sodium-dodecyl-sulfate and Periodontal-Diseases

Topics: Anti-Bacterial Agents; Benzoates; Clinical Trials as Topic; Dental Research; Meta-Analysis as Topic; Mouthwashes; Outcome Assessment, Health Care; Periodontal Diseases; Periodontitis; Research Design; Sodium Dodecyl Sulfate; Tetracycline

Topics: Dentifrices; Humans; Periodontal Diseases; Periodontal Index; Sodium Dodecyl Sulfate; Toothpastes

Porphyromonas gingivalis has been implicated as a major aetiological agent in certain forms of periodontal disease, P. gingivalis is a Gram-negative, asaccharolytic bacterium that obtains energy from the fermentation of amino acids derived from the hydrolysis of host protein. Virulence factors of this bacterium include the capsule, fimbrial adhesins, cytotoxins and extracellular hydrolytic enzymes. A 43 kDa fimbrillin from P. gingivalis has been isolated and characterized. However, there is evidence that a second type of fimbria exists on the surface of P. gingivalis. A putative P. gingivalis fimbrial protein from a membrane preparation has been isolated and identified. This protein was shown to be reactive with sera from patients harbouring P. gingivalis. A 28 kDa protein fragment was purified by anion exchange, gel filtration and reversed-phase chromatography. N-terminal sequence analysis of the 28 kDa protein fragment revealed homology to the fimbrial precursor protein of Dichelobacter nodosus. A peptide corresponding to the N-terminal 26 amino acyl residues of the 28 kDa protein fragment was synthesized and used to raise antibodies to the protein. Western blot analysis after SDS-PAGE of a P. gingivalis membrane preparation using the antibodies raised to the synthetic peptide detected three proteins of 36, 41 and 67 kDa. When protease inhibitors were not included in the extraction procedure only the 36 and 41 kDa bands were detected. It would appear, therefore, that the intact protein has an M(r) of 67 kDa and that the 28, 36 and 41 kDa bands represent protein fragments produced by endogenous proteolytic activity. Based on sequence homology, the 67 kDa protein is possibly a sub-unit of a second P. gingivalis fimbrial type or a surface receptor.

Topics: Adhesins, Bacterial; Animals; Antibodies, Bacterial; Antigens, Bacterial; Bacterial Capsules; Bacterial Outer Membrane Proteins; Bacterial Proteins; Bacteroides; Blotting, Western; Chromatography, Gel; Chromatography, Ion Exchange; Cytotoxins; Electrophoresis, Polyacrylamide Gel; Fimbriae Proteins; Fimbriae, Bacterial; Humans; Hydrolases; Molecular Weight; Peptide Fragments; Periodontal Diseases; Porphyromonas gingivalis; Protease Inhibitors; Protein Precursors; Sequence Homology, Amino Acid; Sodium Dodecyl Sulfate; Virulence

The presence of lipoproteins and lipooligosaccharides in Treponema denticola, an oral spirochaete associated with periodontal diseases, was investigated. T. denticola ATCC 35404 and the clinical isolate GM-1 were metabolically labeled with [3H]-cis-9-octadecenoic acid and extracted with the non-ionic detergent Triton X-114. The extract was phase separated, precipitated with acetone and delipidated to remove non-covalently bound lipid (dLPP). In T. denticola ATCC 35404, sodium dodecyl sulfate polyacrylamide electrophoretic separation followed by autoradiography showed [3H]-cis-9-octadecenoic acid incorporation in bands with apparent molecular masses of 14, 20, 26, 31, 38, 72 and 85 kDa and a broad band running from 113 kDa to the top of the gel. This last band resolved into a 53 kDa [3H]-cis-9-octadecenoic acid band upon heating for 10 min, at 100 degrees C. The structural relationship of the outer sheath major oligomeric polypeptide of strain ATCC 35404 and the 53 kDa protein was demonstrated immunologically. Antibodies against the 113 kDa component of the oligomer cross-reacted with the 53 kDa protein. Proteinase K degraded the [3H]-cis-9-octadecenoic acid bands with the exception of the 14 kDa. The 14 kDa was also the major [3H]-fatty acid labeled compound found in the water phase following phenol-water extraction of whole T. denticola ATCC 35404 cells. This compound was purified from the water phase by gel filtration followed by hydrophobic chromatography. Chemical analysis showed that hexadecanoic acid was the predominant fatty acid bound to T. denticola lipoproteins. In the GM-1 strain [3H]-cis-9-octadecenoic acid incorporation was observed in the 116 kDa and 14 kDa bands. dLPP from strain ATCC 35404 caused an enhanced (0.8-8 micrograms/ml) luminol dependent chemiluminiscence (LDCL) effect in human polymorphonuclear neutrophils (PMN) which could be related to protein concentration. The addition of dLPP to PMN together with FMLP at submaximal concentration (1 microM) resulted in a synergistic activation of LDCL. At 21 micrograms/ml, dLPP also induced lysozyme release by the PMN at approximately 30% of the release induced by the chemotactic peptide at 1 microM. In addition, dLPP (21 micrograms/ml) increased additively the release of lysozyme caused by 1 microM FMLP. The release of beta-glucuronidase was not affected. The modulation of neutrophil activity was abolished by preincubation of dLPP with proteinase K. The purified 14 kDa had no effect on eith

