Invasive differences among Porphyromonas gingivalis strains from healthy and diseased periodontal sites.

BACKGROUND AND OBJECTIVE: The purpose of this study was to determine any difference between Porphyromonas gingivalis isolates from periodontally healthy sites as compared to those from diseased sites with respect to the ability to invade host cells. MATERIAL AND METHODS: Subgingival plaque samples were obtained from periodontally healthy and diseased sites using paper points. P. gingivalis colonies were isolated and tested, using an antibiotic protection assay, for their ability to invade KB cells. P. gingivalis 381 and Escherichia coli MC1061 were used as controls. RESULTS: Mean values of 16.79 +/- 0.86 x 10(3) colony-forming units/mL and 26.14 +/- 2.11 x 10(3) colony-forming units/mL were observed in invasion assays for isolates from periodontally healthy and diseased sites, respectively. P. gingivalis present in diseased sites had significantly greater invasive abilities than strains isolated from healthy sites. No statistical difference was noted between male or female subjects concerning the degree of invasion; isolates from diseased sites from both genders had significantly greater invasion abilities than those from healthy sites. A significant correlation was found between the increased invasive capabilities of P. gingivalis isolates vs. an increased probing depth. CONCLUSION: The increased invasion noted with P. gingivalis isolates from diseased sites vs. healthy sites, and the increased invasive capabilities with increasing probing depth, indicate that P. gingivalis isolates have a varying ability to invade host cells in the periodontal pocket.

Accelerated stability studies of heparin.

The objective of this study was to extend our understanding of the stability of heparin. Sodium heparin, derived from porcine intestinal mucosa, was first incubated in 0.1 N hydrochloric acid and 0.1 N sodium hydroxide at 30 and 60 degrees C and sampled at times ranging from 0 to 1000 h. The absorbance spectra of the products formed under basic conditions showed an ultraviolet maxima at 232 nm associated with chemically catalyzed beta-elimination at the uronic acid residues. The products formed under acidic conditions showed a decreased staining intensity consistent with desulfation and a decrease in molecular weight corresponding to hydrolysis of glycosidic linkages when analyzed by gradient polyacrylamide gel electrophoresis. Heparin samples were next prepared in 10 mM sodium phosphate buffer at pH 7.0 in sealed ampules that had been flushed with nitrogen and incubated at 100 degrees C. Samples taken at times ranging from 0 to 4000 h were then analyzed. Heparin was relatively stable over the first 500 h, after which it rapidly degraded. Heparin, assayed using both anti-factor Xa and anti-factor IIa amidolytic methods retained 80-90% of its activity over the first 500 h, but these activities dropped precipitously, to approximately 6% and approximately 0.5% of the initial activity at 1000 h and 2000 h, respectively. This rapid decomposition began only after the buffering capacity of the solution was overwhelmed by acidic degradants, which caused the pH to decrease. Decomposition processes observed under these conditions included the endolytic hydrolysis of glycosidic linkages and loss of sulfation, particularly N-sulfate groups, and were similar to the degradation processes observed in 0.1 N hydrochloric acid. This study provides initial observations on heparin degradation pathways. More complete, quantitative studies and studies leading to the isolation and characterization of specific degradants are still required.

Preparation and structural characterization of large heparin-derived oligosaccharides.

