stocks contain only one infectious particle per 3,500 to 12,000 virions. Virus derived from a molecular clone of a macrophagetropic provirus will not infect this cell line. We have also cocultivated peripheral blood lymphocyte cultures from HIV-infected individuals with the CD4-LTR/beta-gal indicator cells. In a majority of primary isolates (five of eight), including isolates from asymptomatic patients, rare virus-infected cells that can activate the beta-galactosidase gene are present.

Amino Acid Sequence↗

PubMed1992

Effect of genomic location on expression of beta-galactosidase mRNA controlled by the herpes simplex virus type 1 UL38 promoter.

To examine the effect of genomic location on the details of expression of selected herpes simplex virus promoters, we have constructed recombination vectors for placing such promoters controlling the beta-galactosidase reporter gene into two regions of the viral genome lacking any nearby promoter or regulatory elements. The first vector generates the promoter-beta-galactosidase reporter gene inverted within the locus of the gC (UL44) translational reading frame; the second replaces the LAT promoter and the first 600 bases of the primary transcript in both copies of the RL region. These locations were chosen to obviate any possible influence of upstream but noncontiguous heterologous or homologous DNA sequence elements upon promoter activity. When the reporter gene controlled by the strict late (gamma) UL38 promoter was placed in the gC location, it was significantly less active than in its normal location; in contrast, promoter activity was comparable to wild-type values when the promoter was recombined into the RL region. The low level of activity in the gC location could be partially alleviated by the incorporation of additional DNA sequences upstream of the UL38 promoter. Despite the effect of genomic location upon the level of expression, the kinetics of expression in either location mirrors the wild-type UL38 strict late kinetics of expression. Finally, we used deletional analysis to demonstrate that no more than 29 bases of DNA sequence 5' of the mRNA cap site are required for promoter activity in either location; this result is consistent with earlier results of transient-expression assays and indicates that the UL38 promoter shares general features with other strict late (gamma) herpes simplex virus promoters.

Animals↗

PubMed2001

An immune response after intraocular administration of an adenoviral vector containing a beta galactosidase reporter gene slows retinal degeneration in the rd mouse.

BACKGROUND/AIMS: Retinal degenerations are a leading cause of blindness for which there are currently no effective treatments. This has stimulated interest in the investigation of gene therapy strategies for these diseases in a variety of animal models. A number of attempts have been made to prevent photoreceptor loss in the rd mouse model of retinal degeneration using adenoviral vectors containing either a copy of the missing functional gene or a gene encoding either a neurotrophic factor or an antiapoptotic factor. The authors have previously demonstrated that intraocular administration of an adenoviral vector containing a beta galactosidase gene (AV.LacZ) results in an immune response to viral gene products and beta galactosidase. Here the effect of the immune response on retinal degeneration is examined. METHODS: Juvenile rd mice were injected intravitreally with AV.LacZ and a proportion were depleted of either CD4+ or CD8+ T cells or both. Control animals were injected with PBS. The mice were sacrificed 10 and 20 days post-injection and their eyes embedded in paraffin wax and sectioned. RESULTS: 10 days after intravitreal injection of AV.LacZ, the outer nuclear layer contains an average of 2.5 rows compared with 1.5 in PBS injected animals (p<0.005). The protective effect of AV.LacZ is negated by immune suppression and does not extend beyond 20 days. CONCLUSION: An immune response to vector and transgene products is able to slow degeneration in the rd mouse. This phenomenon should be taken into account when analysing the degeneration in the rd mouse following gene transfer.

Adenoviridae↗

PubMed1973

Small intestinal beta-galactosidase activity in the horse.

Two enzymes having lactase activity are present in the equine small intestine. The first, the digestive enzyme, neutral beta-galactosidase, declines in activity from birth to three years, disappearing completely between 3 and 4 years of age. The other, the soluble lysosomal enzyme, acid beta-galactosidase, having affinity for lactose and a synthetic beta-galactoside, shows a decrease in activity in the first three months of life and thereafter varies little in activity and represents the lactase enzyme in the adult horse. This pattern may parallel the development of lactase activity in many other mammals and in the majority of the world's human population.

Age Factors↗

PubMed1976

beta-Galactosidase from Bacillus stearothermophilus.

