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PubMed1995

An improved method for extraction of mRNA and protein from the fungus Chaetomium gracile with anhydrous hydrogen fluoride.

We previously reported that DNAs directly applicable to restriction analyses and transformation of Escherichia coli can be extracted from fungi and yeasts by use of anhydrous hydrogen fluoride (HF) under a mild condition, 5 min at 0 degrees C [Oshiro, S., Katsura, N., Kitada, K., and Gunge, N. (1987) FEBS Lett. 220, 383-386]. In the present investigation, we examined whether this improved method is also applicable to extraction of RNA and protein from the fungus Chaetomium gracile. The RNA and protein were effectively extracted from the fungus after anhydrous hydrogen fluoride (HF) treatment for a short time (1 min) at 0 degrees C. The extracted poly(A)-enriched mRNA and proteins were fully intact: the mRNA purified by messenger-activated paper with poly(U) directed not only the incorporation of [3H]glycine into polypeptides but also the synthesis in a rabbit reticulocyte lysate cell-free system of proteins reactive to antibodies against the soluble fraction extracted from the fungus by HF. Analyses by gel filtration and polyacrylamide electrophoresis under native conditions showed that dextranase extracted from the fungus by HF under the same conditions had the same molecular weight and electrophretic mobility as the enzyme excreted into the medium. This suggests that the mRNAs and proteins extracted by this method are also applicable to protein synthesis directed in a cell-free system and to enzyme purification from a fungus insusceptible to lytic enzymes. This method provides a pure preparation of mRNA within 5 h and starting materials for protein purification within 1 h.

Animals↗
PubMed1982

Synthesis of glucan on the cell surface of Streptococcus mutans: chemical and scanning electron microscopic studies.

The appearance and continuing growth of extracellular material on Streptococcus mutans HS6 cells in sucrose-containing Merthiolated buffer was observed in a scanning electron microscope and was found to be related to the glucan synthesis on the cell and to adherence of the cell to a smooth surface. Cells grown in broth completely deprived of sucrose by invertase (HS6-IV) had a characteristic, slightly rugged surface structure. On incubation of HS6-IV in the sucrose-containing buffer, a few small globular particles appeared on the surface and grew to an irregular shape (globular to fibrilar) after several hours. The increase in the total glucan content of the cells paralleled the growth of the globular material, to which ferritin-conjugated anti-dextran globulin was found to bind. On the cell surface of cells harvested from conventional broth, both small globular and irregular structures, which possibly formed from sucrose in the broth, existed originally and continued to grow during incubation, along with the material newly appearing on the surface. The accumulation of glucan on the cells resulted in their adherence to a glass surface. The inhibition of growth of the extracellular material on the cells by trypsin, dextranase or anti-glucosyltransferase corresponded to the decrease in glucan synthesis and the loss of adhering ability. These results indicated that the material growing on the cell surface was glucan synthesized by glucosyltransferases.

Adhesiveness↗
PubMed1996

Direct detection of Streptococcus mutans in human dental plaque by polymerase chain reaction.

Streptococcus mutans is an etiological agent in human dental caries. A method for the detection of S. mutans directly from human dental plaque by polymerase chain reaction has been developed. Oligonucleotide primers specific for a portion of the dextranase gene (dexA) of S. mutans Ingbritt (serotype c) were designed to amplify a 1272-bp DNA fragment by polymerase chain reaction. The present method specifically detected S. mutans (serotypes c, e and f), but none of the other mutans streptococci: S. cricetus (serotype a), S. rattus (serotype b), S. sobrinus (serotypes d and g), and S. downei (serotype h), other gram-positive bacteria (16 strains of 12 species of cocci and 18 strains of 12 species of bacilli) nor gram-negative bacteria (1 strain of 1 species of cocci and 20 strains of 18 species of bacilli). The method was capable of detecting 1 pg of the chromosomal DNA purified from S. mutans Ingbritt and as few as 12 colony-forming units of S. mutans cells. The S. mutans cells in human dental plaque were also directly detected. Seventy clinical isolates of S. mutans isolated from the dental plaque of 8 patients were all positive by the polymerase chain reaction. These results suggest that the dexA polymerase chain reaction is suitable for the specific detection and identification of S. mutans.

