Comparison of cultural methods and DNA probe analyses for the detection of Actinobacillus actinomycetemcomitans, Bacteroides gingivalis, and Bacteroides intermedius in subgingival plaque samples.

The purpose of this study was to compare DNA probe analyses to cultural methods for detecting three periodontal pathogens, Actinobacillus actinomycetemcomitans, Bacteroides gingivalis, and Bacteroides intermedius, in human subgingival plaque. Subgingival sites from patients diagnosed as either healthy or showing evidence of gingivitis or juvenile or adult periodontitis were sampled using two paper points. The number of these pathogens from one paper point was determined using microbiologic media and speciated by biochemical tests. Results were then compared to bacterial numbers obtained from the other paper point using species-specific DNA probes. In 60 samples from the disease group, DNA probe analysis demonstrated 100% effectiveness in detecting A. actinomycetemcomitans and B. intermedius and 91% effectiveness in detecting B. gingivalis at culture positive levels (greater than or equal to 10(3) cells). In addition, probe assays frequently identified these pathogens in samples that were culture negative. Probe analysis revealed a better correlation between presence of a pathogen and clinical evidence of disease on an individual patient basis. In contrast, most samples taken from sites of healthy individuals showed undetectable levels of all three pathogens as determined by both techniques. These results suggest that DNA probe technology is at least equivalent and often superior to cultural methods for detecting A. actinomycetemcomitans, B. gingivalis, and B. intermedius in human subgingival plaque samples.

A cross-reactivity study of whole genomic DNA probes for Haemophilus actinomycetemcomitans, Bacteroides intermedius, and Bacteroides gingivalis.

In this study, we evaluated the sensitivity and specificity of whole genomic DNA probes for the periodontal pathogens Haemophilus actinomycetemcomitans, Bacteroides intermedius, and Bacteroides gingivalis. By means of these probes, DNA hybridizations were performed against other organisms found in the oral cavity and organisms previously determined to be genetically similar. All three probes were sensitive to 10(3) cells for their respective organism. The H. actinomycetemcomitans probe cross-reacted with several haemophilus strains, Wolinella, and Campylobacter, indicating that H. actinomycetemcomitans-specific sequences would have to be identified and cloned for accurate detection of this organism in heterogeneous patient samples. Only very low levels of cross-reactivity were observed between the B. intermedius probe and representative black-pigmented Bacteroides. This low level of cross-reactivity did not interfere with the accurate identification of B. intermedius in sample evaluations. The B. gingivalis probe showed no cross-reactivity. Whole genomic probes will be used for the detection of B. intermedius and B. gingivalis in patient samples.

Monensin inhibits catecholamine synthesis in pheochromocytoma cells.

The carboxylic ionophore monensin inhibits the activity of tyrosine 3-monooxygenase and decreases the rate of catecholamine synthesis in pheochromocytoma cells incubated in vitro. The ionophore inhibits dopa production in intact pheochromocytoma cells, but does not itself inhibit tyrosine 3-monooxygenase and does not produce a stable inactivation of the enzyme as assayed in cell-free extracts of the cells. The inhibition of dopa production by monensin is dependent upon extracellular Na+, but does not require extracellular Ca++. This effect of monensin is more pronounced in the presence of pargyline. In the absence of pargyline, monensin also depletes the cells of norepinephrine and increases the accumulation of the deaminated norepinephrine metabolite, dihydroxyphenylglycol. Finally, monensin increases the release of catecholamines from isolated chromaffin granules. These results are consistent with the hypothesis that monensin causes the release of norepinephrine from chromaffin granules into the cytoplasm of pheochromocytoma cells and that the increase in cytoplasmic norepinephrine inhibits tyrosine 3-monooxygenase activity.

Studies on dihydropteridine reductase activity in pheochromocytoma cells.

The activity of dihydropteridine reductase (DPR) in pheochromocytoma cells has been studied. The activity of this enzyme in crude extracts of pheochromocytoma cells is approximately 50 nmol/min/mg protein. This activity is very much greater than the activity of tyrosine 3-monooxygenase (TH) in these extracts and the rate of conversion of tyrosine to DOPA in intact pheochromocytoma cells. Incubation of the cells with 56 mM-K+ or with cholera toxin has previously been shown to increase the rate of catecholamine synthesis and to cause a stable activation of TH in the cells. These treatments do not produce a stable activation of DPR, as assayed in vitro. Methotrexate inhibits DPR activity in vitro with an I50 of approximately 20 microM, but has no effect on the rate of DOPA formation in intact pheochromocytoma cells. Therefore, DPR does not appear to be the rate-limiting enzyme in the pathway of catecholamine synthesis in pheochromocytoma cells. Moreover, the activities of DPR and of TH are not regulated coordinately in these cells.

Tyrosine 3-monooxygenase regulates catecholamine synthesis in pheochromocytoma cells.

Incubation of pheochromocytoma cells with 56 mM K+ or with cholera toxin increases the conversion of [14C]tyrosine to [14C]catecholamines (Chalfie, M., Settipani, L., and Perlman, R. L. (1979) Mol. Pharmacol. 15, 263-270). We have now measured the tyrosine content and the rate of dihydroxyphenylalanine production in these cells. Incubation with 56 mM K+ or with cholera toxin increases the rate of dihydroxyphenylalanine production but decreases the tyrosine content of the cells. We have also measured the uptake of tyrosine into pheochromocytoma cells. The rate of tyrosine uptake is more than 1 order of magnitude greater than the rate of dihydroxyphenylalanine production. Moreover, tyrosine uptake is not affected by cholera toxin and is decreased by approximately 30% in media that contain 56 mM K+. These results provide direct evidence that tyrosine 3-monooxygenase regulates catecholamine synthesis in pheochromocytoma cells and that incubation with 56 mM K+ or with cholera toxin causes the activation of this enzyme in these cells.

Increased spontaneous reversion of certain frameshift mutations in DNA polymerase I deficient strains of Escherichia coli.

A tenfold increase in the spontaneous reversion frequency of two of six lacZ frameshift mutations tested was observed in strains containing the following DNA polymerase I mutations--polA1, polA5, polA6, polAex1, res-3 and resA1. Reconstruction experiments indicated that this increase was not the result of a selective effect. Only a fourfold increase in frameshift mutations was found in strains containing a polA107 mutation. Both the polAex1 and polA107 mutations result in defective 5' to 3' exonuclease activity and do not affect polymerizing activity, but have different effects on frameshift mutation. A polA mutation on the chromosome induced frameshift mutations in a gene on an F episome. None of three auxotrophic mutations studied showed high frequency reversion in the presence of the polA1 or polA6 mutations.