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PubMed1977

[Role of substrate inductors in the mechanism of cortisol action on beta-galactosidase activity in different strains of E. coli k-12 and in rat liver].

A comparative study of the effect of cortisone on the beta-galactosidase synthesis in E. coli K-12 strains with an induced (E. coli 200 PS/Iac), constitutive (E. coli ML-308), and superrepressed (E. coli 200is) type of the enzyme synthesis and in the cells of rat liver demonstrated that the hormone proper had no derepressive effect. An increase of the beta-galactosidase synthesis occurred only in the presence of specific substrate inductors. It is supposed that the principal link in the action mechanism of cortisone on the laclose operon of E. coli and the enzyme production in the cells of the rat liver is preliminary derepression of the genome areas by means of the substrate inductors.

Animals↗
PubMed1977

[Dry medium for determining the beta-galactosidase activity of enterobacteria].

Differential diagnosis of enterobacteria is based on the determination of beta-galactosidase enzyme, hydrolyzing lactose in the nutrient substrates; however, the tests suggested for its determination are time consuming and their use in practice is limited. Dry nutrient medium prepared by the authors containing o-nitrophenyl-betaD-pyranoside was tested on 1625 strains of enterobacteria in comparison with the Le Minor's ONPS test used at present. The results of beta-galactosidase determination by the, mentioned methods proved to coincide (significance level greater than 95%); this permits to recommend the method described--in dry nutrient medium--as a differential-diagnostic test for the identification of enterobacteria.

Culture Media↗
PubMedDate not supplied

[Lactose content and beta-galactosidase activity in yogurt, cheeses and curdled milk made in Brazil].

We studied yogurts and industrialized curdled milk in three different storage times, as well as homemade and Syrian curdled milk and cheeses. Lactose content (gm%) and beta-galactosidase activity were determined in these products. Lactose content was elevated in yogurts and curdled milk with a lactose reduction of about 22% compared to the lactose of cow milk, with a mean and standard desviation (M +/- SD) of 3.81 +/- 0.47 gm%. In the cheeses lactose content was low, between 1.91 and 0.03 g%. beta-galactosidase activity was present in yogurts and curdled milk, with values between 0.58 and 3.61 U in time I and M +/- SD of 0.15 +/- 0.23 U. A significant decrease of beta-galactasidase activity was observed during the storage time. By analyzing and comparing these findings with the literature, we conclude that the products studied, by their low content of lactose (cheeses and yakult) or by the presence of beta-galactasidase activity (yogurts and curdled milk at time I) probably would be tolerated by most hipolactasic persons.

Brazil↗
PubMed1995

Myeloproliferative sarcoma virus directed expression of beta-galactosidase following retroviral transduction of murine hematopoietic cells.

The introduction of genetic sequences into hematopoietic stem cells (HSC) has allowed study of HSC proliferation in vivo by proviral-sequence molecular analysis in the DNA of progeny. Analysis of HSC proliferation could be enhanced by development of a retroviral vector that encodes a reporter gene that allows sensitive detection of transduced cells. We developed a recombinant retrovirus vector encoding the reporter gene lacZ under the transcriptional control of the myeloproliferative sarcoma virus long-terminal repeat (LTR). Bone marrow cells from C3H mice were co-cultured on retrovirus producer cell lines and cultured for growth of colony-forming unit granulocyte/macrophage (CFU-GM) and high proliferative potential colony-forming cells (HPP-CFC) in semisolid media or were transplanted into irradiated recipients. In other experiments, recombinant retrovirus was injected in vivo into the liver of developing fetal rat pups, and circulating hematopoietic cells of the postnatal rats were analyzed for evidence of proviral integration and expression of beta-galactosidase. Expression of lacZ was detected in both CFU-GM and HPP-CFC that were cultured immediately following in vitro infection of mouse bone marrow. Beta-galactosidase activity from the retrovirus was also detected in both marrow cells isolated from reconstituted mice 22 weeks following transplantation as well as in blood cells of postnatal rats transduced in utero with the recombinant retrovirus. This strategy may be especially useful for characterizing proliferation of transduced populations of hematopoietic cells and in the development of protocols for somatic gene therapy.

Animals↗
PubMed1994

Divalent metal ion requirements of a thermostable multimetal beta-galactosidase from Saccharopolyspora rectivirgula.