Animals↗
PubMed1976

The cell surface of Trypanosoma musculi bloodstream forms. II. Lectin and immunologic studies.

Living Trypanosoma musculi bloodstream trypomastigotes were agglutinated specifically with concanavalin A (ConA), wheat germ agglutinin (WGA), soybean agglutinin (SBA), and fucose-binding protein (FBP). The agglutination with these lectins of living cells from which the coat was removed by trypsinization was the same as with intact trypanosomes. Glutaraldehyde or formalin fixation did not affect the results with regard to agglutination with WGA, SBA, and FBP, but lower agglutination with ConA was observed upon fixation. By using a dense iron-dextran marker many fewer ConA marker particles were localized at the fine structural level in the intact than in trypsin-treated trypanosomes. On the basis of the results obtained by agglutination and electron microscopy, it is likely that fixation cross-links intact surface-coat components associated with the ConA binding sites. It is evident from the studies in which lectins were employed that ligands containing alpha-D-mannose, N-acetylglucosamine, N-acetylgalactosamine, and alpha-L-fucose are randomly distributed in the outer surface of the pellicular and flagellar membranes of T. musculi trypomastigotes. Results obtained with alpha-amylase- and dextranase-treated trypanosomes suggested that lectin-binding sugar ligands in the cell surface were not directly associated with alpha-1,4 or repetitive alpha-1,6 glucan-bonded polysaccharide moieties. Similar conclusions can be drawn on the basis of neuraminidase treatment with regard to N-acetylated neuraminic acids. After thorough washing, intact, but not trypsin-treated trypomastigotes were agglutinated specifically with antisera against whole mouse serum and against mouse IgG. Evidently, adsorbed constituents of mouse serum are regular components of the T. musculi surface coat. After incubation in dilute whole mouse serum or in mouse IgG solutions, also the trypsinized cells were agglutinated by the 2 antisera. No such results were obtained with trypsinized cells incubated in serum-free buffers. It was concluded that mouse serum proteins were readily readsorbed on, and firmly bound to the trypsinized cells' surfaces. Specific agglutinations were obtained with trypsinized cells after incubation in dilute rat, rabbit, bovine, and human sera and in solutions of rat and rabbit IgG in reactions with the corresponding antisera. It seems, therefore, that the host serum proteins are adsorbed nonspecifically to the cell surface of trypsinized T. musculi bloodstream forms. When examined by electron microscopy, the intact trypomastigotes were covered by an ununiform, slightly granular, fibrillar extracellular coat, applied to the entire outer lamina of the pellicular and flagellar membranes. No indication of such a coat was noted in the trypsinized organisms. Flocculent surface coat-like matrix could, however, be discerned in cells which, after trypsinization, were incubated in various sera.

Agglutination↗
PubMed1993

Purification and characterization of the extracellular alpha-amylase from Streptococcus bovis JB1.

The extracellular alpha-amylase (1,4-alpha-D-glucanglucanohydrolase; EC 3.2.1.1) from maltose-grown Streptococcus bovis JB1 was purified to apparent homogeneity by ion-exchange chromatography (Mono Q). The enzyme had an isoelectric point of 4.50 and an apparent molecular mass of 77,000 Da, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was rich in acidic and hydrophobic amino acids. The 15-amino-acid NH2-terminal sequence was 40% homologous with the Bacillus subtilis saccharifying alpha-amylase and 27% homologous with the Clostridium acetobutylicum alpha-amylase. alpha-Amylase activity on soluble starch was optimal at pH 5.0 to 6.0. The enzyme was relatively stable between pH 5.5 and 8.5 and at temperatures below 50 degrees C. When soluble potato starch was used as the substrate, the enzyme had a Km of 0.88 mg.ml-1 and a kcat of 2,510 mumol of reducing sugar.min-1.mg of protein-1. The enzyme exhibited neither pullulanase nor dextranase activity and was 40 to 70% as active on amylopectin as on amylose. The major end products of amylose hydrolysis were maltose, maltotriose, and maltotetraose.

