Identification of the human salivary cystatin complex by the coupling of high-performance liquid chromatography and ion-trap mass spectrometry

Human salivary cystatins, five major (S, S1, S2, SA, SN) and two minor (C and D), are multifunctional proteins playing a different role in the oral environment. Salivary cystatin SN is able to effectively inhibit lysosomal cathepsins B, C, H and L and cystatin SA inhibits cathepsins C and L in vitro. These activities suggest, particularly for cystatin SN, an important role in the control of proteolytic events in vivo. Differently, cystatins S are involved, together with statherin, in the mineral balance of the tooth. Due to their distinct role, a reliable method for identification and quantification of the different cystatins, as well as of possible truncated and derived forms, could be helpful for the assessment of the status of the oral cavity. To this purpose high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI MS) was applied to the analysis of human saliva obtained from healthy subjects. All known salivary cystatins, with the exception of cystatin C, were detected. Strong evidence was also obtained for the presence in saliva of post-translational modified isoforms of cystatins, which may be related to donor habits. Cystatin SN and cystatins S, S1 and S2 were well separated by HPLC-ESI MS coupling from other components and thus this approach can be successfully applied to their quantification.     

Detection of Streptococcus anginosus from saliva by real-time polymerase chain reaction

AIMS: The purpose of the present investigation was to assess the salivary levels of Streptococcus anginosus in periodontitis patients. METHODS AND RESULTS: The salivary levels of Strep. anginosus were assessed using real-time polymerase chain reaction (PCR). Streptococcus anginosus was detected in 28 of 37 (75.6%) of periodontitis patients and in three of the 20 (15%) healthy subjects. The mean values for bleeding on probing and probing depth in positive patients were statistically higher than those in negative patients. A significant decrease in Strep. anginosus levels was observed after periodontal treatment. CONCLUSIONS: Although the levels of Strep. anginosus are extremely low, they may reflect the status of periodontal health. SIGNIFICANCE AND IMPACT OF THE STUDY: Real-time PCR is a useful method for obtaining the relative quantities of Strep. anginosus from saliva samples and for monitoring the effect of therapy.     

No effects of pulsed radio frequency electromagnetic fields on melatonin, cortisol, and selected markers of the immune system in man

There is growing public concern that radio frequency electromagnetic fields may have adverse biological effects. In the present study eight healthy male students were tested to see whether or not radio frequency electromagnetic fields as used in modern digital wireless telecommunication (GSM standard) have noticeable effects on salivary melatonin, cortisol, neopterin, and immunoglobulin A (sIgA) levels during and several hours after exposure. In a specifically designed, shielded experimental chamber, the circularly polarized electromagnetic field applied was transmitted by an antenna positioned 10 cm behind the head of upright sitting test persons. The carrier frequency of 900 MHz was pulsed with 217 Hz (average power flux density 1 W/m2). In double blind trials, each test person underwent a total of 20 randomly allotted 4 hour periods of exposure and sham exposure, equally distributed at day and night. The results obtained show that the salivary concentrations of melatonin, cortisol, neopterin and sIgA did not differ significantly between exposure and sham exposure.     

Comparison of oligosaccharides derived from salivary mucin of Japanese secretor and non-secretor individuals of blood group type-A

Comparison of oligosaccharide components derived from salivary mucin was performed between secretor and non-secretor individuals. Salivary mucin was collected from four secretors and three non-secretors having blood group type-A. Compositional analysis showed that the contents of galactose and N-acetylglucosamine in the non-secretor were higher than those in the secretor. The O-linked oligosaccharides obtained by treatment with alkaline borohydride were separated by gel filtration using Sephadex G-50. The results indicated that the size of the type-A active oligosaccharides from the secretor was similar to or smaller than that of the non-secretor. Ion-exchange chromatography showed that the secretors had strong type-A activities in both the neutral and acidic fractions but the non-secretors showed type-A activity mainly in the neutral fraction. These results suggest that compositional differences in blood group substances exist between secretors and non-secretors.     

Salivary proteins and glycoproteins in phlebotomine sandflies of various species, sex and age

Salivary gland proteins were studied in sandflies (Diptera: Psychodidae: Phlebotominae) by electrophoretic techniques. In Phlebotomus duboscqi Neveu-Lemaire the protein concentration was about 30 times higher in females than in males. SDS PAGE revealed eight major bands of 29-62 kDa in salivary gland extracts (SGE) from females, whereas only one band of 57kDa was detected in males. The number of protein components in SGE gradually increased with the age of females. In P. papatasi (Scopoli) the typical electrophoretic pattern was reached in 3-5 days after imago emergence, depending on the temperature at which females were maintained. All major protein components of the female SGE were present in the content of glands. Female SGE were compared in seven colonies of five Phlebotomus and Lutzomyia species; electrophoretic profiles distinguished between species and even between colonies of different geographical origin. In general, the highest variability of major protein components was observed in the 38-48kDa region. Four colonies of the subgenus Phlebotomus (P. duboscqi and P. papatasi) possessed common mobility polypeptides, the highest similarity was found between two colonies of P. papatasi. Other species tested significantly differed, specific prominent bands of 33, 35 and 38kDa were found in P. halepensis Theodor, P. perniciosus Newstead and Lutzomyia longipalpis (Lutz & Neiva), respectively. Glycoproteins in SGE of Lu. longipalpis and P. duboscqi females were identified and analysed using blotting with five lectin conjugates. Specific reaction of lectins ConA and WGA revealed the complex type of N-glycans in the 48 and 53-54kDa glycoproteins present in both species. Similar glycosylation was detected in species-specific bands of the 57-60 and 65-67 kDa in P. duboscqi and Lu. longipalpis, respectively. The high mannose type of glycosylation was found in the 20 and 39 kDa polypeptides of Lu. longipalpis and the 40-42 kDa polypeptides of P. duboscqi. Innate lectin activity specific for aminosugars was detected in SGE of P. duboscqi females using haemagglutination tests with rabbit erythrocytes.     

