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.     

Evaluation and characterization of fetal exposures to low frequency magnetic fields generated by laptop computers

Portable – or “laptop” – computers (LCs) are widely and increasingly used all over the world. Since LCs are often used in tight contact with the body even by pregnant women, fetal exposures to low frequency magnetic fields generated by these units can occur. LC emissions are usually characterized by complex waveforms and are often generated by the main AC power supply (when connected) and by the display power supply sub-system.In the present study, low frequency magnetic field emissions were measured for a set of five models of portable computers. For each of them, the magnetic flux density was characterized in terms not just of field amplitude, but also of the so called “weighted peak” (WP) index, introduced in the 2003 ICNIRP Statement on complex waveforms and confirmed in the 2010 ICNIRP Guidelines for low frequency fields. For the model of LC presenting the higher emission, a deeper analysis was also carried out, using numerical dosimetry techniques to calculate internal quantities (current density and in-situ electric field) with reference to a digital body model of a pregnant woman. Since internal quantities have complex waveforms too, the concept of WP index was extended to them, considering the ICNIRP basic restrictions defined in the 1998 Guidelines for the current density and in the 2010 Guidelines for the in-situ electric field. Induced quantities and WP indexes were computed using an appropriate original formulation of the well known Scalar Potential Finite Difference (SPFD) numerical method for electromagnetic dosimetry in quasi-static conditions.     

Norovirus detection in shellfish using two Real-Time RT-PCR methods

Shellfish are recognized as a potential vehicle of viral diseases. The aim of the present study was to determine the ability of two real-time RT-PCR methods (an in-house method and a commercial kit) for detecting Norovirus (NoV) belonging to genogroups GI and GII in shellfish. The analyses were performed both on a Norovirus Reference Panel (NRP), consisting of synthetic RNA, and on naturally contaminated mussels. For the experiments carried out on the NRP a statistically significant difference (?2=8.03) was shown between the results obtained by the two methods. The in-house real-time RT-PCR allowed the detection of all genotypes belonging to GI and GII, while the commercial kit was not suitable for the detection of the majority of the GI sequences constituting the panel. No significant difference was instead detected in the experiments carried out on shellfish, where the presence of GI was always concomitant with GII. Both methods were suitable for detection of NoV in shellfish, however the in-house real-time RT-PCR method had the advantage of differentiating GI and GII contamination. As regards the shellfish analysed, a considerable frequency of NoV contamination (34.4% of the samples) was detected, with a predominance of NoV GII.     

In Vitro and In Vivo Human Herpesvirus 8 Infection of Placenta

Herpesvirus infection of placenta may be harmful in pregnancy leading to disorders in fetal growth, premature delivery, miscarriage, or major congenital abnormalities. Although a correlation between human herpesvirus 8 (HHV-8) infection and abortion or low birth weight in children has been suggested, and rare cases of in utero or perinatal HHV-8 transmission have been documented, no direct evidence of HHV-8 infection of placenta has yet been reported. The aim of this study was to evaluate the in vitro and in vivo susceptibility of placental cells to HHV-8 infection. Short-term infection assays were performed on placental chorionic villi isolated from term placentae. Qualitative and quantitative HHV-8 detection were performed by PCR and real-time PCR, and HHV-8 proteins were analyzed by immunohistochemistry. Term placenta samples from HHV-8-seropositive women were analyzed for the presence of HHV-8 DNA and antigens. In vitro infected histocultures showed increasing amounts of HHV-8 DNA in tissues and supernatants; cyto- and syncitiotrophoblasts, as well as endothelial cells, expressed latent and lytic viral antigens. Increased apoptotic phenomena were visualized by the terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end-labeling method in infected histocultures. Ex vivo, HHV-8 DNA and a latent viral antigen were detected in placenta samples from HHV-8-seropositive women. These findings demonstrate that HHV-8, like other human herpesviruses, may infect placental cells in vitro and in vivo, thus providing evidence that this phenomenon might influence vertical transmission and pregnancy outcome in HHV-8-infected women.     

Searching the point of no return in Helicobacter pylori life: necrosis and/or programmed death?