Amino Acid Sequence↗
PubMed1994

Glucosyltransferase Mutants of Leuconostoc mesenteroides NRRL B-1355.

Leuconostoc mesenteroides NRRL B-1355 produces dextrans and alternan from sucrose. Alternan is an unusual dextran-like polymer containing alternating alpha(1-->6)/alpha(1-->3) glucosidic bonds. Cultures were mutagenized with UV and ethyl methanesulfonate, and colony morphology mutants were selected on 10% sucrose plates. Colony morphology variants exhibited changes from parent cultures in the production of one or more glucosyltransferases (GTFs) and glucans. Mutants were characterized by measuring resistance of glucan products to dextranase digestion, by electrophoresis, and by high-pressure liquid chromatography of maltose acceptor products generated from sucrose-maltose mixtures. Some mutants produced almost pure fraction L dextran, and cultures exhibited a single principal GTF band on sodium dodecyl sulfate-acrylamide gels. Other mutants produced glucans enriched for alternan. Colony morphology characteristics (size, smoothness, and opacity) and liquid culture properties (clumpiness, color, and viscosity in 10% sucrose medium) were explained on the basis of GTF production. Three principal GTF bands were detected.

Journal Article↗
PubMed1977

Sucrose metabolism in resting-cell suspensions of caries associated and non-caries-associated dental plaque.

Small specimens of cariogenic plaque (CP) and non-cariogenic plaque (NCP) from the same tooth were individually dispersed in buffer, divided equally, and incubated for 45 min with [(14)C]sucrose uniformly labeled either in the glucosyl moiety or the fructosyl moiety. Sucrose metabolism was analyzed periodically during an anaerobic incubation at 37 degrees C. Radiochemical techniques were devised to analyze formation of lactic acid, soluble extracellular polysaccharide, total cell-bound and insoluble products, intracellular polysaccharide, lactic acid from intracellular polysaccharide catabolism, insoluble extracellular glucan, CO(2), total volatile acids, individual volatile acids, and rates of sucrose consumption. The contribution of the glucosyl and fructosyl moieties of sucrose to each metabolic by-product was determined. All of the metabolic data were adjusted to the size of the plaque specimens as determined by colony-forming units, Coulter counter particle counts, and fluorometric protein analyses. Both types of dental plaque transformed from 70 to 80% of the consumed sucrose into lactic acid and cell-bound and insoluble products, primarily intracellular polysaccharide and extracellular glucan. Volatile acids accounted for most of the remaining by-products. CP metabolized significantly more sucrose than NCP and consequently produced significantly higher levels of each metabolic by-product. High levels of Streptococcus mutans were found in CP (averaging 40% of colony-forming units), whereas it was virtually absent in NCP. Actinomyces and S. sanguis levels were distinctly higher in NCP. NCP harbored more anaerobes and dextranase-forming microorganisms than CP.

Actinomyces↗
PubMed1977

Binding of lectins to Streptococcus mutans cells and type-specific polysaccharides, and effect on adherence.

The lectin concanavalin A (Con A) agglutinated the cells of 13 of 15 strains of the seven serotypes of Streptococcus mutans in an 18-h incubation period. Strains of types a, d, f, and g agglutinated within 2 h. Strains of a, d, and f were also agglutinated in 2 h by the castor bean lectin RCA. S. sanguis, S. salivarius, S. bovis, Actinomyces viscosus, A. naeslundii, and Lactobacillus plantarum were agglutinated within 2 h. The S. mutans type f polysaccharide was precipitated by Con A. The a, b, c, d, and e polysaccharides were not precipitated. Glucan from d and e strains of S. mutans and dextran T2000 were also precipitated by Con A. D-glucose inhibited the agglutination of type f cells by Con A and the agglutination of type d cells by D-galactose. The quantity of [acetyl-3H]Con A bound was not proportional to the degree of agglutination. Cells grown in sucrose medium bound more Con A than those grown in glucose medium. After treatment with dextranase, the sucrose-grown cells bound two- to fourfold more Con A. The binding of Con A to the type-specific polysaccharide or to teichoic acid could not be determined by the use of specific antibody due to the binding of Con A to the antibody globulin on the cell surface. Con A bound to S. mutans cells did not inhibit the activity of cell-bound glucosyltransferase, glucan synthesis, and in vitro adherence. Bound Con A also did not inhibit the ability of heat-treated cells to bind glucosyltransferase, synthesize glucan, and produce in vitro adherence.

