A 5 nuclease TaqMan PCR assay was developed for the quantitative detection of the major cariogenic bacteria Streptococcus mutans and Streptococcus sobrinus. The absolute and relative numbers of bacteria were measured by this method. This assay will be useful for quantifying these organisms in oral specimens and for analyzing biofilm formation.

.-The mechanisms by which lipopolysaccharide (LPS) is recognized, and how such recognition leads to innate immune responses, are poorly understood. Stimulation with LPS induces the activation of a variety of proteins, including mitogen-activated protein kinases (MAPKs) and NF-B. Activation of protein tyrosine kinases (PTKs) is also necessary for a number of biological responses to LPS. We used a murine macrophage-like cell line, RAW264.7, to demonstrate that Janus kinase (JAK)2 is tyrosine phosphorylated immediately after LPS stimulation. Anti-Toll-like receptor (TLR)4 neutralization antibody inhibits the phosphorylation of JAK2 and the c-Jun NH2-terminal protein kinase (JNK). Both the JAK inhibitor AG490 and the kinase-deficient JAK2 protein reduce the phosphorylation of JNK and phosphatidylinositol 3-kinase (PI3K) via LPS stimulation. Pharmacological inhibition of the kinase activity of PI3K with LY-294002 decreases the phosphorylation of JNK. Finally, we show that JAK2 is involved in the production of IL-1␤ and IL-6. PI3K and JNK are also important for the production of IL-1␤. These results suggest that LPS induces tyrosine phosphorylation of JAK2 via TLR4 and that JAK2 regulates phosphorylation of JNK mainly through activation of PI3K. Phosphorylation of JAK2 via LPS stimulation is important for the production of IL-1␤ via the PI3K/JNK cascade. Thus JAK2 plays a pivotal role in LPS-induced signaling in macrophages.cytokine; toll-like receptor-4; c-Jun NH 2-terminal kinase

A 5 nuclease TaqMan PCR was developed for the quantitative detection of the periodontopathic bacteria Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. The relative numbers of bacteria were measured by the comparative threshold cycle method. This simplified method is a way of obtaining the relative quantities of these organisms from specimens and of monitoring the effect of therapy.Periodontitis is an inflammation of the supporting tissues of the teeth (4). It is generally accepted that periodontal diseases are infectious diseases caused by oral bacteria (5). Actinobacillus actinomycetemcomitans is a nonmotile, gram-negative, capnophilic, fermentative coccobacillus that has been implicated in the etiology of localized juvenile periodontitis (12,16,18), while Porphyromonas gingivalis is a gram-negative, blackpigmented anaerobe that is strongly implicated as a major pathogen in adult periodontitis (14,19). Several PCR-based systems that use oral specimens for the detection of oral bacterial infections, especially periodontitis, have been reported (1,2,13,15). Most previous diagnostic systems are qualitative and are therefore unsuitable for the evaluation of treatment, as quantitative analysis is essential for monitoring the effect of therapy in treatment trials.The TaqMan assay based on the 5Ј-3Ј exonuclease activity of Taq polymerase has been developed for quantitative detection of DNA (9). Briefly, an oligonucleotide probe that has a reporter fluorescent dye attached to its 5Ј end and a quencher dye attached to its 3Ј end is used for the assay. When the probe hybridizes to its target template, the reporter dye is cleaved by the 5Ј nuclease activity of Taq polymerase and becomes capable of emitting a fluorescent signal, since it is no longer suppressed by the quencher dye (8).This report describes a simple, rapid method for the relative quantification of major periodontopathic bacteria, including A. actinomycetemcomitans and P. gingivalis, in saliva and subgingival plaque. The method uses a TaqMan PCR assay and the comparative threshold cycle (⌬⌬Ct) method. This is the first reported TaqMan method developed for the detection of A. actinomycetemcomitans.The bacterial strains used in this study are listed in Table 1. The strains of A. actinomycetemcomitans and P. gingivalis were cultured as described previously (15,17). Subgingival plaque and saliva samples from patients with periodontitis were prepared as described previously (15).The oligonucleotide primers and probes, designed by using Primer Express (version 1.5) software (Applied Biosystems, Foster City, Calif.), are listed in Table 2. The sequences of the universal primers and a probe for a broad range of bacteria are complementary to highly conserved regions within the 16S rRNA gene (7). The A. actinomycetemcomitans-and P. gingivalis-specific primers and probes were designed from the lktA (6) and 16S rRNA genes, respectively. The specificities of the primers and probes were confirmed by conventional PCR (Table 1) and dot blot analysis with d...

The molecular mechanisms by which pathogen-associated molecular patterns recognized by TLR2, such as peptidoglycan (PGN), induce homotolerance are largely unknown. It was recently reported that IRAK-M negatively regulates TLR signaling. In this study, we elucidate the molecular mechanisms of tolerance induced by PGN, with a focus on the role of IRAK-M. We demonstrate that pretreatment of macrophage RAW264.

We identified a gene (atlA) encoding autolytic activity from Streptococcus mutans Xc. The AtlA protein predicted to be encoded by atlA is composed of 979 amino acids with a molecular weight of 107,279 and has a conserved ␤-1,4-N-acetylmuramidase (lysozyme) domain in the C-terminal portion. Sodium dodecyl sulfate extracts of strain Xc showed two major bacteriolytic bands with molecular masses of 107 and 79 kDa, both of which were absent from a mutant with inactivated atlA. Western blot analysis revealed that the 79-kDa band was derived from the 107-kDa peptide by cleavage of its N-terminal portion. The inactivation of atlA resulted in a marked decrease in autolysis and the formation of very long chains of cells compared to the case for the parent strain. Although both the parent and mutant strains formed biofilms in the presence of sucrose, the biofilms formed by the mutant had a sponge-like architecture with large gaps and contained 30% less biomass than those formed by the parent strain. Furthermore, strain Xc formed glucose-dependent, loose biofilms in the absence of sucrose, but the mutant lost this ability. These results suggest that AtlA may play an important role in biofilm formation by S. mutans. The antibody produced against the C-terminal peptide containing the ␤-1,4-N-acetylmuramidase domain drastically inhibited the autolytic activity of strain Xc. This inhibition was specific among the oral streptococci to S. mutans. These results indicate that the catalytic domain of AtlA is located at the C terminus, suggesting that further characterization of this domain may provide a means to control cariogenic dental plaque formation.