Biomedical subjects

S Kalfas

Publications and source records attributed to S Kalfas.

At least 37 records · Page 2Linked to original sources

PubMed1999

Cryptobacterium curtum gen. nov., sp. nov., a new genus of gram-positive anaerobic rod isolated from human oral cavities.

Novel Eubacterium-like isolates, strains 12-3T and KV43-B, which were isolated from the periodontal pocket of an adult patient with periodontal disease and necrotic dental pulp, respectively, were studied taxonomically and phylogenetically. The morphological and differential biochemical characteristics of these organisms are also described in this paper. These organisms were Gram-positive, anaerobic, non-spore-forming, rod-shaped bacteria that were inert in most of the conventional biochemical tests and closely resembled members of asaccharolytic oral Eubacterium species. On the other hand, protein profiles of whole cells in SDS-PAGE and Western immunoblotting reaction analysis distinguished these isolates from strains of the previously described genus Eubacterium. The G+C content of the DNAs from the novel isolates was 50 and 51 mol%, respectively. The levels of DNA-DNA relatedness to other asaccharolytic oral Eubacterium species, including Eubacterium brachy, Eubacterium lentum, Eubacterium nodatum, Eubacterium timidum, Eubacterium saphenum, Eubacterium minutum and Eubacterium exiguum, was less than 11%. These organisms also exhibited a very low level of reassociation with the DNA of Eubacterium limosum, the type species of the genus Eubacterium. The results of 16S rDNA sequence comparisons revealed that these organisms represent a novel lineage distinct from all previously described genera of Gram-positive, rod-shaped bacteria. On the basis of our results, it is suggested that strains 12-3T and KV43-B should be classified in a new genus and species, for which the name Cryptobacterium curtum gen. nov., sp. nov. is proposed. The type strain of Cryptobacterium curtum is 12-3T (= ATCC 700683T).

Adult↗

PubMed1998

Characterization of the proteinase-dependent cytotoxicity of Porphyromonas gingivalis.

Cytotoxicity of proteins from Porphyromonas gingivalis towards gingival fibroblasts and epithelial cells was studied by methods measuring vital dye uptake, DNA synthesis, release of cytoplasmic components, and apoptosis. Microscopic examination of the cells was also performed for detection of morphological changes. Experiments were made with dialyzed culture supernatants of P. gingivalis as well as supernatant fractions obtained by isoelectric focusing. The main cell damage observed was rounding of the cells and detachment from each other and the underlying surface. The cell-damaging activity correlated with the cystein-dependent proteolytic activity of the various supernatant preparations as well as with the occurrence of gelatinolytic protein bands with molecular weights previously reported for the cysteine proteinases gingipains R and K of P. gingivalis. The activity was abolished by heat treatment or by the addition of cysteine proteinase inhibitors. The results indicate that the main cytotoxic effect towards fibroblasts and epithelial cells is degradation of the intercellular matrix. The gingipains released in P. gingivalis culture supernatants are the responsible factors for this degradation.

Adhesins, Bacterial↗

PubMed1998

Ribotype diversity of Actinomyces with similar intraoral tropism but different types of N-acetyl-beta-D-galactosamine binding specificity.

Sixty-three isolates of Actinomyces naeslundii genospecies 1 and 2 and Actinomyces odontolyticus from three subjects clustered into 22 ribotypes. Unique ribotypes were found in the subjects and within individual tissue sites (bucca, tooth and tongue). A odontolyticus ribotypes shared tongue-specific binding properties, while those of genospecies 1 and 2 from buccal and tooth surfaces shared different types of N-acetyl-beta-D-galactosamine binding specificity.

Acetylgalactosamine↗

PubMed1998

Restriction fragment-length polymorphism analysis of 16S rDNA from oral asaccharolytic Eubacterium species amplified by polymerase chain reaction.

