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At least 1,657 records · Page 92Linked to original sources
PubMed1997

Galactose oxidase-glucan binding domain fusion proteins as targeting inhibitors of dental plaque bacteria.

In order to inhibit the growth of bacteria present in the human oral cavity, a novel system which targets antimicrobial agents to dental plaque has been developed. This system involves a hybrid protein consisting of a peptide expressing the bactericidal properties of galactose oxidase (GAO) fused to the glucan binding domain (GBD) of the Streptococcus mutans glucosyltransferase-S enzyme. A gene encoding GAO from the fungus Fusarium sp. has been inserted into an Escherichia coli expression vector and fused to sequences encoding the GBD, which binds to the glucans synthesized by oral streptococci. Bacterial extracts expressing the hybrid protein were tested for their ability to target the GAO activity to an in vitro plaque model consisting of streptococcal cells bound to microtiter plate wells. The binding of the hybrid protein to the streptococcal cells through its GBD and the dependence of binding on the production of glucans by bacteria were demonstrated. Furthermore, killing of three different species of oral streptococci by bound hybrid protein in conjunction with the galactose-lactoperoxidase-iodide cytotoxic system has been demonstrated. These results suggest a novel strategy for controlling dental plaque formation as well as dental caries in humans.

Cariostatic Agents↗
PubMed2003

Unusual organization for lactose and galactose gene clusters in Lactobacillus helveticus.

The nucleotide sequences of the Lactobacillus helveticus lactose utilization genes were determined, and these genes were located and oriented relative to one another. The lacLM genes (encoding the beta-galactosidase protein) were in a divergent orientation compared to lacR (regulatory gene) and lacS (lactose transporter). Downstream from lacM was an open reading frame (galE) encoding a UDP-galactose 4 epimerase, and the open reading frame had the same orientation as lacM. The lacR gene was separated from the downstream lacS gene by 2.0 kb of DNA containing several open reading frames that were derived from fragmentation of another permease gene (lacS'). Northern blot analysis revealed that lacL, lacM, and galE made up an operon that was transcribed in the presence of lactose from an upstream lacL promoter. The inducible genes lacL and lacM were regulated at the transcriptional level by the LacR repressor. In the presence of glucose and galactose galE was transcribed from its promoter, suggesting that the corresponding enzyme can be expressed constitutively. Lactose transport was inducible by addition of lactose to the growth medium.

Amino Acid Sequence↗
PubMed1991

Protection of gerbils from amebic liver abscess by immunization with the galactose-specific adherence lectin of Entamoeba histolytica.

No protective antigens from Entamoeba histolytica have been previously defined. We tested the ability of the galactose-specific adherence lectin of E. histolytica to elicit a protective immune response in conjunction with Freund's incomplete and complete adjuvants. The gerbil (Meriones unguiculatus) model of an experimental amebic liver abscess was used. Gerbils were immunized intraperitoneally or subcutaneously with 10 micrograms of the affinity-purified lectin in complete Freund's adjuvant and then at 2 and 4 weeks with 10 micrograms of the lectin in incomplete Freund's adjuvant. All of the immunized animals developed antilectin antibody titers of greater than 1/1,024 as measured by a radioimmunoassay. The gerbil antilectin antibodies were shown by Western immunoblotting to be directed to the heavy subunit but not the light subunit of the lectin. Immune gerbil sera inhibited amebic adherence by 100% at a 1/10 dilution. Immune and control gerbils were challenged at 6 weeks by the intrahepatic injection of 5 x 10(5) E. histolytica trophozoites. Four independent trials demonstrated complete protection from amebic liver abscess formation in 67% of lectin-immunized gerbils. Unexpectedly, liver abscess weights were significantly higher in the gerbils that failed to become immune than in the control animals. Our results demonstrate that the galactose lectin is a protective antigen and provide an immune-animal model to study the mechanisms of protection and potential disease exacerbation conferred by the antilectin immune response.

