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PubMedDate not supplied

The influence of CpG motifs on a protein or DNA-based Th2-type immune response against major pollen allergens Bet v 1a, Phl p 2 and Escherichia coli-derived beta-galactosidase.

BACKGROUND: DNA immunization and protein immunization with CpG motifs as adjuvants represent promising approaches in allergen-specific immunotherapy. OBJECTIVE: We investigated the effect of coinjection or prepriming with CpG-ODN on Th2-type responses induced by gene gun and protein immunization. METHODS: BALB/c mice were immunized with the gene gun using plasmid DNA containing the cDNAs coding for the genes of Bet v 1a, Phl p 2 and beta-galactosidase or with the purified Al(OH)(3)-adsorbed proteins. In addition, CpG-ODN were applied by coinjection or by prepriming treatment. Antibody and cytokine responses were measured by ELISA, proliferative and cytotoxic responses were determined by standard labeling procedures. Furthermore, the allergenic activity of sera was measured by passive cutaneous anaphylaxis. RESULTS: Gene gun immunization and protein immunization induced a clear Th2-type response for all antigens. The Th1-promoting effect of CpG-ODN coinjection together with gene gun immunization was restricted to beta-galactosidase as indicated by the increase of IgG2a and a marked expression of IFN-gamma. CpG motifs also increased the specific cytotoxic response against beta-galactosidase. Prepriming with CpG-ODN and gene gun or protein immunization with Bet v 1a exhibited no significant difference to the non-CpG control group. However, sera from mice preprimed with CpG-ODN induced no anaphylaxis with gene gun immunization, but with protein immunization. CONCLUSIONS: The effect of CpG motifs in vivo depends on a variety of parameters like the nature of the antigen and the immunization modality. Furthermore, our studies indicate that a combination of CpG + DNA immunization may be more effective in antagonizing Th2 responses than the combination of CpG + protein immunization.

Adjuvants, Immunologic↗
PubMed1996

Fabry disease: fourteen alpha-galactosidase A mutations in unrelated families from the United Kingdom and other European countries.

The nature of the molecular lesions in the alpha-galactosidase A gene causing Fabry disease in 12 unrelated families from the United Kingdom and 4 from other European countries was determined in order to provide precise heterozygote detection and prenatal diagnosis for these families. The entire alpha-galactosidase A coding region and flanking intronic sequences were analyzed by amplification of genomic DNA and solid-phase direct sequencing or by SSCP analysis followed by solid-phase direct sequencing. Fourteen new mutations were identified including 10 missense mutations (M42V, R49S, C56Y, D92H, D93G, P205T, W236C, W287G, N298H, and W340R), 2 nonsense mutations (Q107X and Q119X) and 2 small deletions (257del18 and 1087del1). Together with the previously reported mutations, a total of 33 lesions in the alpha-galactosidase A gene have been identified in unrelated British families, further documenting the molecular genetic heterogeneity of this disease.

Fabry Disease↗
PubMed1969

Intestinal beta-galactosidases. I. Separation and characterization of three enzymes in normal human intestine.

Previous studies based on work in the rat and preliminary experiments with human intestine have suggested that two beta-galactosidases are present in small intestine, and it is believed that only one of these enzymes is a lactase important for the digestion of dietary lactose. The high prevalence of intestinal lactase deficiency in man prompted more complete study of these enzymes. Human intestinal beta-galactosidases were studied by gel filtration on Sephadex G-200 and Biogel P-300 as well as by density gradient ultracentrifugation. Gel filtration produced partial separation into three peaks of enzyme activity, but much activity against synthetic substrates was lost. Only the trailing peak with specificity for synthetic beta-galactosides was completely separated from the other enzymes. Thus gel filtration was not a suitable preparative procedure for biochemical characterization. Density gradients separated the enzymes more completely, and they were designated according to their sedimentation rates and further characterized. Enzyme I has a molecular weight of 280,000, pH optimum of 6.0, and specificity for lactose of at least five times that for cellobiose or synthetic substrates. A second lactase, enzyme II, possesses slightly greater activity against lactose than for some synthetic substrates and is incapable of splitting cellobiose. Further, it has a lower pH optimum (4.5) and is present in two molecular species (molecular weights 156,000 and 660,000). Enzyme III shows specificity only for synthetic beta-galactosides but has a pH activity curve identical with enzyme I and a molecular weight of 80,000. Whereas human liver and kidney contain a beta-galactosidase with the same biochemical characteristics as intestinal enzyme II, enzymes I and III appear to be peculiar to intestine, and enzyme I most probably represents the lactase of importance in the mucosal digestion of dietary lactose. The following paper considers this further in terms of the biochemical change in intestinal lactase deficiency.