To understand the roles of metal ions on the catalytic properties and thermostability of the thermostable beta-galactosidase of Saccharopolyspora rectivirgula, a thermophilic actinomycete, we have investigated the binding kinetics and requirements of divalent metal ions by equilibrium dialysis, titration, and metal ion buffer techniques. We found that the monomeric multimetal enzyme (M(r) 136,977) had eight specific binding sites for divalent metal ions. These sites were classified as follows: a very tight (class I) site for Ca2+, three tight (class II) sites consisting of two Ca(2+)-specific sites (class IICa) and one Mn(2+)-specific site (class IIMn; Kd for Mn2+, 2.0 nM), and four loose (class III) sites for Mn2+ (Kd, 1.2 microM) and Mg2+ (Kd, 2 microM). Removal of metal ions bound to class II and III sites of the holoenzyme (Ca3Mn5 species; relative Vmax (Vrel), 100%) by a chelating resin at 4 degrees C yielded a less thermostable Ca1 species (Vrel, 1.7%) with a class I Ca2+ ion, removal of which by a chelating resin at 50 degrees C caused a complete irreversible inactivation of the enzyme. Titration studies revealed that stoichiometric binding of Mn2+ to a class IIMn site of the Ca1 species caused a 33-fold activation whereas binding of Ca2+ to class IICa sites had no effect on enzyme activity. Ca1 species could be also activated 8-fold by heating at 60 degrees C for 20 min, suggesting that the catalytically important class II Mn2+ plays important roles in maintaining the native structure essential for activity. Occupation of class III sites by Mg2+ or Mn2+ was of physiological importance to attain sufficient thermostability by which this extracellular beta-galactosidase remained active for a prolonged time at elevated temperatures as was observed during growth of S. rectivirgula.

Binding Sites↗
PubMed1994

Proteolytic processing of ovalbumin and beta-galactosidase by the proteasome to a yield antigenic peptides.

The identification of genes in the class II region of the MHC that are homologous to genes encoding subunits of the proteasome has led to intense interest in the possible role of this enzyme in the proteolytic processing of polypeptide Ags. We have tested the ability of the 20S proteasome to produce peptides that can be presented by class I molecules as targets for killing by OVA-specific and beta-galactosidase-specific CTL clones. Samples of intact OVA and beta-galactosidase were subjected to digestion in vitro by 20S proteasome purified from bovine red cells and the resulting peptide mixtures were fractionated by reverse-phase HPLC. The fractions were tested for their ability to sensitize appropriate mouse target cells for lysis by specific CTL clones. In both cases, components that under all chromatographic conditions eluted with retention times indistinguishable from synthetic peptides representing known epitopes of the naturally processed proteins were found to be able to sensitize the target cells. Moreover, in the case of OVA, the presence of the expected target peptides was demonstrated directly by amino acid sequence and mass spectrometric analysis. The results demonstrate that the pure 20S proteasome is capable of generating antigenic peptides from two proteins for presentation by class I molecules without the participation of additional components of the protein degradation system. This finding is consistent with the hypothesis of proteasome involvement in Ag processing in vivo.

Amino Acid Sequence↗
PubMed1994

Mutations in the lysosomal beta-galactosidase gene that cause the adult form of GM1 gangliosidosis.

Three adult patients with acid beta-galactosidase deficiency/GM1 gangliosidosis who were from two unrelated families of Scandinavian descent were found to share a common point mutation in the coding region of the corresponding gene. The patients share common clinical features, including early dysarthria, mild ataxia, and bone abnormalities. When cDNA from the two patients in family 1 was PCR amplified and sequenced, most (39/41) of the clones showed a C-to-T transition (C-->T) at nucleotide 245 (counting from the initiation codon). This mutation changes the codon for Thr(ACG) to Met(ATG). Mutant and normal sequences were also found in that position in genomic DNA, indicating the presence of another mutant allele. Genomic DNA from the patient in family 2 revealed the same point mutation in one allele. It was determined that in each family only the father carried the C-->T mutation. Expression studies showed that this mutation produced 3%-4% of beta-galactosidase activity, confirming its deleterious effects. The cDNA clones from the patients in family 1 that did not contain the C-->T revealed a 20-bp insertion of intronic sequence between nucleotides 75 and 76, the location of the first intron. Further analysis showed the insertion of a T near the 5' splice donor site which led to the use of a cryptic splice site. It appears that the C-->T mutation results in enough functional enzyme to produce a mild adult form of the disease, even in the presence of a second mutation that likely produces nonfunctional enzyme.