Topics: Antibodies; Autoradiography; Bacterial Proteins; Chemotactic Factors; Chromatography; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Endopeptidase K; Enzyme Inhibitors; Exocytosis; Fatty Acids; Glucuronidase; Humans; Indicators and Reagents; Lipopolysaccharides; Lipoproteins; Luminescent Measurements; Luminol; Muramidase; N-Formylmethionine Leucyl-Phenylalanine; Neutrophil Activation; Neutrophils; Oleic Acid; Oligosaccharides; Palmitic Acid; Periodontal Diseases; Radiopharmaceuticals; Sodium Dodecyl Sulfate; Surface-Active Agents; Treponema; Tritium

By comparison of the cell surface proteins derived from the outer membrane and fibrils from 14 Prevotella intermedia and 19 Prevotella nigrescens strains using SDS and analysed by SDS-PAGE, it was possible to distinguish the two species. A polypeptide of approx. 21 kDa distinguished P. intermedia strains, whereas two polypeptides of approx. 18 and 22 kDa could be used to identify P. nigrescens strains. Four other human oral black pigmented bacterial species (Porphyromonas gingivalis, Prevotella denticola, Prevotella loescheii and Prevotella melaninogenica) did not have the 18-, 21- or 22-kDa polypeptides shown by P. intermedia or P. nigrescens. The cell-associated proteolytic activity of eight strains of P. intermedia, 14 strains of P. nigrescens and one strain of P. gingivalis (W50) was assessed using four chromogenic substrates. The hydrolysis of the substrate GPPNA (indicative of dipeptidyl peptidase IV-like activity) and SAAPPNA (elastase-like activity) by P. intermedia strains varied from 32 to 114 units and 0.5 to 12.6 units of activity respectively, where one unit was defined as the amount of protease enzyme catalysing the formation of 1 nmol of p-nitroaniline under experimental conditions. 37.5% (3 of 8) of P. intermedia strains hydrolysed SAAPPNA (chymotrypsin-like enzyme activity) with activities of between 7 and 12 units. The hydrolysis of GPPNA and SAAAPNA by P. nigrescens strains was 32-149 and 3-16 units, respectively. 57% (8 of 14) of P. nigrescens strains hydrolysed SAAPPPNA with activities ranging from 3 to 8 units. None of the P. intermedia or P. nigrescens strains examined were found to have trypsin-like enzyme activity (BAPNA hydrolysis). The GPPNA and SAAAPNA hydrolytic activity associated with the proteases from Porphyromonas gingivalis W50 was at least twice that of P. intermedia and P. nigrescens strains. The similar peptidase activities of P. intermedia and P. nigrescens against chromogenic substrates cannot be used to differentiate the species, but SDS-PAGE of cell surface protein extracts allowed unambiguous speciation between P. intermedia and P. nigrescens. This simple technique of cell surface protein analysis can be performed in most laboratories and offers a convenient way by which to differentiate the two species.

Topics: Bacterial Outer Membrane Proteins; Cell Extracts; Coloring Agents; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Gingival Diseases; Humans; Peptides; Periodontal Diseases; Phenotype; Prevotella; Prevotella intermedia; Sodium Dodecyl Sulfate

The aim of the study was to examine whether proteolytic artifacts, which result in a loss and poor resolution of protein bands, occur during sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis of cellular proteins from selected proteolytic (Porphyromonas gingivalis, Prevotella nigrescens and Treponema denticola) and non-proteolytic (Fusobacterium nucleatum) bacteria. Conditions to limit or prevent proteolysis were also investigated. Bacterial cells were incubated in solubilizing buffer (SDS+ beta mercaptoethanol) at room temperature for various periods of time before boiling. A control assay consisted of trichloroacetic acid-treated bacterial cells. Cellular proteins were separated by electrophoresis and stained with Coomassie blue. Proteolysis occurred very rapidly in the case of P. gingivalis (< 30 s), whereas a longer incubation time (> 1 h) was required to observe similar effects in P. nigrescens and T. denticola. No proteolysis was observed for F. nucleatum. In all cases, heat (100 degrees C) and low pH (< 4) treatments of bacterial cells could avoid production of proteolytic artifacts. Incorporation of specific protease inhibitors before solubilization of bacteria could also prevent proteolysis. More particularly, N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), iodoacetamide and diisopropylfluorophosphate (50 mM) were highly efficient for P. gingivalis, P. nigrescens and T. denticola, respectively. When outer membranes of P. gingivalis were prepared in the presence of TLCK, numerous additional protein brands, not seen in the absence of TLCK, were detected. The present study suggests that specific protease inhibitors, effective in preventing proteolysis, should be identified and added during cell fractionation and protein purification procedures.