Chromatography↗
PubMed1993

The 67-kD elastin/laminin-binding protein is related to an enzymatically inactive, alternatively spliced form of beta-galactosidase.

We and others have previously shown that a 67-kD cell surface elastin/laminin-binding protein (EBP) is responsible for cell adhesion to elastin and laminin and for mediating the process of elastin fiber assembly, but the nature of this protein was unknown. In this report we provide evidence that a 67-kD catalytically inactive form of beta-galactosidase produced by alternative splicing demonstrates immunological and functional similarity and sequence homology to the 67-kD EBP, suggesting that the two might be the same. Antibody prepared to a synthetic peptide, N-Ac-GSPSAQDEASPL, corresponding to a frame-shift-generated sequence unique to the alternatively spliced form of human beta-galactosidase, also recognized sheep EBP both on Western blotting and in aortic tissue. Furthermore, this synthetic peptide (S-GAL) binds to elastin and laminin, but not to fibronectin, collagen I, or collagen III. Moreover, both tropoelastin and laminin which bind to S-GAL peptide affinity columns can be specifically eluted from them with an excess of free S-GAL peptides. In addition, sequence homology among this splice variant of human beta-galactosidase, sheep EBP, and NH2-terminal sequences of some elastases suggests that these proteins share a common ligand-binding motif that has not been previously recognized.

Alternative Splicing↗
PubMedDate not supplied

Incorporation of beta-galactosidase-expressing endothelial cells into the skeletal muscle microvascular bed of mice.

Cloned murine endothelial cells (cEC) were used as a carrier system for introducing a foreign gene into the microvascular bed of the hind limb of inbred mice. cEC were transfected with a beta-galactosidase-neo fusion construct, which enables both selection for DNA uptake in the presence of G 418 and the staining of cells for beta-galactosidase activity. Transfected cEC adhered and integrated readily into confluent monolayers of nontransfected cEC (up to 26% of total cell number). Seeding lacZ-transfected cEC on explanted arteries revealed rapid adhesion of the cells (within minutes) to the intact endothelium. After injection of 10(6) transfected EC via the femoral artery into the microvascular bed of the hind limb their presence was documented by beta-galactosidase staining after various time periods (1 h to 4 wk). Implanted cEC were detected in numerous elements of the microcirculation both in frozen sections and in squash preparations of the hind limb muscle and in the femoral bone up to 4 wk after the injection. The microvascular bed of skeletal muscle of the mouse as a recipient site for transduced syngeneic endothelial cells is, thus, a suitable experimental model to study various strategies for somatic gene therapy.

Animals↗
PubMed1986

Atypical adult GM1 gangliosidosis: biochemical comparison with other forms of primary beta-galactosidase deficiency.

We studied beta-galactosidase in skin fibroblasts from patients with different forms of beta-galactosidase deficiency: adult GM1 gangliosidosis, type 1 GM1 gangliosidosis, and Morquio B syndrome. Enzyme properties in the adult cases differed from the other disorders and also from normal controls. Genetic hybridization studies indicated that all three forms belong to the same complementation group. Therefore, the adult disorder must be due to a mutation of the structural gene for beta-galactosidase, which is allelic to the mutations in type 1 GM1 gangliosidosis and Morquio B syndrome.

Fibroblasts↗
PubMed1994

The 'prespore-like cells' of Dictyostelium have ceased to express a prespore gene: analysis using short-lived beta-galactosidases as reporters.

