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

Exposure to reversine affects the chondrocyte morphology and phenotype in vitro

Articular chondrocytes derived from osteoarthritic tissues (OA HAC) show a severely reduced chondrogenic commitment. This impairment undermines their use for tissue-engineered cartilage repair, which relies on cell proliferation and growth to meet therapeutic needs, but also on efficient cell plasticity to recover the chondrogenic phenotype. Reversine (Rev), a 2,6-disubstituted purine inhibitor of spindle-assembly checkpoints, was described to convert differentiated mesenchymal cells to their undifferentiated precursors. We hypothesized that Rev exposure could divert OA HAC to more plastic cells, re-boosting their subsequent commitment. HAC were enzymatically released from OA cartilage specimens, expanded for 2 weeks and treated with 5 \u3bcm Rev in dimethylsulphoxide (DMSO) or with DMSO alone for 6 days. Cell growth was assessed using the AlamarBlueTM assay. Cytoskeletal structure, endoproliferation and caspase-3-immunopositivity were assayed by epifluorescence microscopy. The OA HAC chondrogenic performance was evaluated by quantitative reverse transcription-polymerase chain reaction (RT-PCR) for glyceraldehyde-3-phosphate dehydrogenase, Sox9, Aggrecan (Agg), type II collagen (Col2), Ki67, cyclinD1, transforming growth factor-\u3b21 (TGF-\u3b21), -2 and -3, interleukin-1\u3b2 (IL-1\u3b2) and -6 , SMAD3 and -7, and vascular endothelial growth factor. Rev-treated OA HAC recovered polygonal morphology and reduced Ki67 expression and proliferation. Cell-cycle impairment accounted for altered cytoskeletal organization, endoproliferation and apoptosis, whereas a compensatory mechanism sustained the increased cyclinD1 transcript levels. Sox9, Agg and TGFs were overexpressed, but not Col2. IL transcripts were massively downregulated. These events were dose-related and transient. Overall, in spite of a higher Rev-induced transcriptional activity for extracellular matrix components and in spite of a Rev-treated cell phenotype closer to that of the three-dimensional native articular chondrocyte, Rev effects seem unleashed from a full regained chondrogenic potential

Significant modifications of the salivary proteome potentially associated with complications of Down syndrome revealed by top-down proteomics

People with Down syndrome, a frequent genetic disorder in humans, have increased risk of health problems associated with this condition. One clinical feature of Down syndrome is the increased prevalence and severity of periodontal disease in comparison with the general population. Because saliva plays an important role in maintaining oral health, in the present study the salivary proteome of Down syndrome subjects was investigated to explore modifications with respect to healthy subjects. Whole saliva of 36 Down syndrome subjects, divided in the age groups 10-17 yr and 18-50 yr, was analyzed by a top-down proteomic approach, based on the high performance liquid chromatography-electrospray ionization-MS analysis of the intact proteins and peptides, and the qualitative and quantitative profiles were compared with sex- and age-matched control groups. The results showed the following interesting features: 1) as opposed to controls, in Down syndrome subjects the concentration of the major salivary proteins of gland origin did not increase with age; as a consequence concentration of acidic proline rich proteins and S cystatins were found significantly reduced in older Down syndrome subjects with respect to matched controls; 2) levels of the antimicrobial α-defensins 1 and 2 and histatins 3 and 5 were significantly increased in whole saliva of older Down syndrome subjects with respect to controls; 3) S100A7, S100A8, and S100A12 levels were significantly increased in whole saliva of Down syndrome subjects in comparison with controls. The increased level of S100A7 and S100A12 may be of particular interest as a biomarker of early onset Alzheimer's disease, which is frequently associated with Down syndrome

Atherosclerosis, dyslipidemia, and inflammation: the significant role of polyunsaturated fatty acids

Phospholipids play an essential role in cell membrane structure and function. The length and number of double bonds of fatty acids in membrane phospholipids are main determinants of fluidity, transport systems, activity of membrane-bound enzymes, and susceptibility to lipid peroxidation. The fatty acid profile of serum lipids, especially the phospholipids, reflects the fatty acid composition of cell membranes. Moreover, long-chain n-3 polyunsatured fatty acids decrease very-low-density lipoprotein assembly and secretion reducing triacylglycerol production. N-6 and n-3 polyunsatured fatty acids are the precursors of signalling molecules, termed “eicosanoids,” which play an important role in the regulation of inflammation. Eicosanoids derived from n-6 polyunsatured fatty acids have proinflammatory actions, while eicosanoids derived from n-3 polyunsatured fatty acids have antiinflammatory ones. Previous studies showed that inflammation contributes to both the onset and progression of atherosclerosis: actually, atherosclerosis is predominantly a chronic low-grade inflammatory disease of the vessel wall. Several studies suggested the relationship between long-chain n-3 polyunsaturated fatty acids and inflammation, showing that fatty acids may decrease endothelial activation and affect eicosanoid metabolis