Animals↗
PubMed1991

Role of the galactose lectin of Entamoeba histolytica in adherence-dependent killing of mammalian cells.

Entamoeba histolytica extracellular killing of host cells is contact dependent. Adherence to human colonic epithelial cells and mucins is mediated by a galactose-specific lectin. The effect on cytotoxicity of a panel of monoclonal antibodies (MAb) directed against the galactose lectin was tested. As expected, those MAb which inhibited adherence also decreased cytotoxicity. However, one antilectin MAb blocked cytotoxicity after adherence had occurred, indicating that the lectin has a role in cell killing that is distinct from its adherence function.

Adhesiveness↗
PubMed1992

Human T-lymphocyte proliferation, lymphokine production, and amebicidal activity elicited by the galactose-inhibitable adherence protein of Entamoeba histolytica.

We studied human T-lymphocyte responses to the purified Entamoeba histolytica galactose-inhibitable adherence protein. Individuals having serum anti-adherence protein antibodies possess peripheral blood lymphocytes which demonstrate antigen-specific responses to the purified adherence protein (10 micrograms/ml) and whole soluble amebic antigen (100 micrograms/ml). This was determined by incorporation of [3H]thymidine (53,080 and 73,114 dpm, respectively) and by increased production of interleukin-2 and gamma interferon (42.0 and 67.5 U/ml, respectively) (P less than 0.05 for each in comparison with values for control lymphocyte responses). Lymphocytes from antiamebic antibody-positive subjects develop in vitro amebicidal activity only when incubated for 5 days with the purified adherence protein (P = 0.02). In conclusion, the E. histolytica galactose-inhibitable adherence protein elicits an in vitro amebicidal cell-mediated immune response, further supporting the potential for the use of this protein in a subunit amebiasis vaccine.

Animals↗
PubMed1996

Oral immunization with a recombinant cysteine-rich section of the Entamoeba histolytica galactose-inhibitable lectin elicits an intestinal secretory immunoglobulin A response that has in vitro adherence inhibition activity.

The LC3-encoded 52-kDa recombinant protein includes amino acids 758 to 1134 of the 170-kDa subunit of the galactose-inhibitable lectin. Oral immunization of BALB/c mice with the LC3-encoded protein and cholera holotoxin induced an intestinal secretory immunoglobulin A (IgA) response (P < 0.01 compared with the control). There was a negative correlation (P = 0.001) between intestinal anti-LC3 IgA and serum IgA and IgG antibody responses. Intestinal secretions from immunized mice completely inhibited the galactose-specific adherence of axenic trophozoites ot Chinese hamster ovary cells (P < 0.01).

Adhesiveness↗
PubMed1973

Galactose-deficient endotoxin from urinary Escherichia coli.

Analysis of 111 rough strains of Escherichia coli from known urinary infections revealed for the first time that galactose-deficient rough mutants are not exclusively a laboratory phenomenon. They exist naturally with or without an epimerase deficiency and represent natural examples of the key role of galactose in the attachment of "O" side chains to the core of bacterial lipopolysaccharides.

Endotoxins↗
PubMed2004

Identification of the Entamoeba histolytica galactose-inhibitable lectin epitopes recognized by human immunoglobulin A antibodies following cure of amebic liver abscess.