In transgenic strains of Dictyostelium discoideum that express beta-galactosidase under the control of a prespore-specific promoter, only early slugs show reporter confined to the prespore zone. As slugs migrate beta-galactosidase-positive cells accumulate in the prestalk zone; ultimately, there may be so many that the prestalk-prespore boundary is no longer distinguishable (Harwood, A., Early, A., Jermyn, K. and Williams, J. (1991) Differentiation 46, 7-13). It is not clear whether these 'anomalous' reporter-positive cells currently express prespore genes; another possibility is that they are ex-prespore cells that have transformed to prestalk and sorted to the prestalk zone (Sternfeld, J. (1993) Roux Archiv. Dev. Biol. 201, 354-363), while retaining their previously produced reporter. To test the activity of the prespore genes in these cells, we have made prespore reporter constructs whose products decay quickly; these are based on constructs used to investigate protein turnover in yeast (Bachmair, A., Finley, D. and Varshavsky, A. (1986) Science 234, 179-186). In strains bearing such constructs, beta-galactosidase-positive cells do not appear in the prestalk zone. The apparent deterioration of the prestalk/prespore pattern in older slugs is thus an artefact of reporter stability.

Animals↗
PubMed1994

Molecular cloning and nucleotide sequence of the beta-galactosidase gene from Enterobacter cloacae GAO.

The gene encoding a beta-galactosidase from Enterobacter cloacae GAO was cloned and expressed in Escherichia coli. The nucleotide sequence of the insert of a positive clone had an open reading frame of 3084 bp that encoded a polypeptide of 1028 amino acid residues with a calculated molecular mass of 116,677 daltons. The amino acid sequence of beta-galactosidase deduced from the nucleotide sequence, especially the sequence around the putative active site and of the fourteen regions, showed significant homology to beta-galactosidases of other microorganisms, E. coli, Klebsiella pneumoniae, Lactobacillus bulgaricus, and Clostridium acetobutylicum.

Amino Acid Sequence↗
PubMed1994

Galactosylation of cyclodextrins and branched cyclodextrins by alpha-galactosidases.

Transgalactosylated derivatives of cyclodextrins (CDs) and glucosyl and maltosyl CDs (G1- and G2-CDs) were synthesized by alpha-galactosidases from coffee bean and Mortierella vinacea (M. vinacea). The structures of the transfer products were analyzed by FAB-mass, 13C-NMR and methylation. Coffee bean alpha-galactosidase transferred a galactosyl residue not only to side chains of G1-CDs and G2-CDs, but also directly to CD rings. M. vinacea alpha-galactosidase transferred a galactosyl residue only to side chains of G2-CDs.

Carbohydrate Sequence↗
PubMed1995

Purification and some properties of alpha-galactosidase from Penicillium purpurogenum.

alpha-Galactosidase was purified by ion-exchange chromatographies on DEAE-cellulose and SE-cellulose columns from the culture filtrate of Penicillium purpurogenum No. 618. The final preparation was judged homogeneous by SDS-PAGE and its molecular mass and isoelectric point were estimated to be 67 kDa and 4.1, respectively. The N-terminal amino acid sequence of the enzyme was analyzed and aligned with those of other alpha-galactosidases. In addition, the enzyme acted on the stubbed alpha-galactosyl residue connected to the beta-1,4-manno-oligosaccharide chain, indicating that this specificity was quite different from that of Mortierella vinacea alpha-galactosidase.

Amino Acid Sequence↗
PubMed2000

Galactosylation of thiol group by beta-galactosidase.

beta-Galactosidase catalyzed beta-galactosylation not only of a hydroxyl group but also of a thiol group in the condensation reaction of D-galactose and 2-mercaptoethanol. The thio-galactosylation product was confirmed as 2-hydroxyethyl S-beta-D-galactoside on the bases of fast atom bombardment mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance spectorometry. Aspergillus oryzae beta-galactosidase hydrolyzed p-nitrophenyl S-beta-D-galactoside most rapidly among several beta-galactosidases and produced the thio-galactosylation product most efficiently. The Penicillim multicolor enzyme was as effective as the A. oryzae enzyme. However the enzymes from Escherichia coli, Saccharomyces fragilis, Kluyveromyces lactis, and Bacillus circulans galactosylated hydroxyl groups predominantly to produce O-galactoside. The thio-galactoside was synthesized most effectively at a 2-mercaptoethanol concentration of about 1.25 M. Galactose concentration at 0.8-2.8 M did not affect the synthetic yield of the thiogalactoside so greatly.

Aspergillus oryzae↗
PubMed1992

A new ultrasensitive bioluminogenic enzyme substrate for beta-galactosidase.