Novel injectable gel (system) as a vehicle for human articular chondrocytes in cartilage tissue regeneration

We developed a novel injectable carrageenan/fibrin/hyaluronic acid-based hydrogel with in situ gelling properties to be seeded with chondrogenic cells and used for cartilage tissue engineering applications. We first analysed the distribution within the hydrogel construct and the phenotype of human articular chondrocytes (HACs) cultured for 3 weeks in vitro. We observed a statistically significant increase in the cell number during the first 2 weeks and maintenance of cell viability throughout the cell culture, together with the deposition/formation of a cartilage-specific extracellular matrix (ECM). Taking advantage of a new in vivo model that allows the integration between newly formed and preexisting cartilage in immunodeficient mice to be investigated, we showed that injectable hydrogel seeded with human articular chondrocytes was able to regenerate and repair an experimentally made lesion in bovine articular cartilage, thus demonstrating the potential of this novel cell delivery system for cartilage tissue engineering.The authors are grateful to Recco orthopaedic staff members for the collaboration and patients for bioptic material donation as well as to Mrs Daniela Marubbi for histological assistance. This work was supported by funds from the Italian MUR (FIRB-Tissuenet project), the European Union-funded STREP project, HIPPOCRATES (Grant No. NMP3-CT-2003-505758) and the European NoE EXPERTISSUES project (Grant No. NMP3-CT-2004-500283)

Performance of the galactomannan antigen detection test in the diagnosis of invasive aspergillosis in children with cancer or undergoing haemopoietic stem cell transplantation

AbstractSerum galactomannan (GM) antigen detection is not recommended for defining invasive aspergillosis (IA) in children undergoing aggressive chemotherapy or allogeneic haemopoietic stem cell transplantation (HSCT). The ability of the GM test to identify IA in children was retrospectively evaluated in a cohort of children. Test performance was evaluated on samples that were collected during 195 periods at risk of IA. Proven IA was diagnosed in seven periods, all with positive GM test results (true positives, 4%), and possible IA was diagnosed in 15 periods, all with negative GM test results (false negatives, 8%). The test result was positive with negative microbiological, histological and clinical features in three periods (false positives, 1%), and in 170 periods it was negative with negative microbiological, histological and clinical features (true negatives, 87%). The sensitivity was 0.32 and the specificity was 0.98; the positive predictive value was 0.70 and the negative predictive value was 0.92. The efficiency of the test was 0.91, the positive likelihood ratio was 18.3, and the negative likelihood ratio was 1.4. The probability of missing an IA because of a negative test result was 0.03. Test performance proved to be better during at-risk periods following chemotherapy than in periods following allogeneic HSCT. The GM assay is useful for identifying periods of IA in children undergoing aggressive chemotherapy or allogeneic HSCT

Dose-dependent effects of L-Arginine on PROP bitterness intensity and latency and characteristics of the chemical interaction between PROP and L-Arginine

Genetic variation in the ability to taste the bitterness of 6-n-propylthiouracil (PROP) is a complex trait that has been used to predict food preferences and eating habits. PROP tasting is primarily controlled by polymorphisms in the TAS2R38 gene. However, a variety of factors are known to modify the phenotype. Principle among them is the salivary protein Ps-1 belonging to the basic proline-rich protein family (bPRP). Recently, we showed that oral supplementation with Ps-1 as well as its related free amino acids (L-Arg and L-Lys) enhances PROP bitterness perception, especially for PROP non-tasters who have low salivary levels of Ps-1. Here, we show that salivary L-Arg levels are higher in PROP super-tasters compared to medium tasters and non-tasters, and that oral supplementation with free L-Arg enhances PROP bitterness intensity as well as reduces bitterness latency in a dose-dependent manner, particularly in individuals with low salivary levels of both free L-Arg and Ps-1 protein. Supplementation with L-Arg also enhanced the bitterness of caffeine. We also used 1H-NMR spectroscopy and quantum-mechanical calculations carried out by Density Functional Theory (DFT) to characterize the chemical interaction between free L-Arg and the PROP molecule. Results showed that the -NH2 terminal group of the L-ArgH+ side chain interacts with the carbonyl or thiocarbonyl groups of PROP by forming two hydrogen bonds with the resulting charged adduct. The formation of this PROP•ArgH+ hydrogen-bonded adduct could enhance bitterness intensity by increasing the solubility of PROP in saliva and its availability to receptor sites. Our data suggest that L-Arg could act as a 'carrier' of various bitter molecules in saliva

