Analysis of proteins in human gingival crevicular fluid by mass spectrometry.

BACKGROUND AND OBJECTIVE: Gingival crevicular fluid is a bodily fluid transuded from periodontal tissues into the gingival crevice and periodontal pocket, and contains many species of components. Proteins in gingival crevicular fluid have been studied as markers for periodontal diseases. Mass spectrometric analysis is used for the analyses of proteins, lipids, saccharides and metals, and expected as an approach for disease diagnosis. For better analysis of the protein components in gingival crevicular fluid, we investigated proteins in gingival crevicular fluid samples from the healthy gingival crevice and periodontal pocket using mass spectrometry. MATERIAL AND METHODS: Gingival crevicular fluid samples were collected from subjects who gave their informed consent and were periodontally healthy or had diseased pockets. These samples were electrophoretically separated, and each fraction on the gels was analysed by nano liquid chromatography coupled with tandem mass spectrometry. Antimicrobial peptides detected in gingival crevicular fluid were confirmed by western blotting. RESULTS: One hundred and four proteins were detected in gingival crevicular fluid samples from both healthy sites and sites of periodontitis; 64 proteins were contained only in gingival crevicular fluid from healthy sites and 63 proteins were observed only in gingival crevicular fluid from periodontitis sites. These proteins were blood-, cytoskeleton-, immunity-, inflammation- and lipid-related proteins and enzymes. Some proteins, including ceruloplasmin, glycogen phosphorylase, glutathione S-transferase, phosphoglycerate mutase, psoriasin, S100A11 and resistin, were identified for the first time in gingival crevicular fluid. Antimicrobial peptides, such as lactoferrin, α1-antitrypsin, lipocalin, S100A7, S100A8, S100A9 and cathelicidin, were observed by mass spectrometry and western blotting. CONCLUSION: Multiple protein components in gingival crevicular fluid were analysed at the same time using mass spectrometry, and this approach may be useful for the diagnosis of periodontal diseases.

Rapid Discrimination of Mitochondrial DNA Type and Use of Results to Study Mitochondrial Inheritance in Pleurotus spp.

We have reported a simple and rapid method to discriminate species in the genus Pleurotus by analysis of restriction-fragment-length polymorphism of whole-cell DNA, and found that several restriction enzymes gave DNA bands useful in such discrimination, but other enzymes tested did not. In the present study, we report the reason why there were useful and useless enzymes; the effective enzymes digested rDNA into small fragments that did not interfere with the detection of DNA bands useful for discrimination. The origin of these discriminative DNA bands was found to be mitochondrial DNA when the banding profiles of whole-cell DNA, mitochondrial DNA, and nuclear DNA were compared. Consequently, our method could be used for rapid and simple identification of mitochondrial DNA type in the genus Pleurotus. The results were used to study mitochondrial inheritance, and we found that only the nucleus but not the mitochondria migrated during the mating of Pleurotus cornucopiae with P. citrinopileatus.

Source of ATP for hexokinase-catalyzed glucose phosphorylation in tumor cells: dependence on the rate of oxidative phosphorylation relative to that of extramitochondrial ATP generation.

We isolated highly intact and tightly coupled mitochondria from the rat ascites hepatoma cell line AH130 by disruption of the cell membrane by nitrogen cavitation. These isolated mitochondria were found to have essentially the same functional properties as rat liver mitochondria, but unlike the latter, hexokinase (HK) was bound to their membrane. Using the tumor mitochondrial preparation, we examined the source of ATP for phosphorylation of glucose by HK under conditions in which intra- and extramitochondrial ATP-generation systems operated separately or together. Results showed that the membrane-bound HK utilized ATP derived from the most efficiently operating ATP generation system, i.e., oxidative phosphorylation. However, when the rate of extramitochondrial ATP generation was much greater than that of oxidative phosphorylation, HK used ATP from the extramitochondrial ATP-generation system.