A derivative of D-luciferin, D-luciferin-O-beta-galactoside, was synthesized and used as highly sensitive substrate for beta-galactosidase. The substrate was physicochemically characterized. Enzymatic cleavage of the new compound by beta-galactosidase was demonstrated and kinetic constants Km, Vmax, kcat and kcat/Km have been determined. The compound has been proved to be a highly sensitive substrate for beta-galactosidase, permitting a limit of detection of 3.7 x 10(-19) mol of enzyme per assay.

Chemical Phenomena↗
PubMed2000

Use of chemical mutagenesis for the isolation of food grade beta-galactosidase overproducing mutants of bifidobacteria, lactobacilli and Streptococcus thermophilus.

A classical chemical mutagenesis protocol was evaluated for increasing beta-galactosidase production by probiotic bacteria to improve their potential to treat symptoms of lactose malabsorption in humans. Two Bifidobacterium species (B. breve and B. longum) and one strain each of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus were tested by a single exposure to two chemical mutagens, ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). To screen for beta-galactosidase (beta-gal) overproducing mutants, optimized EMS and MNNG mutant pots for each strain were plated on BHI agar containing 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal). Colonies that exhibited a blue color were selected for quantitative beta-gal activities using the o-nitrophenyl-beta-galactoside (ONPG) assay. Seventy-five mutants were obtained out of more than 2 million colonies screened and showed increased beta-galactosidase activities compared with the wild-type strains. EMS gave a higher frequency of beta-gal overproducing mutants than MNNG for three of the four strains, S. thermophilus, B. breve, and B. longum, whereas the frequency of L. delbrueckii ssp. bulgaricus beta-gal mutants was similar with both mutagens. The highest beta-gal increases, when induced during growth in lactose, for mutants of each culture were 137% for L. delbrueckii ssp. bulgaricus; 104% for S. thermophilus; 70% for B. breve; and 222% for B. longum mutants. This food-grade classical approach has the ability to moderately increase beta-gal concentrations in probiotic cultures to improve their potential for treating the symptoms of lactose malabsorption in humans.

Bifidobacterium↗
PubMed1997

Influence of bile on beta-galactosidase activity and cell viability of Lactobacillus reuteri when subjected to freeze-drying.

The effect of bile on beta-galactosidase activity and cell viability was investigated using two strains of Lactobacillus reuteri that were subjected to freeze-drying. In the presence of 0.15% oxgall, beta-galactosidase activity of the whole cells was significantly increased. After lyophilization, the cultures that had been treated with oxgall showed a low survival rate without changes in beta-galactosidase activity. The poor resistance of the cells to damage from freeze-drying might be related to the presence of membranous structures containing simple folds and buds of the cell membrane, as was observed by transmission electron microscopy.

Bile↗
PubMed1995

Removal of the alpha-galactosides of sucrose from soybean meal using either ethanol extraction or exogenous alpha-galactosidase and broiler performance.

Two studies using broiler chicks and one using adult White Leghorn roosters were conducted to determine the influence of stachyose and raffinose (alpha-galactosides of sucrose) present in soybean meal (SBM) on the nutritional value of the meal. In Experiment 1, the addition of four levels (0, .05, .10, or .20 g/kg) of alpha-galactosidase with and without 1 g/kg of invertase to a corn-SBM diet had no effect on weight gain, feed efficiency, protein digestibility, or the digestible energy value of the feed when fed to broiler chicks. However, both enzymes decreased (P < .001) dietary AMEn. In Experiment 2, ethanol extraction and incubation of SBM with alpha-galactosidase decreased the concentrations of the alpha-galactosides of sucrose in SBM from 6.59 to .81 and 1.43%, respectively. However, when broiler chicks were fed semi-purified diets containing SBM, ethanol-extracted SBM, water-incubated SBM, or water plus alpha-galactosidase-incubated SBM, no improvements in weight gain, feed efficiency, or apparent protein digestibility were observed. There was also no improvement in TMEn when the above meals were precision fed to adult White Leghorn roosters (Experiment 3). These results indicate that the removal of up to approximately 90% of the alpha-galactosides of sucrose has no beneficial effect on the nutritional value of SBM for chickens.

Animal Feed↗
PubMed1976

Characterization of T and B antigen-binding cells for beta-galactosidase. III. Independence of antigen-binding cells in normal animals from antigenic stimulation.