Adult↗
PubMed1993

Mutations in acid beta-galactosidase cause GM1-gangliosidosis in American patients.

We describe four new mutations in the beta-galactosidase gene. These are the first mutations causing infantile and juvenile GM1-gangliosidosis to be described in American patients. Cell lines from two patients with juvenile and from six patients with infantile GM1-gangliosidosis were analyzed. Northern blot analysis showed the acid beta-galactosidase message to be of normal size and quantity in two juvenile and four infantile cases and of normal size but reduced quantity in two infantile cases. The mutations are distinct from the Japanese mutations. All are point mutations leading to amino acid substitutions: Lys577-->Arg, Arg590-->His, and Glu632-->Gly. The fourth mutation, Arg208-->Cys, accounts for 10 of 16 possible alleles. Two infantile cases from Puerto Rico of Spanish ancestry are homozygous for this mutation, suggesting that this allele may have come to South America and North America via Puerto Rico. That these mutations cause clinical disease was confirmed by marked reduction in catalytic activity of the mutant proteins in the Cos-1 cell expression system.

Animals↗
PubMed1993

Conversion of vitamin D3 binding protein (group-specific component) to a macrophage activating factor by the stepwise action of beta-galactosidase of B cells and sialidase of T cells.

Highly conserved DBP (human DBP is known as Gc) of serum alpha 2-globulin fraction can be converted to a potent macrophage activating factor by stepwise modification of Gc glycoprotein with beta-galactosidase of B cells and sialidase of T cells. These glycosidases, beta-galactosidase and sialidase, are membrane bound and not soluble in culture medium. Thus, consecutive contact of Gc protein with B cells and T cells, presumably via specific receptors, is required for conversion of Gc glycoprotein to the macrophage activating factor. The essential role of T cell sialidase in macrophage activation was confirmed by the finding that peritoneal nonadherent cells of SM/J mouse, whose T cells are deficient in sialidase activity, were unable to convert Gc protein to the macrophage activating factor and thus did not activate macrophages. Treatment with sialidase of a conditioned medium of lipid metabolite-treated SM/J mouse nonadherent cells efficiently generated the macrophage activating factor. When Gc protein was first treated with soluble or immobilized sialidase and used in a medium for 2 h cultivation of lipid metabolite-treated SM/J mouse nonadherent cells or BALB/c mouse B cells, the resultant conditioned media contained a large amount of the macrophage activating factor. These results support the hypothesis that Gc protein carries a dibranched trisaccharide with galactose and sialic acid termini.

Animals↗
PubMed1993

[Molecular genetics of beta-galactosidase deficiency (GM1-gangliosidosis and Morquio syndrome type B)].

Recent advances in the molecular study of beta-galactosidase deficiency (GM1-gangliosidosis and Morquio syndrome type B) are reviewed. Until now, 14 different mutations have been found in the beta-galactosidase gene in patients with this disorder. Gene mutations are heterogeneous, but common and specific mutations have been identified for three types of protracted clinical course; 51Ile-->Thr mutation for Japanese adult/chronic GM1-gangliosidosis, 201Arg-->Cys for Japanese late infantile/juvenile GM1-gangliosidosis and 273Trp-->Leu for Caucasian Morquio syndrome type B. These phenotype-specific mutant genes produce mutant proteins with significant residual enzyme activity, whereas mutant proteins associated with infantile GM1-gangliosidosis patients show complete loss of enzyme activity. The phenotypic variations of this disorder may be related to different mode of intracellular processing and turnover of mutant enzyme proteins.

Adolescent↗
PubMed1995

Enzymic hydrolysis of raffinose and stachyose in soymilk by alpha-galactosidase from Gibberella fujikuroi.

