A predictive model for astringency based on in vitro interactions between salivary proteins and (-)-Epigallocatechin gallate.

Astringency is an important quality attribute of green tea infusion, and (-)-Epigallocatechin gallate (EGCG) is the main contributor to astringency. Turbidity was used to predict the intensity of astringency for EGCG. The interactions between the selected proteins and EGCG, and the impacts of temperature, pH, protein structure, and EGCG concentration were studied. Mucin was selected as the protein in study for the prediction of EGCG astringency intensity. A predictive model (R2 = 0.994) was developed based on the relationship between the astringency of EGCG and the turbidity of EGCG/mucin mixtures at pH 5.0 and 37 °C. The fluorescence quenching analyses showed the interactions between EGCG and the selected proteins, which induced the reversible protein molecule conformational changes. The interactions were considered as the main reason that causes the astringency of tea infusions. The results provided a biochemical approach to explore the sensory qualities of green tea.

Inhibition Mechanisms of Wine Polysaccharides on Salivary Protein Precipitation.

In this work, high-performance liquid chromatography, fluorescence quenching, nephelometry, and sodium dodecyl sulfate polyacrylamide gel electrophoresis were used to study the effect of polysaccharides naturally present in wine [rhamnogalacturonan II (RG II) and arabinogalactan proteins (AGPs)] on the interaction between salivary proteins (SP) together present in saliva and tannins (punicalagin (PNG) and procyanidin B2). In general, the RG II fraction was more efficient to inhibit SP precipitation by tannins, especially for acidic proline-rich proteins (aPRPs) and statherin/P-B peptide, than AGPs. The RG II fraction can act mainly by a competition mechanism in which polysaccharides compete by tannin binding. However, in the presence of Na+ ions in solution, no RG II effect was observed on SP-tannin interactions. On the other hand, dependent upon the saliva sample as well as the tannin studied, AGPs can act by both mechanisms, competition and ternary (formation of a ternary complex with SP-tannin aggregates enhancing their solubility).

Analytical ultracentrifugation in saliva research: Impact of green tea astringency and its significance on the in-vivo aroma release.

Current saliva testing methods rely on cutting edge yet expensive techniques for the detection and analysis of genetic material, proteins and biomarkers for clinical use. However, these techniques are limited in scope and often cannot be used with complex food materials. We propose an efficient ex-vivo tool for evaluating biologically relevant interactions between food components and human saliva using sedimentation velocity analytical ultracentrifugation (SV-AUC). We evaluated macromolecular content from "unstimulated" (US) and "stimulated" (SS) samples pooled from 5 healthy volunteers. Over 90% of total saliva protein consisted of α-amylase and mucin, and up to 10% was secretory immunoglobulin A (SIgA). It was shown that α-amylase concentration increased upon parafilm stimulation, which lead to a decrease in the viscosity of saliva. Then, we used a simple food system (green tea) to evaluate changes in the salivary protein content caused by green tea polyphenols. It was found that aroma release from green tea is highly influenced by interactions between α-amylase and polyphenol epigallocatechin 3-gallate (EGCG). This interaction was found to increase the viscosity of the salivary bulk, suggested to contribute to astringency, and increased the concentrations of ß-ionone, benzaldehyde and isovaleraldehyde (P < 0.01), suggested to play a significant role in the characteristic flavour of green tea.

Characterization of a glycine-rich protein from Rhipicephalus microplus: tissue expression, gene silencing and immune recognition.

Salivary molecules, as glycine-rich proteins (GRPs), are essential to tick attachment and feeding on the host and are suggested to be involved in the host's immune system evasion, therefore representing natural candidates in the search for protective vaccine antigens. This work shows the molecular characterization of a GRP from Rhipicephalus microplus (RmGRP). The cDNA and putative amino acid sequences were analysed, as well as the transcription level in tick tissues/developmental stages, showing the highest levels of gene expression in 1-day-old larvae and salivary glands of fully engorged females. RmGRP gene silencing resulted in a lower hatching rate of larvae from treated females. In addition, recombinant RmGRP (rRmGRP) was recognized by sera from naturally and experimentally infested bovines, displaying considerable differences among the individuals tested. rRmGRP was recognized by anti-saliva and anti-salivary glands sera, while anti-rRmGRP serum recognized RmGRP in saliva and salivary glands, indicating its secretion into the host. The data collected indicate that RmGRP may present roles other than in the tick-host relationship, especially in embryo development. In addition, the high expression in adult females, antigenicity and presence of shared characteristics with other tick protective GRPs turns RmGRP a potential candidate to compose an anti-tick vaccine cocktail.