Immunity to Entamoeba species intestinal infection is associated with the presence of intestinal IgA antibodies against the parasite's galactose-inhibitable adherence lectin. We determined the epitope specificity of serum and intestinal antilectin IgA antibodies by enzyme-linked immunosorbent assay using overlapping fragments of a recombinant portion of the lectin heavy subunit, designated LC3. These findings were correlated with the effects of epitope-specific murine antilectin immunoglobulin A (IgA) monoclonal antibodies (MAbs) on amebic in vitro galactose-specific adherence. LC3 is a highly antigenic and immunogenic cysteine-rich protein (amino acids [aa] 758 to 1150) that includes the lectin's carbohydrate binding domain. The study subjects, from Durban, South Africa, were recently cured of amebic liver abscess (ALA) with or without concurrent Entamoeba histolytica intestinal infection or were infection free 1 year after cure. We also studied seropositive subjects that were infected with E. histolytica, disease free, and asymptomatic. Serum anti-LC3 IgA antibodies from all study groups exclusively recognized the third (aa 868 to 944) and the seventh (aa 1114 to 1134) LC3 epitopes regardless of clinical status; epitope 6 (aa 1070 to 1114) was also recognized by serum anti-LC3 IgG antibodies. However, IgG antibody recognition of epitope 6 but not 3 or 7 was lost 1 year following cure of ALA. We produced 14 murine anti-LC3 IgA MAbs which collectively recognized five of the seven LC3 epitopes. The majority of the murine MAbs recognized the first epitope (aa 758 to 826), which was not recognized by human IgA antibodies. Interestingly, adherence of E. histolytica trophozoites to CHO cells was inhibited by MAbs against epitopes 1, 3, 4 (aa 944 to 987), and 6 (P < 0.01). The LC3 epitopes recognized by human IgA antibodies (3 and 7) were further characterized by use of overlapping synthetic peptides. We identified four peptides (aa 891 to 903, 918 to 936, 1114 to 1134, and 1128 to 1150) that in linear or cyclized form were recognized by pooled intestinal IgA antibodies and serum IgG antibodies from subjects with ALA and asymptomatic, seropositive infected subjects. This study identifies the lectin epitopes to be studied in an amebiasis subunit vaccine designed to elicit mucosal immunity mimicking that of humans cured of ALA.

Animals↗
PubMed1973

Role of cyclic adenosine 3',5'-monophosphate in the in vivo expression of the galactose operon of Escherichia coli.

Studies of levels of galactokinase in Escherichia coli with mutations affecting synthesis of, or response to, cyclic adenosine 3',5'-monophosphate show that this nucleotide does not play a major role in expression of the galactose operon, causing at most a twofold stimulation. The discrepancy between our in vivo results and the marked stimulation by cyclic adenosine 3',5'-monophosphate in in vitro systems indicates that current cell-free systems lack a factor which allows efficient expression of the galactose operon even in the absence of cyclic adenosine 3',5'-monophosphate or of the binding protein for this nucleotide.

Carbon Isotopes↗
PubMed1973

Genetics and physiology of a tolE mutant of Escherichia coli K-12 and phenotypic suppression of its phenotype by galactose.

The tolE mutation causes tolerance to colicins E2 and E3 as well as other effects on the phenotype of Escherichia coli K-12. The lipopolysaccharide of the mutant shows a reduction in the content of galactose, glucose, and rhamnose. The phenotype of the mutant, including the composition of the lipopolysaccharide, is suppressed by galactose. The map position is shown by the gene order trp-purB-tolE-tolD-galKETO.

Chloramphenicol↗
PubMed1974

Isolation and complementation of mutants in galactose taxis and transport.

By using a new screening method, we have obtained 43 new Escherichia coli mutants defective in ring formation on galactose swarm plates, which score for defects in chemotaxis or transport. They were complemented and compared with mutations previously known to lie in the galactose-binding protein or the beta-methylgalactoside (mgl) permease, or both. The mutations were all found to lie in three genes, called mglA, mglB, and mglC. mglB codes for the gene specifying the binding protein. Based on co-transduction experiments, mglA, mglB, and mglC lie close to each other on the bacterial chromosome.

Alkanesulfonates↗
PubMed1975

Exocellular glycopeptide from a Penicillium charlesii mutant incapable of growth on D-galactose.