Agglutination↗
PubMed1977

Adherence of Veillonella species mediated by extracellular glucosyltransferase from Streptococcus salivarius.

The effect of extracellular products from Streptococcus salivarius on sucrose-dependent adherence to smooth surfaces by other oral bacteria was studied in vitro. Strains of Streptococcus mitis, Streptococcus pyogenes, and Veillonella parvula without innate ability to adhere to a steel wire were able to do so when incubated with sucrose and cell-free culture fluid from S. salivarius strains 9759, 25975, CNII, and MEPI. These culture fluids synthesized more adherent material and water-insoluble glucan than those from Streptococcus mutans C67-1 and seven other S. salivarius strains. Among the S. salivarius strains, glucosyltransferase (GT; dextransucrase, EC 2.4.1.5) activity varied more than 100-fold. Cells of Veillonella and S. mitis S3 that had been incubated in culture fluids from S. salivarius 25975 and 9759, respectively, and then washed adhered upon subsequent incubation with sucrose. This was due to adsorbed GT because (i) the adherence was sensitive to dextranase; (ii) it was observed only with the high-GT culture fluids; (iii) it was dependent on sucrose; and (iv) the washed Veillonella cells synthesized glucan, but not fructan, from sucrose. These results suggest that sucrose-dependent adherence of bacteria without such innate ability can be mediated by (i) entrapment in insoluble glucan synthesized by S. salivarius culture fluids, and (ii) prior adsorption of GT from S. salivarius culture fluids. The possibility that GT formed by high-yield strains of S. salivarius is distributed through the mouth by the action of salivary flow and contributes to sucrose-dependent adherence and plaque formation is considered.

Adsorption↗
PubMed1978

Interaction of glucosyltransferase with the cell surface of Streptococcus mutans.

The partially purified glucosyltransferase (GTF) fraction synthesizing primarily water-insoluble glucans, GTF-A, and the homogeneous fraction synthesizing water-soluble glucans, GTF-B, were utilized to assess the binding of GTF activity to the cell surface of Streptococcus mutans GS-5. Growth of the cells in either Todd-Hewitt broth or a chemically defined medium did not appear to affect the ability of the cells to bind either enzyme fraction. Heat inactivation of the cells did not singificantly reduce the interaction of the enzymes with the cells. Cell surface glucan molecules appear to be involved in GTF binding to the cells because: (i) dextranase or alpha-1,3-glucanase treatment of the cells markedly reduced enzyme binding; (ii) the inclusion of soluble dextrans in the binding assays reduced both GTF-A and GTF-B binding to the cells; and (iii) pretreatment of the cells or the GTF-B fraction with soluble dextrans before binding significantly reduced enzyme binding to the cells. In addition, enzyme binding appears to require a cell surface protein component because Pronase, but not trypsin, treatment of cells reduced enzyme binding. Furthermore, the removal of a portion of the cell surface GTF-glucan complex with 3 N NaCl appears to provide additional binding sites for the enzymes. These results are interpreted in terms of the mechanism of the conversion of extracellular GTF to the cell-associated form.

Binding Sites↗
PubMed1978

Mechanism of coaggregation between Actinomyces viscosus T14V and Streptococcus sanguis 34.

