The gram-negative, oral bacterium Actinobacillus actinomycetemcomitans has been implicated as the causative agent of several forms of periodontal disease in humans. When cultured in broth, fresh clinical isolates of A. actinomycetemcomitans form tenacious biofilms on surfaces such as glass, plastic, and saliva-coated hydroxyapatite, a property that probably plays an important role in the ability of this bacterium to colonize the oral cavity and cause disease. We examined the morphology of A. actinomycetemcomitans biofilm colonies grown on glass slides and in polystyrene petri dishes by using light microscopy and scanning and transmission electron microscopy. We found that A. actinomycetemcomitans developed asymmetric, lobed biofilm colonies that displayed complex architectural features, including a layer of densely packed cells on the outside of the colony and nonaggregated cells and large, transparent cavities on the inside of the colony. Mature biofilm colonies released single cells or small clusters of cells into the medium. These released cells adhered to the surface of the culture vessel and formed new colonies, enabling the biofilm to spread. We isolated three transposon insertion mutants which produced biofilm colonies that lacked internal, nonaggregated cells and were unable to release cells into the medium. Actinobacillus actinomycetemcomitans is a gram-negative, nonmotile coccobacillus that colonizes the human oral cavity (20). A. actinomycetemcomitans has been implicated as the causative agent of several forms of severe periodontal disease, including localized juvenile periodontitis, early-onset periodontitis, and rapidly progressive periodontitis (37). Infrequently, A. actinomycetemcomitans can enter the submucosa and cause nonoral infections, including bacteremias, infective endocarditis, and localized abscesses (17). When cultured in broth, fresh clinical isolates of A. actinomycetemcomitans form extremely tenacious biofilms on surfaces such as glass, plastic, and saliva-coated hydroxyapatite (7, 8, 11, 13-16, 20, 29, 30). Nearly all of the cells grow attached to the surface, while the broth remains clear and is often sterile (7). The dense biofilm that forms on the surface is resistant to removal by agents such as detergents, proteases, heat, sonication, and vortex agitation (7) and exhibits increased resistance to antimicrobial agents compared with that exhibited by cells grown in planktonic form (6). Tight adherence has been shown to play an important role in the ability of A. actinomycetemcomitans to colonize the mouths of rats (9) and probably plays an equally important role in its ability to colonize humans. Tenacious surface attachment is dependent on the presence of long, bundled adhesive pili (fimbriae) that form on the surface of the cell (29). Mutations in flp-1, which encodes the major fimbrial protein subunit, result in cells that fail to produce fimbriae or attach to surfaces (15).Kaplan and Fine have recently shown that A. actinomycetemcomitans biofilm colonies are capable of releasi...

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Knock-out (KO) mice lacking gangliotetraose gangliosides attributable to disruption of the gene for GM2/GD2 synthase [GalNAcT (UDP-N-acetylgalactosamine:GM3/GD3 ␤-1,4-N-acetylgalactosaminyltransferase; EC 2.4.1.92)] are revealing key neural functions for the complex gangliosides of brain. This study has found such animals to be highly susceptible to kainic acid (KA)-induced seizures in terms of both seizure severity and duration. Intraperitoneal injection of 25 mg/kg KA produced status epilepticus for ϳ200 min in normal mice or heterozygotes and more than four times longer in the KO mice. The latter group suffered ϳ30% mortality, which increased to ϳ75% at dosage of 30 mg/kg KA, compared with 10 -14% for the other two genotypes at the latter dosage. Nissl staining and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling assay revealed substantial deterioration of pyramidal neurons attributable to apoptosis in the KO hippocampus, especially the CA3 region. Seizure activity in the KO mouse was only moderately diminished by intraperitoneal injection of GM1 ganglioside, whereas LIGA 20, a semisynthetic analog of GM1, substantially reduced both seizure severity and cell damage. The potency of LIGA 20 was correlated with its enhanced membrane permeability (compared with GM1), as seen in the increased uptake of /Ca2ϩ exchanger located at the inner membrane of the nuclear envelope in KO mice, an exchanger dependent on tight association with GM1 or its analog for optimal activity. These results point to a neuroprotective role for GM1 and its associated exchanger in the nucleus, based on regulation of Ca 2ϩ flux between nucleoplasm and nuclear envelope.

Oocyst walls were purified from unsporulated oocysts of Eimeria tenella. Analysis of the purified material indicated a composition of 67% peptide, 14% lipid, and 19% carbohydrate. The likely physical arrangement of the components places the lipid in a 10 nm thick outer layer, covering a 90 nm thick layer of glycoprotein. The protein component of the structure was dissociated using thiol reagents under denaturing conditions, and was shown to consist of a repeating subunit of approximately 10,000 daltons. The results suggest an explanation for the physical and mechanical resistance of the oocyst wall, as well as possible mechanisms for excystation of sporulated oocysts.

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