Proteomic Analysis of Whole Saliva in Relation to Dental Caries Resistance.

Saliva contains possible biomarkers that are associated with dental caries. The present study aimed to analyse differences in the abundance of proteins in the saliva between caries-positive (CP; N = 15) and caries-free (CF; N = 12) males and to compare differences in the abundance of proteins between two saliva sample fractions (supernatant and pellet). We found 14 differently significantly expressed proteins in the CF group when comparing the supernatant fractions of the CP and CF groups, and three proteins in the pellet fractions had significantly higher expression in the CP group. Our results indicate very specific protein compositions of the saliva in relation to dental caries resistance (the saliva of the CP group mainly contained pellet proteins and the saliva of the CF group mainly contained supernatant proteins). This was the first time that the saliva pellet fraction was analysed in relation to the dental caries status. We detected specific calcium-binding proteins that could have decalcified enamel in the saliva pellet of the CP group. We also observed significantly up-regulated immune proteins in the saliva supernatant of the CF group that could play an important role in the caries prevention. The particular protein compositions of the saliva pellet and supernatant in the groups with different susceptibilities to tooth decay is a promising finding for future research.

Proteomic analysis of peroxynitrite-induced protein nitration in isolated beef heart mitochondria.

Mitochondria are exposed to reactive nitrogen species under physiological conditions and even more under several pathologic states. In order to reveal the mechanism of these processes we studied the effects of peroxynitrite on isolated beef heart mitochondria in vitro. Peroxynitrite has the potential to nitrate protein tyrosine moieties, break the peptide bond, and eventually release the membrane proteins into the solution. All these effects were found in our experiments. Mitochondrial proteins were resolved by 2D electrophoresis and the protein nitration was detected by immunochemical methods and by nano LC-MS/MS. Mass spectrometry confirmed nitration of ATP synthase subunit beta, pyruvate dehydrogenase E1 component subunit beta, citrate synthase and acetyl-CoA acetyltransferase. Immunoblot detection using chemiluminiscence showed possible nitration of other proteins such as cytochrome b-c1 complex subunit 1, NADH dehydrogenase [ubiquinone] iron-sulfur protein 2, elongation factor Tu, NADH dehydrogenase [ubiquinone] flavoprotein 2, heat shock protein beta-1 and NADH dehydrogenase [ubiquinone] iron-sulfur protein 8. ATP synthase beta subunit was nitrated both in membrane and in fraction prepared by osmotic lysis. The high sensitivity of proteins to nitration by peroxynitrite is of potential biological importance, as these enzymes are involved in various pathways associated with energy production in the heart.

Effects of Leukaemia Inhibitory Factor Receptor on the Early Stage of Osteogenic Differentiation of Human Bone Marrow Mesenchymal Cells.

Most people worldwide suffer from dental caries. Only a small part of the population is cariesresistant and the reason for this resistance in unknown. Only a few studies compared the saliva protein composition of persons with carious teeth and persons with no caries. Our study is the first to relate proteomic analysis of the caries aetiology with gender. In this study, we compared the differences in the abundances of proteins in the saliva between cariesresistant and caries-susceptible females and males by nano-liquid chromatography-tandem mass spectrometry (Label-Free Quantitative Proteomics). Our results demonstrate that the observed differences in the protein levels might have an influence on anticaries resistance. A total of 19 potential markers of tooth caries were found, for example proteins S100A8 and annexin A1 with higher expression in the cariessusceptible group in comparison with the caries-free group and mucin-5B, lactoferrin, lysozyme C with higher expression in the caries-free group in comparison with the caries-susceptible group. The presented study is the first complex proteomic and gender project where the saliva protein content of caries-free and caries-susceptible persons were compared by label-free MS. The newly detected potential protein markers of dental caries can be a good basis for further research and for possible future therapeutic use.

Proteomics of human teeth and saliva.

Teeth have been a focus of interest for many centuries--due to medical problems with them. They are the hardest part of the human body and are composed of three mineralized parts--enamel, dentin and cementum, together with the soft pulp. However, saliva also has a significant impact on tooth quality. Proteomic research of human teeth is now accelerating, and it includes all parts of the tooth. Some methodological problems still need to be overcome in this research field--mainly connected with calcified tissues. This review will provide an overview of the current state of research with focus on the individual parts of the tooth and pellicle layer as well as saliva. These proteomic results can help not only stomatology in terms of early diagnosis, identifying risk factors, and systematic control.