HPLC method for measurement of human salivary α-amylase inhibition by aqueous plant extracts.

Control of hyperglycemia is an important treatment in metabolic disorders such as type II diabetes and obesity. α-Amylase, as the first enzyme of glucose release from dietary polysaccharides, is a potential target to identify new sources of novel anti-obesity and anti-diabetic drugs. In this work, different herbal extracts as α-amylase inhibitors were studied by measuring the rate of the cleavage of a maltooligomer substrate 2-chloro-4-nitrophenyl-ß-D-maltoheptoside. Measurement of chromophore containing products after reversed phase HPLC separation was used for α-amylase activity measurement. Rates of hydrolysis catalysed by human salivary α-amylase were determined in the presence and absence of lyophilised water extracts of eleven herbs. Remarkable bioactivities were found for extracts of Cinnamomum zeylanicum Blume (bark), Camellia sinensis L. (leaf), Ribes nigrum L. (leaf), Laurus nobilis L. (leaf), Vaccinium macrocarpon Aiton (fruit) and Syzygium aromaticum L. (bud). Determined IC50 values were in 0.017-41 µg/ml range for these six selected plant extracts. Our results confirm the applicability of this HPLC-based method for the quick and reliable comparison of plants as α-amylase inhibitors.

Effect of Astringent Stimuli on Salivary Protein Interactions Elucidated by Complementary Proteomics Approaches.

The interaction of astringent substances with salivary proteins, which results in protein precipitation, is considered a key event in the molecular mechanism underlying the oral sensation of puckering astringency. As the chemical nature of orally active astringents is diverse and the knowledge of their interactions with salivary proteins rather fragmentary, human whole saliva samples were incubated with suprathreshold and isointensity solutions of the astringent polyphenol (-)-epigallocatechin gallate, the multivalent metal salt iron(III) sulfate, the amino-functionalized polysaccharide chitosan, and the basic protein lysozyme. After separation of the precipitated proteins, the proteins affected by the astringents were identified and relatively quantified for the first time by complementary bottom-up and top-down mass spectrometry-based proteomics approaches. Major salivary target proteins, which may be involved in astringency perception, are reported here for each astringent stimulus.

Gold nanotheranostics: photothermal therapy and imaging of Mucin 7 conjugated antibody nanoparticles for urothelial cancer.

OBJECTIVE: To kill urothelial cancer cells while preserving healthy cells, this study used photothermal therapy (PTT). PTT techniques target urothelial cancer cells using gold nanoparticles (GNPs) and a green light laser. MATERIALS AND METHODS: The GNPs were conjugated with anti-Mucin 7 antibodies, which acted as a probe for targeting tumor cells. Conjugated GNPs were exposed to a green light laser (532 nm) with sufficient thermal energy to kill the transitional cell carcinomas (TCCs). RESULTS: According to our results, nanoparticles conjugated with Mucin 7 antibodies damaged all types of cancer cells (MBT2, T24, 9202, and 8301) at relatively low energy levels (i.e., 500 laser shots at 10 W/cm(2) in power, 1.6 Hz in frequency, and 300 ms in duration). Nonconjugated nanoparticles required 30 W/cm(2) or more to achieve the same effect. Cell damage was directly related to irradiation time and applied laser energy. CONCLUSIONS: The minimally invasive PTT procedure combined with Mucin 7 targeted GNPs is able to kill cancer cells and preserve healthy cells. The success of this treatment technique can likely be attributed to the lower amount of energy required to kill targeted cancer cells compared with that required to kill nontargeted cancer cells. Our in vitro pilot study yielded promising results; however, additional animal studies are required to confirm these findings.

A preliminary characterization of Aglianico (Vitis vinifera L. cv.) grape proanthocyanidins and evaluation of their reactivity towards salivary proteins.

The flavan-3-ol and proanthocyanidin composition of Aglianico seeds and skins were for the first time determined by HPLC-MS in comparison with the international grapes Merlot and Cabernet Sauvignon. Monomers [(+)-catechin C, (-)-epicatechin EC, (-)-epicatechin-3-O-gallate, ECG] and oligomers [B1, B2, B3, B4 dimers and trimer C1] were identified and quantified in grape extracts. In order to evaluate the reactivity towards salivary proteins of model wine solutions of seeds and skins monomeric/oligomeric and polymeric fractions, the Saliva Precipitation Index (SPI) was carried out. Fractions were also analyzed for their mean degree of polymerization (mDP), percentage of galloylation (%G) and of prodelphinidin (%P) by phloroglucinolysis. Aglianico was the most effective in precipitating proteins than Merlot and Cabernet Sauvignon, mainly for the high percentage of galloylation of grape fractions. The mDP and the percentage of ECG in terminal units resulted to significantly contribute to the precipitation of salivary proteins by grape proanthocyanidins.