Proteomics of saliva: personal experience

The salivary proteome is a complex protein mixture resulting from the activity of salivary glands with the contribution of other components that form the oral environment such as oral tissues and micro-organisms. For diagnosis purposes, saliva collection has the great advantage of being an easy and non-invasive technique. Human saliva proteomics have proven to be a novel approach in the search for protein biomarkers for detection of different local and systemic diseases. Currently, more than 1400 salivary proteins have been identified. In the last few years, our research group has extensively studied the salivary proteomics in order to analyse the salivary composition, investigating the major families of proteins present in human and mammalian saliva, the post-translational modifications, the different contributions of glands, the physiological and pathological modifications of saliva. The aim of this report is to present our personal experience in salivary proteomics. In conclusion, salivary proteome analysis represents an important field both for diagnosis and monitoring of various diseases and could be considered a novel approach to prevention of various pathological conditions

A cascade of 24 histatins (histatin 3 fragments) in human saliva. Suggestions for a pre-secretory sequential cleavage pathway

The systematic search by tandem mass spectrometry of human saliva from four different subjects, of 136 possible fragments originated from histatin 3, allowed the detection of 24 different peptides. They include, with the exception of histatin 4, all the known histatin 3 fragments, namely histatins 5-12 and the peptides corresponding to 15-24, 26-32, 29-32 residues, and 13 new fragments corresponding to 1-11, 1-12, 1-13, 5-13, 6-11, 6-13, 7-11, 7-12, 7-13, 14-24, 14-25, 15-25, and 28-32 residues of histatin 3. On the contrary, none of 119 possible fragments of histatin 1, including histatin 2, was detected. The results suggest that the genesis of histatin 3-related peptides, being under the principal action of trypsin-like activities, is probably not a random process but rather follows a sequential fragmentation pathway. Lack of detection of C-terminal fragments, with the exception of 26-32, 28-32, and 29-32 fragments, suggested that arginine 25 should be the first cleavage site, generating histatin 6 and 26-32 fragments. The genesis of 28-32 and 29-32 fragments and histatin 5 should implicate a subsequent exo-protease action. Similarly, lack of detection of fragments having Lys-5 and Arg-6 at the N terminus and Arg-25 at the C terminus strongly suggested that sequences KRKF (11-14 residues) and AKR (4-6 residues) should be the second and the third cleavage sites, respectively. Lys-17 and Arg-22 are not cleaved at all

Dose-Dependent Effects of L-Arginine on PROP Bitterness Intensity and Latency and Characteristics of the Chemical Interaction between PROP and L-Arginine

Genetic variation in the ability to taste the bitterness of 6-n-propylthiouracil (PROP) is a complex trait that has been used to predict food preferences and eating habits. PROP tasting is primarily controlled by polymorphisms in the TAS2R38 gene. However, a variety of factors are known to modify the phenotype. Principle among them is the salivary protein Ps-1 belonging to the basic proline-rich protein family (bPRP). Recently, we showed that oral supplementation with Ps-1 as well as its related free amino acids (L-Arg and L-Lys) enhances PROP bitterness perception, especially for PROP non-tasters who have low salivary levels of Ps-1. Here, we show that salivary L-Arg levels are higher in PROP super-tasters compared to medium tasters and non-tasters, and that oral supplementation with free L-Arg enhances PROP bitterness intensity as well as reduces bitterness latency in a dose-dependent manner, particularly in individuals with low salivary levels of both free L-Arg and Ps-1 protein. Supplementation with L-Arg also enhanced the bitterness of caffeine. We also used 1H-NMR spectroscopy and quantum-mechanical calculations carried out by Density Functional Theory (DFT) to characterize the chemical interaction between free L-Arg and the PROP molecule. Results showed that the -NH2 terminal group of the L-ArgH+ side chain interacts with the carbonyl or thiocarbonyl groups of PROP by forming two hydrogen bonds with the resulting charged adduct. The formation of this PROP\u2022ArgH+ hydrogen-bonded adduct could enhance bitterness intensity by increasing the solubility of PROP in saliva and its availability to receptor sites. Our data suggest that L-Arg could act as a 'carrier' of various bitter molecules in saliva