Reciprocal changes in maternal and fetal metabolism of corticosterone in rat during gestation.

OBJECTIVE: The objective of this study was to investigate the role of 11beta-hydroxysteroid dehydrogenases (11HSD1 and 11HSD2) in determining the fetal concentration of glucocorticoids. METHODS: The expression patterns for mRNA abundance, protein level, and enzyme activities of placental and fetal 11HSD1 and 11HSD2 were assessed from embryonic day 13 (E13) to day 21 (E21; term E22). The transplacental passage of maternal corticosterone and its contribution to fetal glucocorticoids was also studied. RESULTS: Placental 11HSD1 mRNA decreased between days E13 and E14 and then remained at much lower values up to E21. Similarly, NADP+-dependent 11beta-oxidation and 11-reduction were lower in late gestation. In contrast, placental 11HSD2 mRNA and protein decreased between E13 and E21. Dithiothreitol increased the activity of 11HSD2 and the output of 11-dehydrocorticosterone into fetal circulation.The fetal activity of 11HSD1 increased and 11HSD2 decreased between E16 and E21. CONCLUSIONS: The final third of gestation is accompanied by reciprocal changes in placental and fetal metabolism of corticosterone due to changes in 11HSD1 and 11HSD2 not only at the level of transcription but also at a posttranslational level.

Glucocorticoid availability in colonic inflammation of rat.

Recent in vitro studies have shown the involvement of pro-inflammatory cytokines in the regulation of the local metabolism of glucocorticoids via 11beta-hydroxysteroid dehydrogenase type 1 and type 2 (11HSD1 and 11HSD2). However, direct in vivo evidence for a relationship among the local metabolism of glucocorticoids, inflammation and steroid enzymes is still lacking. We have therefore examined the changes in the local metabolism of glucocorticoids during colonic inflammation induced by TNBS and the consequences of corticosterone metabolism inhibition by carbenoxolone on 11HSD1, 11HSD2, cyclooxygenase 2 (COX-2), mucin 2 (MUC-2), tumor necrosis factor alpha (TNF-alpha), and interleukin 1beta (IL-1beta). The metabolism of glucocorticoids was measured in tissue slices in vitro and their 11HSD1, 11HSD2, COX-2, MUC-2, TNF-alpha, and IL-1beta mRNA abundances by quantitative reverse transcription-polymerase chain reaction. Colitis produced an up-regulation of colonic 11HSD1 and down-regulation of 11HSD2 in a dose-dependent manner, and these changes resulted in a decreased capacity of the inflamed tissue to inactivate tissue corticosterone. Similarly, 11HSD1 transcript was increased in colonic intraepithelial lymphocytes of TNBS-treated rats. Topical intracolonic application of carbenoxolone stimulated 11HSD1 mRNA and partially inhibited 11HSD2 mRNA and tissue corticosterone inactivation and these changes were blocked by RU-486. The administration of budesonide mimicked the effect of carbenoxolone. In contrast to the local metabolism of glucocorticoids, carbenoxolone neither potentiates nor diminishes gene expression for COX-2, TNF-alpha, and IL-1beta, despite the fact that budesonide down-regulated all of them. These data indicate that inflammation is associated with the down-regulation of tissue glucocorticoid catabolism. However, these changes in the local metabolism of glucocorticoids do not modulate the expression of COX-2, TNF-alpha, and IL-1beta in inflamed tissue.

Advanced glycation end-product pentosidine accumulates in various tissues of rats with high fructose intake.