The compositions of exocellular saccharide-containing polymers from six mutants of Penicillium charlesii incapable of growing on galactose were investigated. The polymers from the mutants contain a much smaller percentage of galactose than that reported for the peptidophosphogalactomannan (PPGM) from the wild-type organism (Gander et al. 1974). A polymer containing only one galactosyl residue per 49 mannosyl residues was investigated in detail. This polymer is a glycopeptide (peptidomannan) with an amino acid composition similar to that of peptidophospogalactomannan and a mass of about 23,000 daltons. Treatment of peptidomannan with 0.4 N NaOH releases mannan, mannopentaose, mannotetraose, mannotriose, mannobiose, and mannose residues, which are attached to the peptide by O-glycosidic linkage to seryl and threonyl groups. The quantity of glycerol and threitol, derived from mannosyl and internal galactofuranosyl residues, respectively, following Smith degradation, showed that peptidomannan contains 2 mol of internal galactofuranosyl residues per mol of polymer. The polymer contains only 3 mol of (1 yields 5)-linked galactofuranosyl residues per mol of polymer, as described by analysis of the methylation products. Methylation analysis also indicates that the polysaccharide contains primarily (1 yields 2)-linked (67.5%) and (1 yields 6)-linked (20.2%) mannopyranosyl residues. However, acetolysis of the polymer suggests that 37% of the residues are (1 yields 6)-linked. Mannopentaose, mannotetraose, mannotriose, mannobiose, and mannose in a molar ratio of 0.30:0.11:0.15:0.39:0.06, respectively, are released by acetolysis. This result is similar to that obtained with peptidophosphogalactomannan. We conclude that the occurrence of large numbers of galactofuranosyl residues in the major extracellular glycopeptide is not an obligatory requirement for glycopeptide formation.

Amino Acids↗
PubMed1979

Lactose metabolism in Streptococcus lactis: phosphorylation of galactose and glucose moieties in vivo.

Starved cells of Streptococcus lactis ML3 grown previously on lactose, galactose, or maltose were devoid of adenosine 5'-triphosphate contained only three glycolytic intermediates: 3-phosphoglycerate, 2-phosphoglycerate, and phosphoenolpyruvate (PEP). The three metabolites (total concentration, ca 40 mM) served as the intracellular PEP potential for sugar transport via PEP-dependent phosphotransferase systems. When accumulation of [14C]lactose by iodoacetate-inhibited starved cells was abolished within 1 s of commencement of transport, a phosphorylated disaccharide was identified by autoradiography. The compound was isolated by ion-exchange (borate) chromatography, and enzymatic analysis showed that the derivative was 6-phosphoryl-O-beta-D-galactopyranosyl (1 leads to 4')-alpha-D-glucopyranose (lactose 6-phosphate). After maximum lactose uptake (ca. 15 mM in 15 s) the cells were collected by membrane filtration and extracted with trichloroacetic acid. Neither free nor phosphorylated lactose was detected in cell extracts, but enzymatic analysis revealed high levels of galactose 6-phosphate and glucose 6-phosphate. The starved organisms rapidly accumulated glucose, 2-deoxy-D-glucose, methyl-beta-D-thiogalactopyranoside, and o-nitrophenyl-beta-D-galactopyranoside in phosphorylated form to intracellular concentrations of 32, 32, 42, and 38.5 mM, respectively. In contrast, maximum accumulation of lactose (ca. 15 mM) was only 40 to 50% that of the monosaccharides. From the stoichiometry of PEP-dependent lactose transport and the results of enzymatic analysis, it was concluded that (i) ca. 60% of the PEP potential was utilized via the lactose phosphotransferase system for phosphorylation of the galactosyl moiety of the disaccharide, and (ii) the residual potential (ca. 40%) was consumed during phosphorylation of the glucose moiety.

Biological Transport, Active↗
PubMed1989

Two transcribing activities are involved in expression of the Streptomyces galactose operon.