The use of intracellular alpha-galactosidase from Gibberella fujikuroi to remove raffinose and stachyose in soymilk was studied. The optimum conditions for the enzymic hydrolysis of raffinose and stachyose was pH 5.5 to 6.0 at 55 degrees C. Alpha-galactosidase showed optimum activity at pH 5.0 and 50 degrees C with the substrate p-nitrophenyl-alpha-D-galacto-pyranoside (PNGP). The enzyme showed no detectable loss of activity when held more than 8 hr at 50 degrees C. Thin layer chromatography (TLC) revealed the following composition of oligosaccharides in local soybean variety: sucrose, 5.53%; raffinose, 1.95%; and stachyose, 6.1%. Investigation by TLC showed complete hydrolysis of raffinose and stachyose in 3 hr. HPLC analysis of hydrolyzate indicated complete hydrolysis of stachyose, and more than 60% hydrolysis of raffinose in 2.5 hr.

Chromatography, High Pressure Liquid↗
PubMed1995

Organ-specific binding system for beta-galactosidase in the male reproductive tract.

This study reports on the binding of beta-galactosidase obtained from different organs of the rat urogenital tract to membranes of these organs. Homologous and cross binding saturation assays indicated that: (1) high-affinity sites that recognize fructose-6-phosphate derivates (FPR) are present in spermatozoa from the rete testis, epididymal membranes and testes, although the latter may reflect binding to testicular spermatozoa; (2) the membranes of the other organs studied do not have FPR; (3) the FPR of the epididymis does not recognize enzymes purified from other organs of the reproductive tract. These results suggest that the FPR-binding system belongs to a peculiar transport route that permits maturing spermatozoa to acquire hydrolytic enzymes secreted by the epididymal epithelium. In the epididymis and seminal vesicles more than 50% of the enzymatic activity of beta-galactosidase was recovered in cytosol, suggesting that the enzyme is located mainly in the secretory fluid of these organs.

Animals↗
PubMed1977

[Lysosomal neuraminidase, beta-galactosidase and beta-N-acetylglucosaminidase of regenerating liver tissue].

Neuraminidase (EC 3.2.1.18) beta-galactosidase (EC 3.2.1.23) and beta-N-acetylglucosaminidase (EC 3.2.1.30) were studied in normal and regenerating rat liver. All these glycosidases were shown to be predominantly localized in lysosome rich fraction. The activity of lysosomal neuraminidase in regenerating rat liver increased 24 hours after partial hepatectomy, whereas those of beta-galactosidase and beta-N-acetylglucosaminidase decreased. The presence of inhibitor of neurominidase in regenerating liver homogenate was shown. Actinomycin D and cycloheximide were shown to have no significant effect on lysosomal neuraminidase in regenerating rat liver.

Acetylglucosaminidase↗
PubMedDate not supplied

Aryl beta-galactosidase from Sclerotium rolfsii: physiological and biochemical studies.

Production of beta-galactosidase by Sclerotium rolfsii NCIM 1084 was studied under submerged fermentation conditions. The enzyme was produced extracellularly and constitutively on glucose. The enzyme production was enhanced when galactose, raffinose, cellobiose, sucrose, xylose, maltose, cellulose and pectin were used as carbon sources. Cellulose and diammonium hydrogen phosphate were best carbon and nitrogen sources, respectively. Surfactants such as Sag, Paraffin oil, Tween 20 and Tween 80 increased the enzyme production. Maximum yield of beta-galactosidase obtained was 3.8-4.2 nkat/ml. The optimum pH, optimum temperature and molecular weight of the beta-glactosidase were 2.7, 60 degrees C and 2,21,000 daltons, respectively. The enzyme is an aryl beta-glactosidase and did not hydrolyse lactose. The Km value for o-nitrophenyl beta-D-galactoside was 3.7 mM. Galactose and 2-mercaptoethanol inhibited the enzyme.

Ascomycota↗
PubMed1997

Fabry disease: thirty-five mutations in the alpha-galactosidase A gene in patients with classic and variant phenotypes.