The slowly metabolized proteins of the extracellular matrix, typically collagen and elastin, accumulate reactive metabolites through uncontrolled non-enzymatic reactions such as glycation or the products arising from the reaction of unsaturated long chain fatty acid metabolites (possessing aldehydic groups). A typical example of these non-enzymatic changes is the formation of advanced glycation end-products (AGEs), resulting from the reaction of carbohydrates with the free amino group of proteins. The accumulation of AGEs and the resulting structural alterations cause altered tissue properties (increased stiffness, reduced elasticity) that contribute to their reduced catabolism and to their aging. Posttranslational nonenzymatic modifications of the proteins of the extracellular matrix (the formation of a typical AGE product--pentosidine) were studied in three types of tissue of three rat strains subjected to a high-fructose diet. Chronic (three-week) hyperglycemia (resulting from fructose loading) caused a significant increase in pentosidine concentration mainly in the aorta and skin of the three rat strains (Lewis, Wistar and hereditary hypertriglyceridemic rats).

Cloning and expression of chicken 20-hydroxysteroid dehydrogenase.

The ligand specificity and activation of steroid receptors depend considerably on the enzymatic activities involved in local pre-receptor synthesis and the metabolism of the steroids. Several enzymes in particular, steroid dehydrogenases have been shown to participate in this process. Here we report the isolation of 20-hydroxysteroid dehydrogenase (ch20HSD) cDNA from chicken intestine and the distribution of ch20HSD mRNA and 20-reductase activity in various avian tissues. Using a reverse transcription PCR and comparison with the known sequences of mammalian 20betaHSDs, we have isolated a new ch20HSD cDNA. This cDNA predicted 276 amino acid residues that shared about 75% homology with mammalian 20betaHSD. Sequences specific to the short-chain dehydrogenase/reductase superfamily (SDR) were found, the Gly-X-X-X-Gly-X-Gly cofactor-binding motif (residues 11-17) and the catalytic activity motif Tyr-X-X-X-Lys (residues 193-197). The cDNA coding for ch20HSD was expressed in Escherichia coli by placing it under isopropylthiogalactoside (IPTG) inducible control. Both the IPTG cells of E. coli and the isolated recombinant protein reduced progesterone to 20-dihydroprogesterone, corticosterone to 20-dihydrocorticosterone and 5alpha-dihydrotestosterone to its 3-ol derivative. The 20-reductase and 3-reductase activities of ch20HSD catalyzed both 3alpha/beta- and 20alpha/20beta-epimers. The mRNA transcripts of ch20HSD were found in the kidney, colon, and testes; weaker expression was also found in the heart, ovaries, oviduct, brain, liver, and ileum. 20-Reductase activity has been proven in tissue slices of kidney, colon, ileum, liver, oviduct, testis, and ovary; whereas the activity was nearly absent in the heart and brain. A similar distribution of 20-reductase activity was found in tissue homogenates measured under V(max) conditions. These results suggest that chicken 20HSD is the latest member of the SDR superfamily to be found, is expressed in many avian tissues and whose precise role remains to be determined.

Intestinal inflammation modulates expression of 11beta-hydroxysteroid dehydrogenase in murine gut.

The effect of glucocorticoids is controlled at the pre-receptor level by the activity of 11beta-hydroxysteroid dehydrogenase (11HSD). The isoform 11HSD1 is an NADP+ -dependent oxidoreductase, usually reductase, that amplifies the action of glucocorticoids due to reduction of the biologically inactive 11-oxo derivatives cortisone and 11-dehydrocorticosterone to cortisol and corticosterone. The NAD+ -dependent isoform (11HSD2) is an oxidase that restrains the effect of hormones due to 11beta-oxidation of cortisol and corticosterone to their 11-oxo derivatives. Although the immunosuppressive and anti-inflammatory effects of glucocorticoids are well known, the relationship between inflammation and local metabolism of glucocorticoids is not well understood. In this study, we demonstrated that colitis induced by dextran sulfate sodium modulates colonic 11HSD1. Experimentally induced intestinal inflammation stimulated colonic NADP+ -dependent but not NAD+ -dependent 11HSD activity. Colonic 11HSD1 mRNA was increased, whereas 11HSD2 mRNA was not changed. Additional parallel studies revealed a similar pattern of 11HSD1 mRNA induction in mesenteric lymph nodes and intestinal intraepithelial lymphocytes, but not in spleen and peritoneal macrophages. These data suggest that inflammation modulates local metabolism of glucocorticoid and support the notion that pre-receptor regulation of endogenous corticosteroids might play a role in inflammatory processes.