The Streptomyces galactose operon is transcribed from two independently regulated promoters: galP1, located at the 5' end of the operon and responsible for galactose-dependent transcription of the operon, and galP2, an internal constitutive promoter. We identified and partially separated two distinct transcribing activities involved in expression of this operon. Using RNA polymerase from Streptomyces lividans and Streptomyces coelicolor partially purified by chromatography on heparin-agarose and DNA-cellulose, we detected activities capable of initiating transcription in vitro specifically from either galP1 or galP2. Circumstantial evidence suggests that the activity for galP2 transcription is a holoenzyme species associated with the previously described sigma 28 protein (referred to here as sigma C). The galP1-transcribing activity is more difficult to evaluate. This activity may correspond to a holoenzyme species associated with sigma A (formerly sigma 35), although other possibilities are discussed. This would be the second reported example of a catabolite-controlled gene in Streptomyces species expressed from multiple promoters recognized by different holoenzyme forms. This may indicate that the involvement of RNA polymerase heterogeneity in gene expression in Streptomyces species is a more general strategy for regulation than the specialized gene expression seen in Escherichia coli.

Base Sequence↗
PubMed1995

The posttranslational modification of phosphoglucomutase is regulated by galactose induction and glucose repression in Saccharomyces cerevisiae.

The enzyme phosphoglucomutase functions at a key point in carbohydrate metabolism. In this paper, we show that the synthesis of the major isoform of yeast phosphoglucomutase, encoded by the GAL5 (PGM2) gene, is regulated in a manner that is distinct from that previously described for other enzymes involved in galactose metabolism in the yeast Saccharomyces cerevisiae. Accumulation of this isoform increased four- to sixfold when the culture experienced either glucose depletion or heat shock. However, heat shock induction did not occur unless the cells were under glucose repression. This nonadditive increase in expression suggests that the regulatory mechanisms controlling the heat shock induction and glucose repression of the GAL5 gene are functionally related. We previously demonstrated that phosphoglucomutase is modified by a posttranslational Glc-phosphorylation reaction. We now show that this posttranslational modification, like phosphoglucomutase expression itself, is also regulated by galactose induction and glucose repression. Finally, no evidence was found to indicate that the Glc-phosphorylation of phosphoglucomutase alters its enzymatic activity under the conditions examined.

Base Sequence↗
PubMed1997

Catabolite inactivation of the galactose transporter in the yeast Saccharomyces cerevisiae: ubiquitination, endocytosis, and degradation in the vacuole.

When Saccharomyces cerevisiae cells growing on galactose are transferred onto glucose medium containing cycloheximide, an inhibitor of protein synthesis, a rapid reduction of Gal2p-mediated galactose uptake is observed. We show that glucose-induced inactivation of Gal2p is due to its degradation. Stabilization of Gal2p in pra1 mutant cells devoid of vacuolar proteinase activity is observed. Subcellular fractionation and indirect immunofluorescence showed that the Gal2 transporter accumulates in the vacuole of the mutant cells, directly demonstrating that its degradation requires vacuolar proteolysis. In contrast, Gal2p degradation is proteasome independent since its half-life is unaffected in pre1-1 pre2-2, cim3-1, and cim5-1 mutants defective in several subunits of the protease complex. In addition, vacuolar delivery of Gal2p was shown to be blocked in conditional end3 and end4 mutants at the nonpermissive temperature, indicating that delivery of Gal2p to the vacuole occurs via the endocytic pathway. Taken together, the results presented here demonstrate that glucose-induced proteolysis of Gal2p is dependent on endocytosis and vacuolar proteolysis and is independent of the functional proteasome. Moreover, we show that Gal2p is ubiquitinated under conditions of glucose-induced inactivation.

Azides↗
PubMed1968

Osmotic remedial response in a galactose-negative mutant of Saccharomyces cerevisiae.

We investigated an osmotic remedial mutant of Saccharomyces which was deficient in galactose-1-phosphate uridyl transferase. Both the rate of growth and the transferase activity of the mutant culture were dependent on the osmotic activity of the growth medium. Organic and ionic solutes proved to be equally effective in inducing the osmotic remedial response. The galactose pathway enzymes were separable by ultracentrifugation, indicating that a stable interenzyme complex was not formed. The results were consistent with the hypothesis that the corrective effect occurs at the tertiary or quaternary level of organization in an environmentally sensitive protein. The possibility that the osmotic remedial response represents an effect of translation is discussed.

Cell-Free System↗