Restriction fragment-length polymorphism (RFLP) analysis of 16S rDNA amplified by polymerase chain reaction was used to generate restriction profiles of the type strains of oral asaccharolytic Eubacterium species, that is, Eubacterium brachy, Eubacterium exiguum, Eubacterium lentum, Eubacterium minutum, Eubacterium nodatum, Eubacterium saphenum, Eubacterium timidum and 33 asaccharolytic Eubacterium strains isolated from oral sites. The 16S rRNA gene sequences from isolated genomic DNA samples were amplified by polymerase chain reaction (PCR). PCR products were purified and characterized by single digestions with 7 restriction endonucleases. Among the 7 endonucleases, HpaII was found to discriminate the respective reference strains. Twenty-three isolates, out of 33, were assigned to one of the reference species, on the basis of their restriction profiles by digestion with HpaII. The remaining 10 isolates could not be assigned to any of the established species and constituted 4 distinct groups, each of which may be a new species.

DNA, Bacterial↗

PubMed1997

Characterization of the lactoferrin-dependent inhibition of the adhesion of Actinobacillus actinomycetemcomitans, Prevotella intermedia and Prevotella nigrescens to fibroblasts and to a reconstituted basement membrane.

Lactoferrin was previously shown to inhibit the adhesion of A. actinomycetemcomitans, P. intermedia and P. nigrescens to human cells. Lactoferrin was also shown to competitively inhibit the binding of these bacteria to the basement membrane protein laminin. The present study aimed to determine the type of interactions inhibited by lactoferrin. Lactoferrin binds to fibroblast monolayers and Matrigel, a reconstituted basement membrane, through ionic interactions. The adhesion of A. actinomycetemcomitans to these substrata was mainly dependent on the ionic strength of the environment. P. intermedia and P. nigrescens also adhere to fibroblasts mainly by ionic interactions, while their adhesion to Matrigel seems to be mediated by specific mechanisms. Lectin-type interactions were not found to be involved in the binding of these bacteria to the substrata. Treatment of either A. actinomycetemcomitans or fibroblasts with lactoferrin decreased the adhesion in a dose-dependent manner, while lactoferrin treatment of Matrigel alone had no adhesion-counteracting effect. Adhesion of P. intermedia and P. nigrescens to Matrigel was not significantly affected by the ionic strength, but the presence of lactoferrin inhibited the adhesion. Lactoferrin bound to Matrigel, P. intermedia and P. nigrescens was rapidly released, while lactoferrin bound to A. actinomycetemcomitans and fibroblasts was retained. These findings indicate that lactoferrin-dependent inhibition of the adhesion of A. actinomycetemcomitans, P. intermedia and P. nigrescens to fibroblasts and Matrigel can involve binding of lactoferrin to both the bacteria and substrata. The decreased adhesion may be due to blocking of both specific adhesin-ligand as well as non-specific charge-dependent interactions.

Aggregatibacter actinomycetemcomitans↗

PubMed1997

Fluoride and mutans streptococci levels in plaque on aged restorations of resin-modified glass ionomer cement, compomer and resin composite.

The use of fluoride-releasing restoratives such as glass ionomer cements (GICs) has increased during the last decade. The antibacterial effect of released fluoride is thought to be a possible caries-preventive effect of these restorations. In this study fluoride concentrations in plaque on 1-year old resin-modified GIC, compomer and resin composite restorations were compared intraindividually and related to the occurrence of caries-associated bacteria. Plaque from class III restorations of the three restorative materials and from a proximal enamel surface in 18 individuals was analysed. Low fluoride levels were detected in all the samples, while the resin-modified GIC samples showed significantly higher amounts. The distribution of oral streptococci, mutans streptococci and lactobacilli did not differ significantly among the surfaces and did not correlate to the fluoride levels in the samples. A good correlation was found between the counts of mutans streptococci in saliva and their proportions in the plaque. The results indicate that the fluoride concentrations released in vivo from 1-year-old restoratives are not high enough to affect the plaque levels of the caries-associated bacteria mutans streptococci and lactobacilli.

Adult↗

PubMed1996

Degradation of lactoferrin by periodontitis-associated bacteria.