Capillary electrophoresis of peptides and proteins with plug of Pluronic gel.

Electromigration capillary methods are promising techniques in proteomics and they are still under research. We used a partial filling approach, i.e. a combination of gel and non-gel separation mechanisms in a single dimension. We tried using an interesting gel, Pluronic F 127, which can be considered as a surfactant capable of self-association both with isotropic and anisotropic gels. The Pluronic was inserted inside the capillary as a plug at the start of the capillary, and it provided separation at the first time. Separation by this gel was achieved according to molecular weight and/or hydrophobicity. The applicability of this method was demonstrated in the separation of real samples-peptides arising from collagen after CNBr or collagenase cleavage and albumin after trypsin cleavage (peptide mapping). Some peptides and proteins were selectively retained by the Pluronic gel. These interactions with the gel did not depended on their molecular weight alone, but they probably depend on a combination of both principles. It was confirmed that capillary electrophoresis with Pluronic plug can give us another new separation option, complementary to free solution capillary electrophoresis. The CE method presented here, consisting of a partial filling approach with combine gel and non-gel separation mechanisms seemed to be a promising method for the separation of complex mixtures of peptides.

Corticosterone metabolism in chicken tissues: evidence for tissue-specific distribution of steroid dehydrogenases.

Glucocorticoids influence the function of numerous tissues. Although there are a very large number of studies that have investigated the local metabolism of glucocorticoids in mammals, the knowledge of this metabolism in birds is limited. The local concentration of corticosterone is critical for both glucocorticoid- and mineralocorticoid-dependent activity, and we have therefore carried out studies of corticosterone metabolism in various chicken organs. It was found that corticosterone was metabolized to 20-dihydrocorticosterone, and in some tissues also to 11-dehydrocorticosterone and 11-dehydro-20-dihydrocorticosterone. The activity of 20-hydroxysteroid dehydrogenase (20HSD), responsible for the transformation of corticosterone to 20-hydroxy derivatives, was abundant in the kidney and intestine, with lower levels in the liver and testis. Low levels of 20HSD were detected in the brain and ovaries. In contrast, 11-hydroxysteroid dehydrogenase (11HSD) activity was only found in the kidney and intestine. No activity was observed in the brain, testis, or ovaries. The treatment of chickens with estrogens stimulated 20HSD activity in the kidney, intestine, and oviduct and 11HSD activity in the liver and oviduct. Kinetic studies for corticosterone yielded an apparent Km for 11HSD in the nanomolar (Km = 21 +/- 5 nmol.l(-1)) and for 20HSD in the micromolar range (Km = 3.7 +/- 0.3 micromol.l(-1)). When progesterone or 5alpha-dihydrotestosterone were used instead of corticosterone, the tissues reduced the former to 20beta-dihydroprogesterone and the latter to both 5alpha,3alpha- and 5alpha,3beta-dihydrotestosterone. The data presents the first evidence for corticosterone metabolism via 11beta-, 3alpha/3beta-, and 20beta-hydroxysteroid dehydrogenases in various chicken organs and provide support for the theory of prereceptor modulation of glucocorticoid signals in avian tissues.

Separation of poly(amidoamine) (PAMAM) dendrimer generations by dynamic coating capillary electrophoresis.

The separation of compounds possessing amino groups (peptides, proteins, polyamino compounds) by capillary zone electrophoresis suffers from the interaction (sticking) of these solutes with the capillary wall. This sticking can result in the absence or incomplete separation of compounds or even in their retention in the capillary. Polyamidoamine (PAMAM) dendrimers are a class of spherical polymers with primary amino groups at the surface. These compounds can be separated reasonably well at acidic pH but not at neutral pH. A new method based on the dynamic coating of the capillary was developed for the separation of these compounds at pH 7.4. The method comprises separation in a fused-silica capillary (57 cm total length, 50 cm to the detector, ID 75 microm) and a background electrolyte consisting of a Tris-phosphate buffer (50 mmol/L, pH 7.4) and 0.05% (w/v) polyethyleneimine. This system is suitable for the separation of 7 generations of dendrimers (generations 0-6). The dynamic coating agent (polyethyleneimine) also improves the separation at acid pH.