Preparation of nitrophorin 7(Δ1-3) from Rhodnius prolixus without start-methionine using recombinant expression in Escherichia coli.

The heterologous recombinant expression of proteins in Escherichia coli without start-methionine is a common problem. The nitrophorin 7 heme properties and function strongly depend on the accurate N-terminal amino acid sequence. Leading protein expression into the periplasm by fusion with the leader peptide pelB yields functional protein; however, the folded protein sticks to the cell debris. Therefore, the periplasmic fraction was dissolved in guanidinium chloride and folded by a drop-in method. Separation from impurities including residual pelB-nitrophorin 7 required establishing an unconventional chromatographic technique using calcium-loaded Chelating Sepharose as cation exchanger and elution by a linear CaCl2 gradient.

Using the RosettaSurface algorithm to predict protein structure at mineral surfaces.

Determination of protein structure on mineral surfaces is necessary to understand biomineralization processes toward better treatment of biomineralization diseases and design of novel protein-synthesized materials. To date, limited atomic-resolution data have hindered experimental structure determination for proteins on mineral surfaces. Molecular simulation represents a complementary approach. In this chapter, we review RosettaSurface, a computational structure prediction-based algorithm designed to broadly sample conformational space to identify low-energy structures. We summarize the computational approaches, the published applications, and the new releases of the code in the Rosetta 3 framework. In addition, we provide a protocol capture to demonstrate the practical steps to employ RosettaSurface. As an example, we provide input files and output data analysis for a previously unstudied mineralization protein, osteocalcin. Finally, we summarize ongoing challenges in energy function optimization and conformational searching and suggest that the fusion between experiment and calculation is the best route forward.

Marked increase in PROP taste responsiveness following oral supplementation with selected salivary proteins or their related free amino acids.

The genetic predisposition to taste 6-n-propylthiouracil (PROP) varies among individuals and is associated with salivary levels of Ps-1 and II-2 peptides, belonging to the basic proline-rich protein family (bPRP). We evaluated the role of these proteins and free amino acids that selectively interact with the PROP molecule, in modulating bitter taste responsiveness. Subjects were classified by their PROP taster status based on ratings of perceived taste intensity for PROP and NaCl solutions. Quantitative and qualitative determinations of Ps-1 and II-2 proteins in unstimulated saliva were performed by HPLC-ESI-MS analysis. Subjects rated PROP bitterness after supplementation with Ps-1 and II-2, and two amino acids (L-Arg and L-Lys) whose interaction with PROP was demonstrated by (1)H-NMR spectroscopy. ANOVA showed that salivary levels of II-2 and Ps-1 proteins were higher in unstimulated saliva of PROP super-tasters and medium tasters than in non-tasters. Supplementation of Ps-1 protein in individuals lacking it in saliva enhanced their PROP bitter taste responsiveness, and this effect was specific to the non-taster group.(1)H-NMR results showed that the interaction between PROP and L-Arg is stronger than that involving L-Lys, and taste experiments confirmed that oral supplementation with these two amino acids increased PROP bitterness intensity, more for L-Arg than for L-Lys. These data suggest that Ps-1 protein facilitates PROP bitter taste perception and identifies a role for free L-Arg and L-Lys in PROP tasting.

Unique thrombin inhibition mechanism by anophelin, an anticoagulant from the malaria vector.

Anopheles mosquitoes are vectors of malaria, a potentially fatal blood disease affecting half a billion humans worldwide. These blood-feeding insects include in their antihemostatic arsenal a potent thrombin inhibitor, the flexible and cysteine-less anophelin. Here, we present a thorough structure-and-function analysis of thrombin inhibition by anophelin, including the 2.3-Å crystal structure of the human thrombin·anophelin complex. Anophelin residues 32-61 are well-defined by electron density, completely occupying the long cleft between the active site and exosite I. However, in striking contrast to substrates, the D50-R53 anophelin tetrapeptide occupies the active site cleft of the enzyme, whereas the upstream residues A35-P45 shield the regulatory exosite I, defining a unique reverse-binding mode of an inhibitor to the target proteinase. The extensive interactions established, the disruption of thrombin's active site charge-relay system, and the insertion of residue R53 into the proteinase S(1) pocket in an orientation opposed to productive substrates explain anophelin's remarkable specificity and resistance to proteolysis by thrombin. Complementary biophysical and functional characterization of point mutants and truncated versions of anophelin unambiguously establish the molecular mechanism of action of this family of serine proteinase inhibitors (I77). These findings have implications for the design of novel antithrombotics.