The degradation of human lactoferrin by putative periodontopathogenic bacteria was examined. Fragments of lactoferrin were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and measured by densitometry. The degradation of lactoferrin was more extensive by Porphyromonas gingivalis and Capnocytophaga sputigena, slow by Capnocytophaga ochracea, Actinobacillus actinomycetemcomitans and Prevotella intermedia, and very slow or absent by Prevotella nigrescens, Campylobacter rectus, Campylobacter sputorum, Fusobacterium nucleatum ssp. nucleatum, Capnocytophaga gingivalis, Bacteroides forsythus and Peptostreptococcus micros. All strains of P. gingivalis tested degraded lactoferrin. The degradation was sensitive to protease inhibitors, cystatin C and albumin. The degradation by C. sputigena was not affected by the protease inhibitors and the detected lactoferrin fragments exhibited electrophoretic mobilities similar to those ascribed to deglycosylated forms of lactoferrin. Furthermore a weak or absent reactivity of these fragments with sialic acid-specific lectin suggested that they are desialylated. The present data indicate that certain bacteria colonizing the periodontal pocket can degrade lactoferrin. The presence of other human proteins as specific inhibitors and/or as substrate competitors may counteract this degradation process.

Aggregatibacter actinomycetemcomitans↗

PubMed1996

Laminin binding to a heat-modifiable outer membrane protein of Actinobacillus actinomycetemcomitans.

The interaction of Actinoabacillus actinomycetemcomitans with the basement membrane protein, laminin, was examined in a 125I-labeled protein-binding assay. The binding of laminin increased by lowering the pH. The ability to bind laminin was decreased in cells at the stationary phase of growth and by the presence of blood in the culture medium. Laminin binding to this bacterium was saturable, and the affinity constant was 4.6 nM. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot (ligand blot) analysis of cell-envelope and outer membrane of A. actinomycetemcomitans displayed a 125I-laminin-reactive protein band with a molecular weight of 29 k. The laminin-binding protein was the previously described outer membrane protein A of A. actinomycetemcomitans. It was identified by its heat-modifiable property, detergent-solubility profile and reactivity with outer membrane protein A-specific polyclonal antiserum. At acidic pH, 25I laminin bound to several cell-envelope components of A. actinomycetemcomitans, but at neutral pH, laminin bound only to the heat-modifiable protein. Despite the existence of the laminin-binding protein, cells grown in blood-containing media did not bind laminin. Several mammalian proteins interfered with laminin-bacterial interaction, including lactoferrin, which binds to the same bacterial protein that inhibited and displaced the laminin-bacterial interaction.

Aggregatibacter actinomycetemcomitans↗

PubMed1995

Lactoferrin interaction with Actinobacillus actinomycetemcomitans.

The interaction of lactoferrin with Actinobacillus actinomycetemcomitans was examined in a 125I-labeled protein binding assay. The binding of human and bovine lactoferrins reached maximum within 1 h. Lactoferrin binding to the bacterium was pH-dependent and reversible. Scatchard analysis indicated the existence of two different types of binding sites on the bacterium, one with a high affinity constant k alpha approximately 8.8 x 10(-7) M) and the other with a low one (k alpha approximately 1.8 x 10(-6) M). Bacteria in the exponential phase of growth showed higher binding than cells in the stationary phase. Bacteria grown in medium containing serum and/or lysed erythrocytes bound lactoferrin to a lesser extent. Heat-inactivated serum, lysed erythrocytes and other proteins such as mucin and laminin inhibited lactoferrin binding to A. actinomycetemcomitans in a competitive binding assay. Sodium dodecyl sulfate polyacrylamide-gel electrophoresis and Western blot analysis of the cell envelope as well as the outer membrane of A. actinomycetemcomitans revealed lactoferrin-reactive protein bands at 29 kDa and 16.5 kDa. The 29-kDa band displayed a heat-modifiable lactoferrin-reactive form with a molecular weight of 34 kDa. Neither proteinase K-treated cell envelope nor lipopolysaccharide of this bacterium showed reactivity with lactoferrin. These data suggests a specific interaction of lactoferrin with outer membrane proteins of A. actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans↗

PubMed1995

Effect of acetate on sorbitol fermentation by oral lactobacilli.