Corticosterone transfer and metabolism in the dually perfused rat placenta: effect of 11beta-hydroxysteroid dehydrogenase type 2.

Although rat is the most widely used model of glucocorticoid programming of the fetus, the role of rat placental 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) in the transplacental pharmacokinetics of the naturally occurring glucocorticoid, corticosterone, has not yet been fully elucidated. In this study, expression of 11beta-HSD2 in the rat placenta on two different gestation days (16 and 22) was examined using quantitative RT-PCR and Western blotting, and dually perfused rat term placenta was employed to evaluate its functional capacity to transfer and metabolize corticosterone. Marked decrease in placental expression of 11beta-HSD2 toward term was observed on both mRNA and protein levels. In perfusion studies, increasing maternal corticosterone concentration from 3 to 200 nM resulted in the fall of 11beta-HSD2 conversion capacity from 64.3 to 16.3%, respectively. Enzyme saturation occurred at about 50 nM substrate concentration. When delivering corticosterone (3 or 100 nM) from the fetal side, a similar decline of 11beta-HSD2 conversion capacity was observed (66.5% and 48.5%, respectively). Addition of carbenoxolone (10 or 100 microM), a non-specific 11beta-HSD inhibitor, to maternal perfusate decreased conversion capacity from 66.7 to 12.6 or 8.1%, respectively. Similarly potent inhibitory effect was observed in feto-maternal studies. Neither saturation nor inhibition of 11beta-HSD2 was associated with transformation of corticosterone in metabolites other than 11-dehydrocorticosterone. These data suggest that 11beta-HSD2 is the principal enzyme controlling transplacental passage of corticosterone in rats and is able to eliminate corticosterone in both maternal and fetal circulations.

Corticosteroid regulation of colonic ion transport during postnatal development: methods for corticosteroid analysis.

Many mammalian species including human are immature at birth and undergo major developmental changes during suckling and weaning period. This problem is also conspicuous for the gastrointestinal tract that undergoes abrupt transitions coinciding with birth and weaning. This review deals with the maturation of ion transport functions in colon, the intestinal segment that plays an important role in sodium and potassium absorption and secretion. The purpose of the present review is to summarize the mechanism of sodium and potassium transport pathways and show how these transport processes change postnatally and how hormones, particularly corticosteroids, modify the pattern of development. Finally we describe some of the ways, how to analyze corticosteroid metabolism in target tissue.

Capillary electrophoretic separation of proteins and peptides by ion-pairing with heptanesulfonic acid.

Heptanesulfonic acid as ion-pairing agent was used for the separation of mixtures of low and high molecular mass peptides/proteins by capillary electrophoresis. The separation conditions used were: capillary 37 cm (30 cm to the detector) x 75 microm i.d., voltage 10 kV, phosphate buffer 50 mmol/l, ion-pairing agent heptanesulfonic acid at three different concentrations, namely, 0, 20 or 100 mmol/l, pH 2.5. The separation reflected the ion-pairing equilibria between the ion-pairing agent and the peptide/protein analytes. The influence of ion-pairing on sample mobility (running time) was more pronounced in case of the higher-molecular peptides as compared to the low molecular ones. This difference offers the possibility to separate low and high molecular peptides/proteins that under the absence of the ion-pairing agent would co-migrate. The principle of this approach was demonstrated on a randomly selected set of peptides/proteins; the practical applicability was demonstrated on a set of CNBr peptides arising from a naturally occurring mixture of collagen types I and III.

Preparative procedures and purity assessment of collagen proteins.

Collagens represent a large family (25 members identified so far) of closely related proteins. While the preparative procedures for the members that are ubiquitous and present in tissues in large quantities (typically fibre and network forming collagens types I, II, III, IV and V) are well established, the procedures for more recently discovered minor collagen types, namely those possessing large non-collagenous domain(s) in their molecule, are mostly micropreparative and for some collagenous proteins even do not exist. The reason is that the proof of their existence is based on immunochemical staining of tissue slices and nucleic database searching. Methods of preparation and identification of constituting alpha-polypeptide chains as well as collagenous and non-collagenous domains are also reviewed. Methods for revealing non-enzymatic posttranslational modifications (particularly of the fibre forming collagen types) are briefly described as well.