Several strains of thermophilic aerobic spore-forming bacilli synthesize beta-galactosidase (EC 3.2.1.23) constitutively. The constitutivity is apparently not the result of a temperature-sensitive repressor. The beta-galactosidase from one strain, investigated in cell-free extracts, has a pH optimum between 6.0 and 6.4 and a very sharp pH dependence on the acid side of its optimum. The optimum temperature for this enzyme is 65 degrees C and the Arrhenius activation energy is about 24 kcal/mol below 47 degrees C and 16 kcal/mol above that temperature. At 55 degrees C the Km is 0.11 M for lactose and 9.8 X 10(-3) M for 9-nitrophenyl-beta-D-galactopyranoside. The enzyme is strongly product-inhibited by galactose (Ki equals 2.5 X 10(-3) M). It is relatively stable at 50 degrees C, losing only half of its activity after 20 days at this temperature. At 60 degrees C more than 60% of the activity is lost in 10 min. However, the enzyme is protected somewhat against thermal inactivation by protein, and in the presence of 4 mg/ml of bovine serum albumin the enzyme is only 18% inactivated in 10 min at 60 degrees C. Its molecular weight, estimated by disc gel electrophoresis, is 215 000.

Aerobiosis↗

PubMed2001

His-391 of beta-galactosidase (Escherichia coli) promotes catalyses by strong interactions with the transition state.

His-391 of beta-galactosidase (Escherichia coli) was substituted by Phe, Glu, and Lys. Homogeneous preparations of the substituted enzymes were essentially inactive unless very rapid purifications were performed, and the assays were done immediately. The inactive enzymes were tetrameric, just like wild-type beta-galactosidase and their fluorescence spectra were identical to the fluorescence spectrum of wild-type enzyme. Analyses of two of the substituted enzymes that were very rapidly purified to homogeneity and rapidly assayed while they were still active (at only a few substrate concentrations so that the data could be rapidly obtained), showed that the kinetic values were very similar to the values obtained with the same enzymes that were only partially purified. This showed that the kinetics were not affected by the degree of purity and allowed kinetic analyses with partially purified enzymes so that large numbers of points could be used for accuracy. The data showed that His-391 is a very important residue. It interacts strongly with the transition state and promotes catalysis by stabilizing the transition state. Activation energy differences (deltadelta G(S) double dagger), as determined by differences in the kcat/Km values, indicated that substitutions for His-391 caused very large destabilizations (22.8-35.9 kJ/mol) of the transition state. The importance of His-391 for transition state stabilization was confirmed by studies that showed that transition state analogs are very poor inhibitors of the substituted enzymes, while inhibition by substrate analogs was only affected in a small way by substituting for His-391. The poor stabilities of the transition states caused significant decreases of the rates of the glycolytic cleavage steps (galactosylation, k2). Degalactosylation (k3) was not decreased to the same extent.

Amino Acid Substitution↗

PubMed1975

The action of beta-galactosidase (Escherichia coli) on allolactose.

The parameters involved in the action of beta-galactosidase (EC 3.2.1.23) (Escherichia coli) on allolactose, the natural inducer of lac operon in E. coli, were studied. At low allolactose concentrations only galactose and glucose were formed, while at high allolactose concentrations transgalactolytic oligosaccharides were also produced. Detectable amounts of lactose were not formed. The V and Km values (49.6 U/mg and 0.00120 M, respectively) indicated that allolactose is as good if not a better substrate of beta-galactosidase as lactose. The pH optimum with allolactose (7.8-7.9) as well as its activation by K+ (as compared to activation by Na+) were similar to the case with lactose as substrate. The alpha-anomer of allolactose was hydrolyzed about two times as rapidly as was the beta-anomer.

Escherichia coli↗

PubMed1975

Flow kinetics of beta-galactosidase chemically attached to nylon tubing.

Beta-Galactosidase (EC 3.2.1.23) has been attached covalently to the inner surface of nylon tubing. An experimental study has been made of the flow kinetics for the hydrolysis of o-nitrophenylgalactose, the substrate concentration and flow rate being varied. The results were analyzed in the light of the theoretical treatment of Kobayashi and Laidler, three different methods of analysis being employed. It is concluded that at the lower substrate concentrations and flow rates employed, the reactions are largely diffusion controlled; with increase in flow rate and substrate concentration the width of the Nernst diffusion layer decreases, and there is found to be less diffusion control. The values of Km(app) vary with flow rate VF, being linear in VF-1/3, and the value extrapolated to very high flow rate agrees well with the Km value for beta-galactosidase in free solution. The theory and results are shown to provide guidelines for the design of open tubular heterogeneous enzyme reactors for industrial, biomedical, and analytical applications.