The rate of acid production and end-products from sorbitol were measured under anaerobic conditions in washed-cell suspensions of oral strains of Lactobacillus casei subsp. casei and Lactobacillus casei subsp. rhamnosus. The enzymatic activities were assayed in cell extracts of these strains. The cells fermented sorbitol to lactate, formate, ethanol and acetate under anaerobic conditions. Exposure of the cells to air (oxygen) led to inactivation of pyruvate formate-lyase and inhibition of anaerobic sorbitol fermentation. In the presence of acetate, air-exposed cells fermented sorbitol with a concomitant consumption of acetate and production of ethanol and lactate. Acetate also enhanced acid production from sorbitol in cells kept under anaerobic conditions and resulted in formation of lactate and ethanol. Cell extracts of all the strains had NADH-coupled acetate-reducing activity, which consisted of sequential reactions of acetate kinase, phosphotransacetylase, acylating aldehyde dehydrogenase and alcohol dehydrogenase. These findings indicate that oral lactobacilli can utilize acetate as an electron acceptor for maintaining their intracellular redox balance during anaerobic sorbitol fermentation in the absence of pyruvate formate-lyase activity.

Acetate Kinase↗

PubMed1995

Phosphorylating enzymes involved in glucose fermentation of Actinomyces naeslundii.

Enzymatic activities involved in glucose fermentation of Actinomyces naeslundii were studied with glucose-grown cells from batch cultures. Glucose could be phosphorylated to glucose 6-phosphate by a glucokinase that utilized polyphosphate and GTP instead of ATP as a phosphoryl donor. Glucose 6-phosphate was further metabolized to the end products lactate, formate, acetate, and succinate through the Embden-Meyerhof-Parnas pathway. The phosphoryl donor for phosphofructokinase was only PPi. Phosphoglycerate kinase, pyruvate kinase, and acetate kinase coupled GDP as well as ADP, but P(i) compounds were not their phosphoryl acceptor. Cell extracts showed GDP-dependent activity of phosphoenolpyruvate carboxykinase, which assimilates bicarbonate and phosphoenolpyruvate into oxaloacetate, a precursor of succinate. Considerable amounts of GTP, polyphosphate, and PPi were found in glucose-fermenting cells, indicating that these compounds may serve as phosphoryl donors or acceptors in Actinomyces cells. PPi could be generated from UTP and glucose 1-phosphate through catalysis of UDP-glucose synthase, which provides UDP-glucose, a precursor of glycogen.

Acetate Kinase↗

PubMed1995

Inhibitory effect of lactoferrin on the adhesion of Actinobacillus actinomycetemcomitans and Prevotella intermedia to fibroblasts and epithelial cells.

Adhesion of the periodontitis-associated bacteria Actinobacillus actinomycetemcomitans and Prevotella intermedia to monolayers of fibroblasts, HEp-2, KB and HeLa cells was quantified with radiolabeled bacteria. Bacterial adhesion was also examined microscopically with Giemsa-stained non-radioactive preparations. The degree of bacterial adherence was dependent on the growth phase of the bacteria. Strains at the exponential phase adhered to a greater extent than those at the stationary phase of growth. Both human and bovine lactoferrins competitively inhibited the adhesion of A. actinomycetemcomitans and P. intermedia to all tested cell monolayers. The inhibitory effect was dose-dependent in the concentration range 0.5-2500 micrograms/ml and not related to the bacterial growth phase. In the presence of lactoferrin, decreased association of bacteria with the cell monolayers was also found by microscopic examination of the preparations. The present findings indicate that lactoferrin may prevent the establishment of bacteria in periodontal tissues through adhesion-counteracting mechanisms in addition to its bacteriostatic and bactericidal properties.