Comparison of the oral bacterial flora in saliva from a healthy subject and two periodontitis patients by sequence analysis of 16S rDNA libraries

The oral bacterial flora in the saliva from two patients with periodontitis and from a periodontally healthy subject were compared using a sequence analysis of 16S rDNA libraries without cultivation. 16S rDNAs were amplified from salivary DNA by PCR and cloned. Randomly selected clones were partially sequenced. On the basis of sequence similarities, the clones were classified into several clusters corresponding to the major phylum of the domain Bacteria. The major phylum in the libraries was the low G+C Gram-positive bacteria. There was no clonal sequence affiliated with periodontopathic bacteria in the salivary sample from the healthy subject, while a number of periodontal pathogens such as Campylobacter rectus, Prevotella intermedia, Porphyromonas gingivalis and Treponema socranskii were detected in the salivary samples from the patients with periodontitis. In addition, a number of previously uncharacterized and uncultured microorganisms were recognized. These organisms may have some role in periodontal disease. This study reveals some potential for a molecular-biological technique to analyze the oral microflora associated with periodontal disease, including previously uncharacterized and uncultured microorganisms, without cultivation.     

Phenol oxidising enzymes in the grain aphid’s saliva

Sucrose-agarose gels and sucrose liquid diets were used to study the phenol oxidising enzymes in the salivary secretions of the grain aphid, Sitobion avenae (Fabricius). Activity indicating the presence of two oxidoreductases, polyphenol oxidase (PPO) and peroxidase (Px), was found. Both enzymes were present in the aphids stylet sheath (gelling saliva) but only polyphenol oxidase activity was found in the halos around sheaths and thus in watery saliva. Electrical penetration graphs (EPG) revealed that the secretion of these enzymes into the gels, by an individual aphid, was associated with its probing activity observed during penetration of the epidermal and mesophyll tissues. The grain aphids PPO, secreted in its saliva reacted with a range of phenolic compounds. As most of these phenolics occur naturally in cereals, the grain aphid could modify its host-plants phenolic composition. The importance of the grain aphids polyphenol oxidase and peroxidase in detoxifying cereal phenolics is discussed.     

Rhodnius prolixus salivary nitrophorins display heme-peroxidase activity

Rhodnius prolixus is a blood feeding triatomine bug that contains salivary nitric oxide bound to hemoproteins previously named nitrophorins. Nitrophorins, in addition to storing and transporting NO, have two other functions such as anti-histaminic and anti-clotting (displayed by nitrophorin 2 only). Additionally, nitrophorins display a thiol oxidase reaction, where cysteine is oxidized to cystine with the production of hydrogen peroxide. In this paper the heme-peroxidase reaction of nitrophorins is described. The heme moiety of nitrophorins is destroyed by addition of cysteine or hydrogen peroxide. No biliverdin is produced during this reaction. We have also found that during the thiol oxidase reaction, nitrophorins can destroy norepinephrine, conferring an additional vasodilatory competence for this class of salivary molecules.     

Chemical Composition and Allelopathic Potential of Essential Oils Obtained from Acacia cyanophylla Lindl. Cultivated in Tunisia

Acacia cyanophylla Lindl . (Fabaceae), synonym Acacia saligna (Labill .) H. L.Wendl ., native to West Australia and naturalized in North Africa and South Europe, was introduced in Tunisia for rangeland rehabilitation, particularly in the semiarid zones. In addition, this evergreen tree represents a potential forage resource, particularly during periods of drought. A. cyanophylla is abundant in Tunisia and some other Mediterranean countries. The chemical composition of the essential oils obtained by hydrodistillation from different plant parts, viz., roots, stems, phyllodes, flowers, and pods (fully mature fruits without seeds), was characterized for the first time here. According to GC‐FID and GC/MS analyses, the principal compound in the phyllode and flower oils was dodecanoic acid ( 4 ), representing 22.8 and 66.5% of the total oil, respectively. Phenylethyl salicylate ( 8 ; 34.9%), heptyl valerate ( 3 ; 17.3%), and nonadecane (36%) were the main compounds in the root, stem, and pod oils, respectively. The phyllode and flower oils were very similar, containing almost the same compounds. Nevertheless, the phyllode oil differed from the flower oil for its higher contents of hexahydrofarnesyl acetone ( 6 ), linalool ( 1 ), pentadecanal, α‐terpineol, and benzyl benzoate ( 5 ) and its lower content of 4 . Principal component and hierarchical cluster analyses separated the five essential oils into four groups, each characterized by its main constituents. Furthermore, the allelopathic activity of each oil was evaluated using lettuce (Lactuca sativa L.) as a plant model. The phyllode, flower, and pod oils exhibited a strong allelopathic activity against lettuce.