Diffusion↗

PubMed1994

Developmentally regulated Gb3 galactosyltransferase and alpha-galactosidase determine Shiga toxin receptors in intestine.

The receptor for Shiga toxin on rabbit intestinal microvillus membranes (MVMs) has been identified as a developmentally regulated glycolipid, globotriaosylceramide [galactose alpha 1-4 galactose beta 1-4 glucose beta 1-1 ceramide (Gb3)]. MVM Gb3 levels increase markedly in the third week of life, concomitant with fluid secretory responses to the toxin. To study mechanisms controlling developmental regulation of MVM Gb3, we measured the specific synthetic Gb3 galactosyltransferase and degradative alpha-galactosidase activities and subcellular distribution of Gb3 at various ages. Quantitative high-performance liquid chromatography demonstrated a similar developmental pattern in both microsomal and MVM Gb3, indicating that late expression of MVM Gb3 is not due to delayed migration of Gb3 from the microsomal to the MVM. The specific Gb3 galactosyltransferase activity increased with age, with a sharp increase seen at 18 days of age, whereas alpha-galactosidase activity followed an inverse pattern. Thus, regulation of both synthetic and degradative pathways for Gb3 appears to explain the observed changes in Gb3 levels with age. The nature of the signals for developmental regulation of Gb3 levels is unknown, as are the physiological consequences of altered MVM glycolipid composition other than mediating response to Shiga toxin.

Aging↗

PubMedDate not supplied

Beta-galactosidase-tagged adventitial myofibroblasts tracked to the neointima in healing rat vein grafts.

OBJECTIVE: Myofibroblasts are present transiently in normal healing wounds. However, they have been found to persist in the stroma of neoplasms, fibrotic conditions and other pathological settings. In rat vein grafts, we have observed the prolonged presence of myofibroblasts. Our aim was to determine the origin of myofibroblasts in vein grafts. METHODS: Epigastric vein to femoral artery grafts were microsurgically placed in male Lewis rats and harvested. Neointimal development, cellular death and proliferation, and cell phenotypes were analyzed using immunohistochemistry and light and electron microscopy. To follow cellular movement in the vessel wall, vein grafts were transfected with replication-defective adenovirus containing the gene encoding beta-galactosidase (n = 50), and harvested at 1, 2, 3, 4, 5, 6, 7, 14 and 28 days. Grafts were analyzed after X-gal staining. RESULTS: Myofibroblasts were detected in the outer adventitia at 4 days, in the media at 1 week and in the developing neointima at 2 weeks. Cells tagged using adenoviral beta-galactosidase demonstrated adventitia to neointima cell migration. CONCLUSIONS: Although there may be other sources of myofibroblasts in this model, the adventitia has been shown to be an origin of myofibroblasts which subsequently migrate through the vessel wall to the neointima during graft remodeling and contribute to neointimal formation.

Adenoviridae↗

PubMed2004

Comparative in vitro expression study of four Fabry disease causing mutations at glutamine 279 of the alpha-galactosidase A protein.

Fabry disease is an X-linked lysosomal storage disorder caused by the deficiency of alpha-galactosidase A that results in the accumulation of neutral sphingolipids. We report a novel point mutation in exon 6, Q279K, carried by an asymptomatic child with a family history of classic Fabry disease. Moreover, we comparatively study the in vitro expression and enzyme activity of Q279K and three other already described mutants in glutamine 279. The Q279K, Q279H and Q279R mutants transfected in COS-1 cells expressed no activity while the residual enzyme activity of the Q279E mutant represented 10% of wild type value. Western blot analysis demonstrated a differential behavior of the mutant proteins: Q279K and Q279H persisted as the inactive 50-kD precursor, indicating that these mutations may affect the normal processing of the enzyme, while the Q279R mutant was not detected probably due to an unstable protein which is rapidly degraded. The in vitro expression studies of the novel Q279K mutation were confirmed by Western blot analysis performed in the patient's lymphocytes which revealed the alpha-galactosidase A precursor of 50 kD but not the processed form.