Aggregatibacter actinomycetemcomitans↗

PubMed1994

Effect of lactoferrin on interaction of Prevotella intermedia with plasma and subepithelial matrix proteins.

A lactoferrin-binding protein with an estimated molecular mass of 57 kDa was identified in the cell envelope of Prevotella intermedia by gel electrophoresis and Western-blot analysis. Peroxidase-labeled bovine lactoferrin and human lactoferrin showed similar specific binding to this protein. Whole cells of P. intermedia were also examined for interactions with 5 125I-labeled plasma and subepithelial matrix proteins. A high degree of binding was found with fibronectin, collagen type I and type IV and laminin, whereas a moderate interaction was detected with fibrinogen. The ability of bovine lactoferrin to affect the interactions of the above proteins with P. intermedia was examined. In the presence of unlabeled bovine lactoferrin, a dose-dependent inhibition of binding was observed with all 5 proteins tested. Unlabeled bovine lactoferrin also dissociated the bacterial complexes with these proteins. The complexes with laminin or collagen type I were more effectively dissociated than fibronectin or fibrinogen, whereas the interaction with collagen type IV was affected to a lesser extent. A strain-dependent variation in the effect of bovine lactoferrin was observed. These data establish the presence of a specific lactoferrin-binding protein in the cell envelope of P. intermedia. The ability of lactoferrin to inhibit the binding of some plasma and subepithelial matrix proteins to P. intermedia could be a protective mechanism against the establishment of this pathogen in the periodontal pocket.

Animals↗

PubMed1994

The role of the succinate pathway in sorbitol fermentation by oral Actinomyces viscosus and Actinomyces naeslundii.

The sorbitol fermentation by Actinomyces viscosus and Actinomyces naeslundii was studied with washed sorbitol-grown cells. The fermentation was followed by titration of acids produced at pH 7.0 under anaerobic conditions. Metabolic end-products and intracellular levels of NAD, NADH and glycolytic intermediates during the fermentation were also analyzed. Cell extracts were examined for certain enzyme activities. Bicarbonate was required for acid production from sorbitol and from a mixture of glucose and sorbitol. Malate and fumarate could also support the acid production of A. viscosus. The main end-products were succinate and lactate but not ethanol. Cell extracts showed no activities of alcohol and aldehyde dehydrogenases, but they had activities of malate dehydrogenase and fumarate reductase. In the absence of bicarbonate, malate or fumarate, the intracellular NADH/NAD ratio increased and the levels of 3- and 2-phosphoglycerate and phosphoenolpyruvate decreased. The results indicate that oral sorbitol-fermenting actinomyces lack the ethanol pathway that can contribute to NADH oxidation. To maintain intracellular redox balance during anaerobic sorbitol fermentation, these bacteria can oxidize surplus NADH through a succinate pathway.

Actinomyces↗

PubMed1994

Initial catabolism of sorbitol in Actinomyces naeslundii and Actinomyces viscosus.

The initial steps of sorbitol catabolism were studied in 4 strains of Actinomyces naeslundii and Actinomyces viscosus that had been isolated from human dental plaque. Cell-free extracts were prepared from cells grown in the presence of either sorbitol, xylitol or glucose. The extracts from all strains grown on sorbitol had nicotinamide adenine dinucleotide-linked dehydrogenase activities for sorbitol and xylitol and reduced nicotinamide adenine dinucleotide-linked reductase activities for fructose and xylulose. Two of the strains also exhibited these activities when grown in the presence of xylitol, and all glucose-grown cells lacked them. The results indicate that sorbitol metabolism in oral actinomyces involve oxidation of sorbitol to fructose by an inducible enzyme, nicotinamide adenine dinucleotide-linked sorbitol dehydrogenase. This step is followed by the phosphorylation of fructose with guanosine triphosphate as a main phosphoryl donor. Thus, the initial catabolic pathway of sorbitol in A. naeslundii and A. viscosus is different from those described for other oral bacteria.