Carbohydrates inhibit salivary proteins precipitation by condensed tannins.

Condensed tannins are a group of polyphenols that are associated with the astringency sensation, as they readily interact and precipitate salivary proteins. As this interaction is affected by carbohydrates, the aim of this work was to study the effect of some carbohydrates used in the food industry [arabic gum (AG), pectin, and poligalacturonic acid (PGA)] on the salivary proteins/grape seed procyanidins interaction. This was assessed monitoring the salivary proteins that remain soluble in the presence of condensed tannins with the addition of carbohydrates (HPLC) and analysis of the respective precipitates (SDS-PAGE). The results show that pectin was the most efficient in inhibiting protein/tannin precipitation, followed by AG and PGA. The results suggest that pectin and PGA exert their effect by formation of a ternary complex protein/polyphenol/carbohydrate, while AG competes with proteins for tannin binding (competition mechanism). The results also point out that both hydrophilic and hydrophobic interactions are important for the carbohydrate effects.

A systematic overview of the medicinal importance of sanguivorous leeches.

Leeches are a class of segmented invertebrates, known for their blood-feeding habits and used in phlebotomy to treat various ailments since antiquity. In Europe, medicinal leeches have recently been rediscovered and are used by maxillofacial and other microsurgeons to aid salvage of compromised venous engorged tissue and amputations, such as digits, ears, and nasal tips. Because of their important salivary components, blood-sucking (sanguivorous) leeches, such as Hirudo medicinalis and related species, have engendered great interest from pharmaceutical companies searching for anticoagulants to prevent blood clotting during microsurgeries. Scientific research reveals that the beneficial effects of leeching, in addition to decongestion, include injection of a cocktail of several medicinally useful bioactive molecules present in their saliva. Owing to its therapeutic potential, the research is continuing as many new salivary compounds are being isolated and synthesized.

Salivary transcriptome of the North American medicinal leech, Macrobdella decora.

A variety of bioactive proteins from medicinal leeches, like species of Hirudo , have been characterized and evaluated for their potential therapeutic biomedical properties. However, there has not previously been a comprehensive attempt to fully characterize the salivary transcriptome of a medicinal leech that would allow a clearer understanding of the suite of polypeptides employed by these sanguivorous annelids and provide insights regarding their evolutionary origins. An Expressed Sequence Tag (EST) library-based analysis of the salivary transcriptome of the North American medicinal leech, Macrobdella decora, reveals a complex cocktail of anticoagulants and other bioactive secreted proteins not previously known to exist in a single leech. Transcripts were identified that correspond to each of saratin, bdellin, destabilase, hirudin, decorsin, endoglucoronidase, antistatin, and eglin, as well as to other previously uncharacterized predicted serine protease inhibitors, lectoxin-like c-type lectins, ficolin, disintegrins and histidine-rich proteins. This work provides a lens into the richness of bioactive polypeptides that are associated with sanguivory. In the context of a well-characterized molecular phylogeny of leeches, the results allow for preliminary evaluation of the relative evolutionary origins and historical conservation of leech salivary components. The goal of identifying evolutionarily significant residues associated with biomedically significant phenomena implies continued insights from a broader sampling of blood-feeding leech salivary transcriptomes.

Human saliva forms a complex film structure on alumina surfaces.

Films formed from saliva on surfaces are important for the maintenance of oral health and integrity by protection against chemical and/or biological agents. The aim of the present study was to investigate adsorbed amounts, thickness, and structure of films formed from human whole saliva on alumina surfaces by means of in situ ellipsometry, neutron reflectivity, and atomic force microscopy. Alumina (Al2O3, synthetic sapphire) is a relevant and interesting substrate for saliva adsorption studies as it has an isoelectric point close to that of tooth enamel. The results showed that saliva adsorbs rapidly on alumina. The film could be modeled in two layers: an inner and dense thin region that forms a uniform layer and an outer, more diffuse and thicker region that protrudes toward the bulk of the solution. The film morphology described a uniformly covering dense layer and a second outer layer containing polydisperse adsorbed macromolecules or aggregates.