Animals↗

PubMed1997

Localization of beta-D-galactosidase activity in semithin epon sections of embryonic tissues using differential interference contrast optics.

In the present study we describe a method for the histochemical demonstration of beta-D-galactosidase activity on tissue sections processed for light microscopy at high resolution. 5-Bromo-indolyl-beta-D-galactopyranoside (Bluo-Gal) was utilized as an indigogenic method for the demonstration of Escherichia coli beta-D-galactosidase reporter gene activity whose expression was studied in a transgenic line where the enzyme, with a nuclear localization signal (nlacZ), is under the transcriptional control of a striated muscle-specific promoter. At the light-microscopic level, by using Differential Interference Contrast (DIC) optics, the reaction product was detected as precipitates in the form of fine birefringent crystals. These were located around and inside the nuclei of beta-gal-expressing cells. This simple method allows an easy and rapid identification of few or even one labeled cell(s) within large microscopic fields (whole embryos) and the labeled cell(s) can be evaluated both morphologically and quantitatively.

Animals↗

PubMed1976

Separation of beta-galactosidases and beta-glucosidases from human liver.

Various fractions obtained from normal human liver homogenates by gel chromatography and selective adsorption and elution from insolubilized concanavalin A were compared as to their beta-galactosidase and beta-glucosidase activities. The high-molecular-weight acidic beta-galactosidase form was converted into the smaller major form by sodium dodecyl sulfate treatment. Electrophoresis and electrofocusing on acrylamide slabs revealed, in addition to the two major isoenzyme forms (acid and neutral), another five minor bands with enzymatic activity.

Galactosidases↗

PubMed1988

A reliable method combining horseradish peroxidase histochemistry with immuno-beta-galactosidase staining.

A sensitive combination of horseradish peroxidase (HRP) tracing and immunohistochemistry was used by Rye et al. [J Histochem Cytochem (1984) 32:1145] in a search for the origins of neurotransmitter- and neuromodulator-containing nerve fibers in brain. In this combination, peroxidase as a marker in immunohistochemistry was thought to yield a homogeneous brown immunoreaction product of diaminobenzidine, different from the black granular reaction product of retrogradely transported HRP, which is visualized by the tetramethylbenzidine (TMB) reaction and subsequent stabilization. A neuron that exhibits both kinds of reaction products in its cytoplasm in sections subjected to combination staining is referred to as a double-labeled cell. With a combined HRP and corticotropin-releasing factor (CRF) immunoperoxidase-antiperoxidase (PAP) method, the first set of experiments showed "false" double-labeled cells in the pyramidal cell layer of rat cerebral cortex, but only rarely in the subcortical areas, possibly because of the use of one enzyme system in two different histochemical procedures. This limitation of the double-staining technique prompted us to demonstrate an alternate combination of HRP tracing and immunohistochemistry in the second set of experiments by employing two previously described independent enzyme systems: HRP as a retrograde tracer and beta-galactosidase as a marker for immunohistochemical demonstration of CRF. A homogeneous blue reaction product indicated immuno-beta-galactosidase staining, and a granular black or brown reaction product labeled retrogradely transported HRP in double-labeled cells in subcortical regions. Neither double labeling nor "false" double labeling was seen in pyramidal cells of cerebral cortex. These findings suggest that application of two independent enzyme systems in a combined HRP and immunohistochemical method may be useful for investigating in origins of peptidergic fibers in brain when the combination of HRP histochemistry and the PAP method appears to be inappropriate.

Animals↗

PubMed2001

Induction of stable prenatal tolerance to beta-galactosidase by in utero gene transfer into preimmune sheep fetuses.