Evaluation of peptide electropherograms by multivariate mathematical-statistical methods. I. Principal component analysis.

Depository effects in slowly metabolised proteins, typically glycation or the estimation of products arising from the reaction of unsaturated long-chain-fatty acid metabolites (possessing aldehydic groups) are very difficult to assess owing to their extremely low concentration in the protein matrix. In order to reveal such alterations we applied deep enzymatic fragmentation resulting in a set of small peptides, which, if modified, are likely to change their electrophoretic properties and can be visualised on the resulting profile. Peptide maps of collagen (a mixture of collagen types I and III digested by bacterial collagenase) were applied as the model protein structure for detecting the nonenzymatic posttranslational changes originating during various physiological conditions like high fructose diet and hypertriglyceridemic state. Capillary electrophoresis in acidic media (sodium phosphate buffer, pH 2.5) was used as the separation method capable of (partial) separation of over 60 peptide peaks. Two to 13 changes were revealed in the profiles obtained reflecting the physiological conditions of the animals tested. Combination of peptide profiling with subsequent t-test evaluation of individual peak areas and principal component analysis based on cumulative peak areas of individual sections of the electropherograms allowed to determine in which section (part) of the electropherogram the physiological state indicating changes occurred. Simultaneously it was possible to reveal the qualitative differences between the four physiological regimes investigated (i.e., which regime affects the collagen molecules most and which affects them least). The approach can be used as guidance for targeted preseparation of the very complex peptide mixture.

Mixtures of nonionic and anionic surfactants: interactions with low-molecular-mass homopeptides.

The interaction between low molecular-mass homopeptides and mixtures of nonionic and anionic surfactants has been assessed by using reversed-phase thin-layer chromatography. The relative strength of interaction for mixtures of sodium dodecylsulfate and tridecylalcohol diglycolate (GNX) at the molar ratios of 8:2, 6:4, 4:6 and 2:8 has been calculated and its relationship with the physicochemical parameters (number of amino acid units, hydrophobicity, side chain bulkiness, electronic characteristics) of peptides has been computed by stepwise regression analysis. Each peptide interacted with each surfactant mixture the strength of interaction markedly depending on both the character of the peptide and the composition of the surfactant mixture. The hydrophobicity and electronic properties of the amino acid units exerted the highest influence on the strength of interaction at the highest concentration of the nonionic surfactant (GNX) whereas the number of amino acid units in the peptide molecule and the bulkiness of the amino acid side chain governed the strength of interaction at the lowest concentration of GNX.

Binding of substituted phenol and aniline derivatives to the corn protein zein studied by high-performance liquid chromatography.

The interaction of 12 substituted phenol, three aminophenol and four substituted aniline derivatives with the corn protein zein was studied on zein-coated silica and alumina stationary phases by high-performance liquid chromatography using bidistilled water as mobile phase. Solutes were eluted from the zein-coated supports with different retention times indicating that they bind to the protein with different forces. They were more strongly retained on silica-based than on alumina-based support proving that the original adsorptive character of the support remains even after impregnation. The retention of solutes on both zein-coated stationary phases significantly depended on the steric and electronic parameters of solutes and was independent of the calculated and measured lipophilicity parameters, indicating that hydrophobic forces are not included in the interaction of zein with these class of solutes. It has been concluded that the interaction is governed by steric and electrostatic forces.

Effect of molecular parameters on the binding of phenoxyacetic acid derivatives to albumins.

The interaction of 12 phenoxyacetic acid derivatives with human and serum albumin as well as with egg albumin was studied by charge-transfer reversed-phase (RP) thin-layer chromatography (TLC) and the relative strength of interaction was calculated. Each phenoxyacetic acid derivative interacted with human and bovine serum albumins whereas no interaction was observed with egg albumin. Stepwise regression analysis proved that the lipophilicity of the derivatives exert a significant impact on their capacity to bind to serum albumins. This result supports the hypothesis that the binding of phenoxyacetic acid derivatives to albumins may involve hydrophobic forces occurring between the corresponding apolar substructures of these derivatives and the amino acid side chains.