AIMS: Ultrastructural and molecular studies to support the hypothesis of programmed cell death in Helicobacter pylori were conducted. METHODS AND RESULTS: Evidence of programmed death in H. pylori is provided through electron microscopic detection and cytochemical labelling of electrondense bodies (EDB), containing packaged DNA in coccoid cells, resembling micronuclei of apoptotic eukaryotic cells. This morphological evidence is also supported by DNA cleavage in homogeneous fragments of about 100 base pairs. Programmed cell death was observed in H. pylori cultures at 37 degrees C, with a maximum of 37.5% of EDB coccoid cells after 7 days. The non-permissive temperature of 4 degrees C anticipated this process, with 40% of EDB coccoid forms within 3 days, and it remained substantially unaffected during the observation time of 14 days. CONCLUSION: In these experiments, deprivation of nutrients and a non-permissive temperature acted as a powerful trigger for programmed cell death. SIGNIFICANCE AND IMPACT OF THE STUDY: Helicobacter pylori bacterial populations, under stressing stimuli, can respond with programmed cell suicide as a means of species preservation.     

HIV-1 Tat-based vaccines: from basic science to clinical trials

Vaccination against human immunodeficiency virus (HIV)-1 infection requires candidate antigen(s) (Ag) capable of inducing an effective, broad, and long-lasting immune response against HIV-1 despite mutation events leading to differences in virus clades. The HIV-1 Tat protein is more conserved than envelope proteins, is essential in the virus life cycle and is expressed very early upon virus entry. In addition, both humoral and cellular responses to Tat have been reported to correlate with a delayed progression to disease in both humans and monkeys. This suggested that Tat is an optimal target for vaccine development aimed at controlling virus replication and blocking disease onset. Here are reviewed the results of our studies including the effects of the Tat protein on monocyte-derived dendritic cells (MDDCs) that are key antigen-presenting cells (APCs), and the results from vaccination trials with both the Tat protein or tat DNA in monkeys. We provide evidence that the HIV-1 Tat protein is very efficiently taken up by MDDCs and promotes T helper (Th)-1 type immune responses against itself as well as other Ag. In addition, a Tat-based vaccine elicits an immune response capable of controlling primary infection of monkeys with the pathogenic SHIV89.6P at its early stages allowing the containment of virus spread. Based on these results and on data of Tat conservation and immune cross-recognition in field isolates from different clades, phase I clinical trials are being initiated in Italy for both preventive and therapeutic vaccination.     

Chromosomal evolution and speciation in the Gyratrix hermaphroditus species complex (Platyhelminthes,Kalyptorhynchia): karyometric and morphological analyses of fifteen fresh-water populations from Western Europe

Fifteen fresh-water populations of the Gyratrix hermaphroditus species complex from Western Europe (Italy, Spain, France) were karyometrically analysed. All shared the same chromosome number 2n = 4. Three distinct karyotypes were recognized on the basis of the different values of the centromeric index of chromosome 2, respectively metacentric, intermediate between meta- and submetacentric, and sub-telocentric. The morphology of chromosome 1 (an isobrachial metacentric) and the length ratio of chromosomes were on the contrary constant in all the populations investigated.Differences in the size of male cuticular organs were observed accompanying the karyological differentiation.The fifteen populations are interpreted as representing at least three sibling-species.Chromosomal evolution and phylogenetical relationships with the marine species of the Gyratrix hermaphroditus complex are discussed.     

Vascular endothelial growth factor enhances in vitro proliferation and osteogenic differentiation of human dental pulp stem cells

Mesenchymal stem cells (MSC), isolated from dental tissues, are largely studied for future application in regenerative dentistry. In this study, we used MSC obtained from human dental pulp (DPSC) of normal impacted third molars that, when cultured in lineage-specific inducing media, differentiate into osteoblasts and adipocytes (evaluated by Alizarin Red S and Red Oil O stainings, respectively), thus showing a multipotency. We confirmed that DPSC, grown under undifferentiating conditions, are negative for hematopoietic (CD45, CD31, CD34, CD144) and positive for mesenchymal (CD29, CD90, CD105, CD166, CD146, STRO-1) markers, that underwent down-regulation when cells were grown in osteogenic medium for 3 weeks. In this condition, they also exhibit an increase in the expression of osteogenic markers (RUNX-2, alkaline phosphatase) and extracellular calcium deposition, whereas the expression of receptors (VEGFR-1 and -2) for vascular endothelial growth factors (VEGF) and related VEGF binding proteins was similar to that found in undifferentiated DPSC. Exposure of DPSC growing under undifferentiating or osteogenic conditions to VEGF-A165 peptide (10-40 ng/ml) for 8 days dose- and time-dependently increased the number of proliferating cells without inducing differentiation towards endothelial lineage, as evaluated by the lack of expression of specific markers (CD31, CD34, CD144). Additionally, exposure of DPSC cultured in osteogenic medium to VEGF-A165 for a similar period enhanced cell differentiation towards osteoblasts as evaluated after 14 and 21 days by Alizarin Red S staining and alkaline phosphatase activity quantification. These findings may have clinical implications possibly facilitating tissue repair and remodeling.