The successful transduction of hematopoietic stem cells and long-term (28 months) transgene expression within the hematopoietic system following the direct injection of high-titer retroviral vectors into preimmune fetal sheep was previously demonstrated. The present studies extended these analyses for 40 months postinjection and evaluated whether the longevity of transgene expression in this model system was the result of induction of prenatal tolerance to the transgene product. The intraperitoneal injection of retroviral vectors into preimmune sheep fetuses transduces thymic epithelial cells thought to present antigen and thus define self during immune system development. To directly demonstrate induction of tolerance, postnatal sheep were boosted with purified beta-galactosidase and showed that the peripheral blood lymphocytes from in utero-transduced sheep exhibited significantly lower stimulation indices to transduced autologous cells than did control animals and that the in utero-transduced sheep had a reduced ability to mount an antibody response to the vector-encoded beta-galactosidase protein compared with control sheep. Collectively, our results provide evidence that the direct injection of retroviral vectors into preimmune sheep fetuses induces cellular and humoral tolerance to the vector/transgene products and provide an explanation for the duration and stability of transgene expression seen in this model. These results also suggest that even relatively low levels of gene transfer in utero may render the recipient tolerant to the exogenous gene and thus potentially permit the successful postnatal treatment of the recipient. (Blood. 2001;97:3417-3423)

Animals↗

PubMed2005

Characterization of gana-1, a Caenorhabditis elegans gene encoding a single ortholog of vertebrate alpha-galactosidase and alpha-N-acetylgalactosaminidase.

BACKGROUND: Human alpha-galactosidase A (alpha-GAL) and alpha-N-acetylgalactosaminidase (alpha-NAGA) are presumed to share a common ancestor. Deficiencies of these enzymes cause two well-characterized human lysosomal storage disorders (LSD)--Fabry (alpha-GAL deficiency) and Schindler (alpha-NAGA deficiency) diseases. Caenorhabditis elegans was previously shown to be a relevant model organism for several late endosomal/lysosomal membrane proteins associated with LSDs. The aim of this study was to identify and characterize C. elegans orthologs to both human lysosomal luminal proteins alpha-GAL and alpha-NAGA. RESULTS: BlastP searches for orthologs of human alpha-GAL and alpha-NAGA revealed a single C. elegans gene (R07B7.11) with homology to both human genes (alpha-galactosidase and alpha-N-acetylgalactosaminidase)--gana-1. We cloned and sequenced the complete gana-1 cDNA and elucidated the gene organization.Phylogenetic analyses and homology modeling of GANA-1 based on the 3D structure of chicken alpha-NAGA, rice alpha-GAL and human alpha-GAL suggest a close evolutionary relationship of GANA-1 to both human alpha-GAL and alpha-NAGA. Both alpha-GAL and alpha-NAGA enzymatic activities were detected in C. elegans mixed culture homogenates. However, alpha-GAL activity on an artificial substrate was completely inhibited by the alpha-NAGA inhibitor, N-acetyl-D-galactosamine.A GANA-1::GFP fusion protein expressed from a transgene, containing the complete gana-1 coding region and 3 kb of its hypothetical promoter, was not detectable under the standard laboratory conditions. The GFP signal was observed solely in a vesicular compartment of coelomocytes of the animals treated with Concanamycin A (CON A) or NH4Cl, agents that increase the pH of the cellular acidic compartment. Immunofluorescence detection of the fusion protein using polyclonal anti-GFP antibody showed a broader and coarsely granular cytoplasmic expression pattern in body wall muscle cells, intestinal cells, and a vesicular compartment of coelomocytes.Inhibition of gana-1 by RNA interference resulted in a decrease of both alpha-GAL and alpha-NAGA activities measured in mixed stage culture homogenates but did not cause any obvious phenotype. CONCLUSIONS: GANA-1 is a single C. elegans ortholog of both human alpha-GAL and alpha-NAGA proteins. Phylogenetic, homology modeling, biochemical and GFP expression analyses support the hypothesis that GANA-1 has dual enzymatic activity and is localized in an acidic cellular compartment.

Animals↗

PubMed1992

Establishment of a host-vector system in Lactobacillus helveticus with beta-galactosidase activity as a selection marker.

A host-vector system was established in Lactobacillus helveticus with beta-galactosidase activity as a selection marker. Plasmid pBG10 was constructed by joining the beta-galactosidase gene from L. bulgaricus, the promoter region of the erythromycin resistance gene from pAM beta 1, the replication region of pBR329, and the replication region of the L. helveticus cryptic plasmid pLJ1. L. helveticus SBT2195 (Lac- mutant), transformed with pBG10, was selected on skim milk plates. The structural gene of alpha-amylase (1536 bp) from Bacillus licheniformis, inserted downstream of the promoter region of the erythromycin resistance gene of pBG10, was expressed in L. helveticus SBT2195. Plasmid pBG10 is a food-grade and expression vector in L. helveticus.

Base Sequence↗