Actinomyces viscosus T14V and Streptococcus sanguis 34 coaggregate by a mechanism which is not inhibited by 1 M NaCl, is dextran independent, requires calcium, is pH dependent with an optimum at pH 8.0 to 8.5, and appears to require the interaction of a protein or glycoprotein on A. viscosus with a carbohydrate on S. sanguis. The coaggregation is inhibited more than 80% by 0.01 M lactose, 0.02 M beta-methyl-D-galactoside, or 0.05 M D-galactose; inhibition of coaggregation was less than 10% in 0.1 M alpha-methyl-D-galactoside, melibiose, maltose, cellobiose, sucrose, and a number of monosaccharides. At very high concentrations of enzyme, protease from S. griseus destroyed the reactive site on A. viscosus but not on S. sanguis. Both were totally resistant to dextranase. Periodate (0.01 M; pH 4) inactivated both bacteria. The ability of S. sanguis to coaggregate with A. viscosus was not destroyed by phenol-water extraction at 65 degrees C for 15 min. When the bacteria were cultured under specified conditions, the coaggregation was highly reproducible. Under the same conditions, T14AV, the avirulent mutant of A. viscosus T14V, did not coaggregate with S. sanguis 34. Electron microscopic studies of coaggregates, labeled immunochemically with antibody to A. viscosus, indicated that fibrils on A. viscosus may be involved in the coaggregation.

Actinomyces↗
PubMed1981

Isolation and characterization of Streptococcus mutans mutants defective in adherence and aggregation.

A method was developed which enriched for mutants of Streptococcus mutans that exhibit defects in adherence to glass, aggregation, or both. Mutants were isolated from derivatives of strains PS14 (serotype c) and 6715 (serotype g) after mutagenesis with either ethyl methane sulfonate or nitrous acid. Cell survival after mutagenesis was kept above 1 to 2% to enhance the probability that mutants resulted from single mutational events. A total of 117 mutants were isolated; they also displayed non-wild-type colony morphology on mitis salivarius agar. These mutants were examined for (i) adherence and aggregation after overnight growth in sucrose-containing medium, (ii) aggregation of nongrowing cells in the presence of 200 microgram of sucrose per ml or 20 microgram of dextran per ml, and (iii) dextranase production on blue dextran agar plates. Although we isolated mutants which exhibited a variation from the parent strain in only one of the traits tested, the majority of mutants exhibited defects in two or more characteristics. Thirty-eight stable mutants of independent origin were categorized into 13 separate phenotypic groups.

Cell Adhesion↗
PubMed1985

Cloning and expression of two Streptococcus mutans glucosyltransferases in Escherichia coli K-12.

Chromosomal DNA from Streptococcus mutans strain MFe28 (serotype h) was cloned in the bacteriophage vector lambda L47.1. Two classes of recombinants were found which expressed glucosyltransferase activity in phage plaques: (i) gtfS, which expressed a glucosyltransferase synthesizing a water-soluble, dextranase-sensitive glucan, and (ii) gtfI, which expressed a primer-dependent glucosyltransferase synthesizing an insoluble glucan.

Bacteriophage lambda↗
PubMed1993

Streptococcus mutans fructosyltransferase (ftf) and glucosyltransferase (gtfBC) operon fusion strains in continuous culture.

Three glucosyltransferases (GTFs), which catalyze the formation of water-insoluble adherent glucans, and fructosyltransferase (FTF), which synthesizes fructans, are believed to contribute to the pathogenic potential of Streptococcus mutans. Study of the regulation of expression of GTF and FTF has been difficult because of the complexity and number of exoenzymes produced by this bacterium. By using continuous chemostat culture to control environmental conditions, chloramphenicol acetyltransferase (CAT) operon fusions were utilized to measure transcriptional activity of the ftf and gtfBC gene promoters. Expression of these operon fusions was differentially regulated in response to culture pH and growth rate and during transition states between growth domains. Furthermore, the addition of sucrose to steady-state cultures resulted in significant increases in CAT specific activities for both fusions. In a few cases, GTF and FTF enzyme specific activities did not parallel those of the corresponding CAT fusion activities; this lack of correspondence was likely due to posttranscriptional events controlling enzyme secretion and enzyme activity, as well as to the differential expression of dextranase(s) and fructan hydrolase by S. mutans. These results clearly demonstrate that the extracellular polymer synthesis machinery of S. mutans is regulated in a complex manner. The use of operon fusions in combination with chemostat culture is a viable approach to analyzing gene expression in S. mutans and will be helpful in defining the molecular mechanisms underlying regulation of expression of virulence attributes under conditions that may more closely mimic those in dental plaque.