Porcine mucosal heparin was partially depolymerized with heparin lyase I and then fractionated into low-molecular-weight (< 5000) and high-molecular-weight (> 5000) oligosaccharides by pressure filtration. The high-molecular-weight oligosaccharide mixture (approximately 50 wt% of the starting heparin) also contained intact heparin. This intact polymer complicates oligosaccharide purification. Thus, the low-molecular-weight fraction was used to prepare homogeneous oligosaccharides for structural characterization. The low-molecular-weight oligosaccharide mixture was first fractionated by low-pressure gel permeation chromatography into size-uniform mixtures of disaccharides, tetrasaccharides, hexasaccharides, octasaccharides, decasaccharides, dodecasaccharides, tetradecasaccharides and higher oligosaccharides. Each size-fractionated mixture was then purified on the basis of charge by repetitive semi-preparative strong-anion-exchange high-performance liquid chromatography. This approach has led to the isolation of 14 homogeneous oligosaccharides from disaccharide to tetradecasaccharide. The purity of these heparin-derived oligosaccharides was determined by gradient polyacrylamide gel electrophoresis, analytical strong-anion-exchange high-performance liquid chromatography, capillary electrophoresis and one-dimensional nuclear resonance spectroscopy. The structure of these oligosaccharides was established using 600 MHz two-dimensional nuclear resonance spectroscopy. The spectral methods used included homonuclear correlation spectroscopy, nuclear Overhauser effect spectroscopy and heteronuclear multiple quantum coherence spectroscopy. The 1H/1H connectivities of the protons of each sugar residue in an oligosaccharide were established by two-dimensional homonuclear correlation spectroscopy, while 1H/13C assignments were made using 1H inverse detection. One- and two-dimensional nuclear resonance spectroscopic analysis of these heparin oligosaccharides showed two closely related groups of heparin-oligosaccharides are afforded by enzymatic depolymerization of heparin. One group is fully sulphated, having the structures delta UAp2S(1[-->4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp2S( 1]n-->4)-alpha- D-GlcNpS6S, where delta UAp is 4-deoxy-alpha-L-threo-hex-4-eno-pyranosyluronic acid, GlcNp is 2-deoxy-2-aminoglucopyranose, IdoAp is idopyranosyluronic acid, S is sulphate and n = 0-6. The other group of oligosaccharides differ in that they contain beta-D-glucuronic acid in place of the alpha-L-iduronic acid residue nearest to the reducing end. The present study describes the isolation and structural elucidation of seven new oligosaccharides: an octasaccharide, two decasaccharides, two dodecasaccharides and two tetradecasaccharides. The utility of two-dimensional nuclear resonance spectroscopy to determine the structure of complex heparin oligosaccharides is also illustrated.

Action pattern of polysaccharide lyases on glycosaminoglycans.

The action pattern of polysaccharide lyases on glycosaminoglycan substrates was examined using viscosimetric measurements and gradient polyacrylamide gel electrophoresis (PAGE). Heparin lyase I (heparinase, EC 4.2.2.7) and heparin lyase II (no EC number) both acted on heparin in a random endolytic fashion. Heparin lyase II showed an ideal endolytic action pattern on heparan sulphate, while heparin lyase I decreased the molecular weight of heparan sulphate more slowly. Heparin lyase III (heparitinase, EC 4.2.2.8) acted endolytically only on heparan sulphate and did not cleave heparin. Chondroitin ABC lyase (chondroitinase ABC, EC 4.2.2.4) from Proteus vulgaris acted endolytically on chondroitin-6-sulphate (chondroitin sulphate C) and dermatan sulphate at nearly identical initial rates, but acted on chondroitin-4-sulphate (chondroitin sulphate A) at a reduced rate, decreasing its molecular weight much more slowly. Two chondroitin AC lyases (chondroitinase AC, both EC 4.2.2.5) were examined towards chondroitin-4- and -6-sulphates. The exolytic action of chondroitin AC lyase A from Arthrobacter aurescens on both chondroitin-4- and -6-sulphates was demonstrated viscosimetrically and confirmed using both gradient PAGE and gel permeation chromatography. Chondroitin AC lyase F from Flavobacterium heparinum (Cytophagia heparinia) acted endolytically on the same substrates. Chondroitin B lyase (chondroitinase B, no EC number) from F.heparinum acted endolytically on dermatan sulphate giving a nearly identical action pattern as observed for chondroitin ABC lyase acting on dermatan sulphate.

Monoclonal antibodies prepared against heparin lyase I and their reactivity toward heparin lyase I, II and III.

1. Six different monoclonal IgG mouse antibodies to heparin lyase I from Flavobacterium heparinum were prepared. 2. The monoclonal antibodies were used to detect heparin lyases I, II and III by dot-blotting immunoassay and by Western blotting. 3. Individual antibodies showed different reactivity toward the three heparin lyases. 4. The reactivity of two of the monoclonal antibodies was destroyed by exposing heparin lyases to sodium dodecyl sulfate. 5. The antibodies can be used to rapidly distinguish between the three heparin lyases.