Influence of wine pectic polysaccharides on the interactions between condensed tannins and salivary proteins.

Alpha-amylase, a major human salivary protein, and IB8c, a representative of the proline-rich proteins, were obtained by isolation from saliva and by solid-phase synthesis, respectively. The interactions between these proteins and condensed tannins isolated from grape seeds were studied at different protein and tannin concentrations by measuring their aggregation. Pectic polysaccharides were isolated from wine, and their effect on protein tannin aggregation was assessed. The results presented in this study showed that the most acidic fractions of arabinogalactan proteins have the ability to inhibit the formation of aggregates between the grape seed tannins and the two different salivary proteins. Rhamnogalacturonan II has the same ability toward alpha-amylase but not IB8c under the conditions of the present study. Polysaccharides show effects at concentrations at which they are present in wine, which could mean an influence in wine astringency. The interaction between condensed tannins and alpha-amylase is differently affected by ionic strength when compared with IB8c.

A glycine- and glutamate-rich protein is female salivary gland-specific and abundant in the malaria vector Anopheles dirus B (Diptera: Culicidae).

Before transmission, malaria parasites reside in the salivary glands of their female mosquito hosts. Saliva proteins assist in blood feeding and also may influence the ability of mosquitoes to transmit malaria. We attempted to identify and isolate cDNAs encoding proteins expressed at a high level in the salivary glands of a malaria vector, Anopheles dirus B Peyton and Harrison (= An. cracens) (Diptera: Culicidae). A major protein with an estimated molecular mass of 35 kDa and an isoelectric point (pI) of approximately 4 was detected on a two-dimensional (2D) gel. Internal peptide sequences of the protein spot showed high similarity to sequences present in the conserved C-terminal domain of glycine- and glutamate (GE)-rich proteins. A full-length cDNA encoding this protein was isolated from a salivary gland cDNA library of female An. dirus B. The cDNA encoded a 256-residue protein with a calculated molecular mass of 25.4 kDa and a pI of 3.9. BLAST analysis confirmed that it is a member of the GE-rich family. Compositional and sequence analysis of this and other family members revealed a highly acidic N-terminal region of variable length and low sequence conservation and a well conserved C-terminal domain containing 10 identical residues across the 13 known members of the gene family in mosquitoes. The An. dirus B GE-rich transcript was detected by reverse transcription-polymerase chain reaction (PCR) only in the female salivary glands, indicating that this protein is female saliva-specific. The GE-rich proteins may function as a salivary lubricant to facilitate blood feeding.

Tick histamine-binding proteins and related lipocalins: potential as therapeutic agents.

Tick histamine-binding proteins bind histamine with high affinity and specificity. This is attained by a novel binding mechanism, whereby histamine is sequestered within a binding cavity of the lipocalin fold. The histamine binding proteins and related protein family members are currently under investigation as potential therapeutic agents for the treatment of various diseases, including conjunctivitis, allergic rhinitis, carcinoid syndrome and rheumatoid arthritis. While these proteins show great therapeutic potential, they are part of a diverse family of tick lipocalin proteins, some of which have been implicated in tick-host rejection and host pathogenesis. As such, the therapeutic mining of tick lipocalins should be considered within the framework of the rest of the family.

In vitro studies on the effect of sodium tripolyphosphate on the interactions of stain and salivary protein with hydroxyapatite.

OBJECTIVES: To study properties of sodium tripolyphosphate (STP) relevant to inhibition or removal of dental stain in vitro. METHODS: The effects of STP and other phosphates on adsorption of a dietary chromogen (black tea polyphenol) and salivary protein to hydroxyapatite (HA) powder were studied by analysing loss of protein or tea stain from solutions mixed with HA or HA pre-treated with the test agents. The effects on desorption of protein and stain from HA were studied by analysis of water or solutions of test agents mixed with HA or HA pre-treated with saliva or tea solution. RESULTS: At concentrations and pH representative of those likely to occur in the mouth, STP inhibited adsorption of salivary protein and black tea polyphenol to, and desorbed these substances from, HA surfaces. Adsorption and desorption of protein and stain were not influenced by pH of the STP solutions but adsorption varied with concentration. STP showed equivalent effectiveness with respect to salivary protein adsorption and desorption as a longer-chain condensed phosphate. The inhibitory activity of HA-bound STP on adsorption of salivary protein and stain resisted extensive washing. CONCLUSIONS: STP is likely to be an effective agent for inhibiting and removing dental stain, whether bound directly to mineralised surfaces or indirectly via salivary pellicle.