Topics: Artifacts; Bacterial Outer Membrane Proteins; Bacterial Proteins; Bacteriological Techniques; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Fusobacterium nucleatum; Periodontal Diseases; Porphyromonas gingivalis; Protease Inhibitors; Reproducibility of Results; Sodium Dodecyl Sulfate; Time Factors; Treponema

Our previous studies have shown that pyrophosphate (PPi), the anticalculus component of tartar-control dentifrices, inhibits the growth of organisms associated with coronal and root surface caries. The purposes of this investigation were to: 1) determine if periodontal pathogens are similarly susceptible to the growth-inhibitory properties of PPi; and 2) determine if combinations of pyrophosphate-sodium dodecyl sulfate (PPi-SDS) inhibit growth synergistically. Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, Eikenella corrodens, and Campylobacter rectus (formerly Wolinella recta) were cultured in appropriate enriched media under anaerobic conditions. Inhibition assays were performed in tubes containing media supplemented with PPi and/or SDS. A range of concentrations of PPi and SDS in 2-fold increments was employed, with each concentration assayed in triplicate. Minimal inhibitory concentration (MIC) analyses revealed all of the bacteria were susceptible to PPi and SDS, with MICs of 0.67% (25 mM) and 0.01% w/v respectively. Combination studies with PPi-SDS showed much greater growth inhibition against P. gingivalis and A. actinomycetemcomitans than achieved with the agents individually. Determination of fractional inhibitory concentration indices indicated a synergistic growth-inhibitory effect. Under the constraints of the conditions employed, these studies demonstrate the efficacy of PPi-SDS combinations in inhibiting the growth of periodontal pathogens. It is conceivable that these compounds may have clinical benefit as a subgingival irrigant.

Topics: Aggregatibacter actinomycetemcomitans; Bacteria; Campylobacter; Colony Count, Microbial; Diphosphates; Dose-Response Relationship, Drug; Drug Synergism; Eikenella corrodens; Fusobacterium nucleatum; Humans; Periodontal Diseases; Porphyromonas gingivalis; Sodium Dodecyl Sulfate

Crevicular fluid (CF) samples were collected by paper strips from healthy and diseased sites. The molecular distribution of fibrin and fibronectin in CF and plasma samples was investigated using SDS-polyacrylamide gel electrophoresis and specific immunoglobulins. Intact fibrin was found in all CF samples. In addition several bands with both lower and higher molecular weights than intact fibrin were seen. These bands were not present in the plasma samples. The low molecular weight bands are suggested to represent degradation products of fibrin. The high molecular bands could be fibrin-fibronectin complexes or fragments of large fibrin polymers. Fibronectin degradation products, but not intact fibronectin, were seen in both healthy and diseased samples.

Topics: Blood; Electrophoresis, Polyacrylamide Gel; Fibrin; Fibrin Fibrinogen Degradation Products; Fibrinogen; Fibronectins; Gingival Crevicular Fluid; Humans; Immunoblotting; Periodontal Diseases; Periodontium; Sodium Dodecyl Sulfate

Gingival crevicular fluid (GCF) is being analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) in our laboratory. We wish to characterize the major protein species in GCF and to investigate their association with the progression of disease. Laser densitometry of SDS/PAGE provides estimates of log molecular weight (LMW) and relative abundance for each band in each sample. Our aim was to develop a method for the refinement of these data into clusters which could be treated as distinct protein species, whose relation to disease progression could be assessed. A reproducibility study showed that the estimated LMW would fall within 3% of the true LMW 95% of the time. The method clusters the estimated LMWs of each band in each sample so that clusters form at LMWs which occurred frequently. The data from each band in each sample are thus refined into cluster number and relative abundance. Application of the technique to data from the reproducibility study showed that clusters formed around the individual components in the mixture, with little misclassification. The technique was then applied to two data sets: from SDS/PAGE of 104 GCF samples from 74 adolescents without progressive periodontal disease, and from 2 patients suffering from advanced progressive disease. The clusters appeared accurately to reflect the appearance of the gels, and clear differences were observed between the two data sets. The method will enable changes in the composition of biological fluids to be associated with external factors such as disease status and should be widely applicable.

Topics: Adolescent; Adult; Body Fluids; Densitometry; Electrophoresis, Polyacrylamide Gel; Gingiva; Humans; Male; Models, Biological; Molecular Weight; Periodontal Diseases; Sodium Dodecyl Sulfate; Statistics as Topic

Topics: Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Humans; Immunoelectrophoresis; Parotid Gland; Periodontal Diseases; Periodontal Index; Saliva; Salivary Proteins and Peptides; Sodium Dodecyl Sulfate; Vitamin D-Binding Protein

Collagenase activity was demonstrated by direct incubation to be present in human periodontal ligament. This activity was found in only one of two populations of ligament, i.e., those ligaments taken from teeth in which their attachment site was at least 2.5 mm apical to the cementoenamel junction. The collagenase was demonstrated to be of host origin because it degraded collagen into 3/4 and 1/4 alpha chain fragments characteristic of mammalian collagenases. The enzyme was shown to be inhibited in the presence of EDTA and to have a pH optimum of 7.5.

Topics: Collagen; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Humans; Microbial Collagenase; Periodontal Diseases; Periodontal Ligament; Sodium Dodecyl Sulfate; Trypsin