Antigen-binding cells to beta-galactosidase were enumerated in thymus and spleen of mice of different ages and found to remain at a constant frequency throughout life. Thymic beta-galactosidase-binding cells (ZBCs),6 representing T-binding cells, showed no appreciable fluctuation, whereas splenic ZBCs, a mixture of T- and B-binding cells, were slightly depressed at birth but found at normal frequencies by 1 week of age. In addition, germfree mice, both within the 1st week after birth and after maturity, had nearly as many binding cells as did conventionally reared age-matched mice. These results considered together suggest that the ability of cells of both T and B origin to recognize antigen, as revealed by their ability to bind beta-galactosidase, arise independently of specific or nonspecific antigenic stimulation, as part of the normal ontogenic sequence of steps of differentiation. This is consistent with the theory of clonal precommitment of T and B cells.

Aging↗
PubMed1975

Amino acid sequence of beta-galactosidase. IV. Sequence of an alpha-complementing cyanogen bromide peptide, residues 3 to 92.

Intracistronic alpha-complementation between a cyanogen bromide digest of beta-galactosidase and an extract of the lac Zminus operator-proximal deletion mutant M15 was used to monitor the purification of a cyanogen bromide peptide (CB2) responsible for the complementation. Key steps in the purification were ion exchange chromatography on carboxymethylcellulose and sulfopropyl-Sephadex in the presence of urea, and Sephadex gel filtration. CB2 contains residues 3 to 92 of beta-galactosidase. Its sequence is: Ile-Thr-Asp-Ser-Leu-Ala-Val-Val-Leu-Gln-Arg-Arg-Asp-Trp-Glu-Asn-Pro-Gly-Val-Thr-Gln-Leu-Asn-Arg-Leu-Ala-Ala-His-Pro-Pro-Phe-Ala-Ser-Trp-Arg-Asn-Ser-Glu-Glu-Ala-Arg-Thr-Asp-Arg-Pro-Ser-Gln-Gln-Leu-Arg-Ser-Leu-Asn-Gly-Glu-Trp-Arg-Phe-Ala-Trp-Phe-Pro-Ala-Pro-Glu-Ala-Val-Pro-Glu-Ser-Trp-Leu-Glu-Cys-Asp-Leu-Pro-Glu-Ala-Asp-Thr-Val-Val-Val-Pro-Ser-Asn-Trp-Gln-Met. Thus no more than 1/13 of the beta-galactosidase polypeptide chain, starting 2 residues from the NH2 terminus, is necessary for alpha-complementation with M15 as alpha-acceptor.

Amino Acid Sequence↗
PubMed2000

Retinoic acid receptor status in mouse spleen during a primary immune response against beta-galactosidase.

OBJECTIVE: Evaluation of the dynamics of all-trans retinoic acid receptor binding properties in mouse spleen nuclear extracts during a primary immune response against beta-galactosidase. METHODS: Female BALB/c mice, aged between 5 and 6 weeks were immunized intradermally into the shaved back (4 spots each) with 100 microg beta-galactosidase in 100 microl sterile phosphate buffered saline (pH 7.2) and blood was taken by tail bleeding on days 0 (preimmune serum), 4 and 6. Production of antibody in serum and the detection of cytokines (IL-4, IFN-gamma) from proliferation supernatants were determined by ELISA. Antigen-specific proliferation assay of isolated spleen cells was based on [3H]-thymidine incorporation measured in a liquid scintillation counter. Both, the maximal binding capacity (Bmax) and the affinity (Ka) of all-trans retinoic acid nuclear receptors (RAR) were evaluated according to Brtko (1994). RESULTS AND CONCLUSIONS: Injection of beta-galactosidase induced the first detectable antibody responses on day 4 (IgM) and on day 6 (IgG). These points of time, reflecting the early and the mature immune response served to measure the antigen-specific proliferation and production of IL-4 and IFN-gamma in the supernatants of the proliferation cultures as well as all-trans retinoic acid receptor (RAR) binding characteristics in spleen nuclear proteins. The RAR Bmax was significantly (P<0.05) decreased only at the time of the first specific IgG antibody production. CONCLUSIONS: The data obtained indicate the involvement of RAR in the late phase of an in vivo immune response.

Animals↗