BACKGROUND: Fabry disease, an X-linked inborn error of glycosphingolipid catabolism, results from mutations in the alpha-galactosidase A (alpha-Gal A) gene located at Xq22.1. To determine the nature and frequency of the molecular lesions causing the classical and milder variant Fabry phenotypes and for precise carrier detection, the alpha-Gal A lesions in 42 unrelated Fabry hemizygotes were determined. MATERIALS AND METHODS: Genomic DNA was isolated from affected probands and their family members. The seven alpha-galactosidase A exons and flanking intronic sequences were PCR amplified and the nucleotide sequence was determined by solid-phase direct sequencing. RESULTS: Two patients with the mild cardiac phenotype had missense mutations, I9IT and F113L, respectively. In 38 classically affected patients, 33 new mutations were identified including 20 missense (MIT, A31V, H46R, Y86C, L89P, D92Y, C94Y, A97V, R100T, Y134S, G138R, A143T, S148R, G163V, D170V, C202Y, Y216D, N263S, W287C, and N298S), two nonsense (Q386X, W399X), one splice site mutation (IVS4 + 2T-->C), and eight small exonic insertions or deletions (304del1, 613del9, 777del1, 1057del2, 1074del2, 1077del1, 1212del3, and 1094ins1), which identified exon 7 as a region prone to gene rearrangements. In addition, two unique complex rearrangements consisting of contiguous small insertions and deletions were found in exons 1 and 2 causing L45R/H46S and L120X, respectively. CONCLUSIONS: These studies further define the heterogeneity of mutations causing Fabry disease, permit precise carrier identification and prenatal diagnosis in these families, and facilitate the identification of candidates for enzyme replacement therapy.

Chromosome Mapping↗
PubMed1997

Effect of silkworm hemolymph on the expression of E. coli beta-galactosidase in insect cell lines infected with recombinant baculoviruses.

The effects of silkworm hemolymph on the expression of foreign genes by recombinant baculoviruses in cell lines were studied. The expression efficiency of beta-galactosidase by recombinant virus containing the E. coli lacZ gene at various concentrations of hemolymph and FBS was determined in BmN and Sf cell lines. The addition of hemolymph to the medium containing FBS accelerated the expression of beta-galactosidase by recombinant viruses in both cells. It was more effective in BmN cells than in Sf cells. Hemolymph was most effective in enhancing virus multiplicity under conditions of 5% FBS.

Animals↗
PubMedDate not supplied

Construction and characterization of bicistronic retroviral vectors encoding the multidrug transporter and beta-galactosidase or green fluorescent protein.

Multidrug resistance (MDR) can be conferred by overexpression of the adenosine triphosphate-driven multidrug transporter P-glycoprotein (Pgp) known as MDR1. Thus, two potential applications of the MDR1 gene that may be useful in gene therapy are the protection of bone marrow cells from the cytotoxic effects of chemotherapy regimens in cancer patients and its possible use as an in vivo selectable gene when linked to a therapeutic gene. In this study, we have designed two retroviral bicistronic expression vectors by linking the MDR1 gene to the reporters known as beta-galactosidase and the red-shifted green fluorescent protein (GFP). We report the creation of stable producer cell lines that synthesize virus particles carrying the MDR-internal ribosomal entry site (IRES)-lacZ and the MDR-IRES-GFP transgenes. These transcriptional fusions allow coordinate expression of Pgp and the reporter gene product to easily mark the MDR phenotype. Using the MDR-IRES-lacZ retrovirus, we demonstrate that periodic pulses of cytotoxic drug selection with a Pgp substrate enable sustained, long-term expression of the reporter beta-galactosidase in otherwise unstable transductants. We have also incorporated the improved features of GFP as a reporter gene into our MDR-IRES-GFP retrovirus. This vector allows rapid and specific identification of MDR1 gene transfer and expression in living cells either by fluorescence microscopy or by fluorescence-activated cell sorter analysis. These two MDR/reporter gene systems should be useful for in vivo studies, the evaluation of the potential of the MDR1 gene in gene therapy applications, and as a monitor of the selective efficacy of its MDR phenotype.

3T3 Cells↗
PubMed1998

Beta-galactosidase in immobilized cells of gherkin Cucumis sativus L.

Cell suspensions of gherkin (Cucumis sativus L.) were permeabilized by Tween-80, and immobilized by glutaraldehyde. Beta-galactosidase showed pH optimum at 4.9 and temperature optimum at 58 degrees C. The enzyme catalysed hydrolysis was linear for 3 h with 60-68% conversion of the substrate. The cells characterized by high beta-galactosidase activity and stability on long-term storage showed valuable technological properties.

Catalysis↗