Actinomyces↗

PubMed1993

Killing of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus by human polymorphonuclear leukocytes in serum and saliva.

The ability of polymorphonuclear leukocytes from human peripheral blood to kill Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus was examined with fresh isolates and laboratory strains from each species (5 strains within each group) under different conditions. Bacterial cells were mixed with a polymorphonuclear leukocyte suspension in the presence of either active serum or heat-inactivated serum or active serum together with sterile-filtered saliva. Surviving bacteria were determined by counting the number of bacterial colony-forming units in the mixtures after a 60-min incubation at 37 degrees C. Mixtures without polymorphonuclear leukocytes served as controls for the evaluation of the degree of killing of the bacteria. In general, A. actinomycetemcomitans resisted phagocytic killing to a greater extent than H. aphrophilus, and the killing of the former species mainly depended on the presence of heat-labile serum components, probably complement factors. Laboratory strains of A. actinomycetemcomitans were more easily killed than fresh isolates. The presence of saliva in the reaction mixtures decreased the degree of killing. However, strain-dependent variations in the killing were found under either condition. The leukotoxic activity of A. actinomycetemcomitans strains, determined by a [51Cr]-release assay, was not correlated with the resistance of these strains to the phagocytic killing. The results point out a strain-dependent difference in the ability of A. actinomycetemcomitans to evade the inflammatory response associated with polymorphonuclear leukocytes. This difference may constitute a potential virulence factor for this periodontopathogen. Furthermore, the leukotoxicity of the strains is not the main determinant that modifies the interaction of A. actinomycetemcomitans with human neutrophils.

Aggregatibacter actinomycetemcomitans↗

PubMed1993

Polymorphonuclear leukocyte chemiluminescence induced by Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus in serum and saliva.

The ability of different strains of Actinobacillus actinomycetemcomitans (A.a.) and Haemophilus aphrophilus (H.a.) to trigger activation of an oxidative burst in human polymorphonuclear leukocytes (PMNL) was examined by measuring the luminol-amplified light emission--chemiluminescence (CL)--from these cells. Bacterial cells were incubated with PMNL from one healthy subject, in the presence of either active serum, heat-inactivated serum, saliva, or saliva and active serum. In the presence of active serum, all five H.a. strains and two out of five A.a. strains triggered a CL response. The CL induced in the presence of heat-inactivated serum was considerably less than that achieved with fresh serum. In the presence of only saliva, all strains induced considerably weaker CL responses than those induced in the presence of saliva with active serum. In the presence of serum, intracellular reactions appeared to be the main source of CL, while addition of saliva and active serum increased the extracellular CL. The results indicate that strain-dependent differences exist among A.a. strains in their ability to trigger the oxygen-dependent bactericidal mechanisms of human PNML. In contrast, the CL patterns of H.a. strains were equivalent. Various factors in the environment, such as activated complement and salivary compounds, affect the interaction of these species with neutrophils.

Aggregatibacter actinomycetemcomitans↗

PubMed1992

Detection of bacterial interaction with lactoferrin by an enzyme-linked ligand binding assay (ELBA).

An enzyme-linked ligand binding assay (ELBA) was devised to measure the interaction between bacteria and human (H) or bovine (B) lactoferrin (Lf) linked to horseradish peroxidase. Reagents were calibrated for optimum colour development with o-phenylenediamine as chromophore and organisms that were either positive or negative in a radioisotope-labelled ligand binding assay (RLBA) with 125I-Lf. Good correlation of Lf binding (r = 0.89) was found between ELBA and RLBA with 169 randomly selected strains of Escherichia coli. A semi-quantitative scoring system for ELBA, corresponding to a similar system for RLBA, was established and shown to be valid for 517 strains from seven species of bacterial pathogens. ELBA was used to measure bacterial Lf binding-saturation and displacement kinetics and shown to be comparable with RLBA. ELBA may be a suitable method for examining the binding of Lf to bacteria without the need for radioactive isotopes.

Animals↗