Extracellular Space↗
PubMed1969

Modified substrates and modified products as inducers of carbohydrases.

Cellobiose and isomaltose are both inducers and repressors of cellulase and dextranase, respectively. The repression can be avoided by supplying the disaccharide slowly. This has been done by use of palmitate and acetate esters which are hydrolyzed by esterases of the growing organism to yield the inducer. Sucrase yields, also, are greatly increased (to 80 times) by substituting sucrose monopalmitate for sucrose in the culture medium.

Aspergillus↗
PubMed1983

Purification and characterization of a bacteriocin from Bacteroides fragilis.

A bacteriocin produced by Bacteroides fragilis 1356 was purified from culture medium and characterized. The spectrum of the inhibitory activity of this bacteriocin was species specific. The bacteriocin was recovered from the initial stages of purification as a complex, greater than 2 X 10(7) daltons in mass, containing protein, lipid, and carbohydrate. The dissociation of this complex by 6.0 M guanidine hydrochloride permitted further purification of the bacteriocin by removal of lipid and carbohydrate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the purified bacteriocin was homogeneous, with a relative molecular weight of 5,000. The activity of the purified bacteriocin was not affected by RNase, DNase, phospholipase A, pancreatic lipase, or dextranase, but was destroyed by trypsin, proteinase K, heat (80 degrees C, 30 min), or a pH below 5 or above 8. Amino acid analysis indicated a predominance of acidic and polar amino acids.

Amino Acids↗
PubMed2003

Coronary flow-induced inotropism is modulated by binding of dextrans to the endothelial luminal surface.

In isolated perfused guinea pig hearts, coronary flow causes a positive inotropic effect [positive coronary flow-induced effect (+CFIE)] that could be altered by dextrans (Dx) in the coronary perfusion solution. To test this possibility, Dx of 20, 40, 70, and 500 kDa were infused and found to modulate +CFIE; however, when Dx infusion was terminated, the effect persisted, i.e., was irreversible/nonwashable, suggesting that Dx may bind to luminal endothelial lectinic structures. This hypothesis was tested when Dx [with fluorescent traces (D*)] bound to the vessel wall was hydrolyzed by dextranase infusion and washout of D* fragments completely reverted the +CFIE, and it was found that bound D* to be displaced by free Dx required concentrations 50-100 times that used during binding. In addition, dose-response curves for Dx on +CFIE show that the higher the Dx molecular mass, the lesser the concentration required to have an effect. Because a large Dx molecule has a greater number polymeric glucose branches, it can bind to a larger number of endothelial lectinic sites, requiring a lower concentration to affect +CFIE. Our results suggest that luminal endothelial lectinic structures are part of the flow-sensing assembly.

Albumins↗
PubMed1999

Pulsatile peptide release from multi-layered hydrogel formulations consisting of poly(ethylene glycol)-grafted and ungrafted dextrans.

Multi-layered hydrogel formulations consisting of poly(ethylene glycol)-grafted dextran (PEG-g-Dex) and ungrafted Dex were investigated as a model of pulsatile drug release. In these formulations, it is considered that the grafted PEG domains act as a drug reservoir dispersed in the Dex matrix based on aqueous polymer two-phase systems. The formulations exhibited surface-controlled degradation by dextranase, and insulin release was observed in a pulsatile manner because of the multi-layered structure: PEG-g-Dex hydrogel layers containing insulin and insulin-free Dex hydrogel layers. Thus, it is suggested that the multi-layered hydrogel formulations using PEG-g-Dex and Dex are feasible for chronopharmacological drug delivery systems.

Animals↗