Genetic Variability for Grain Components Related to Nutritional Quality in Spelt and Common Wheat.

Spelt (Triticum aestivum ssp. spelta) is part of the so-called ancient wheats. These types of wheats are experiencing a revival as they have been proposed to be healthier than conventional wheat. However, the given healthier condition of spelt is not substantiated by solid scientific evidence. The objective of this study was to analyze the genetic variability for several grain components, related to nutritional quality (arabinoxylans, micronutrients, phytic acid) in a set of spelt and common wheat genotypes to determinate if spelt is potentially healthier than common wheat. The results obtained indicated that within the compared species, there is a significant variation in the nutritional compounds, and it is not truthful and accurate to state that one species is healthier than the other. Within both groups, genotypes showing outstanding values for some traits were detected, which could be used in breeding programs to develop new wheat cultivars with good agronomic performance and nutritional quality.

Testing commercial biopreservative against spoilage microorganisms in MAP packed Ricotta fresca cheese.

Ricotta fresca cheese is susceptible to secondary contamination and is able to support the growth of pathogens or spoilage psychotrophic bacteria during storage. The aim of the present study was to evaluate which among three commercial biopreservatives was suitable to be used to control the growth of spoilage microorganisms in sheep's milk MAP ricotta fresca cheese. 144 Ricotta fresca cheese samples were inoculated either with the bioprotective culture Lyofast FPR 2 (including Enterococcus faecium, Lactobacillus plantarum e Lactobacillus rhamnosus) or Lyofast CNBAL (Carnobacterium spp) or the fermentate MicroGARD 430. Not inoculated control and experimental ricotta were MAP packed (30% CO2 and 70% N2) and stored at 4 °C. Triplicate samples were analyzed after 5 h and 7, 14 and 21 days after inoculation for total bacterial count, mesophilic lactic acid bacteria, Enterobacteriaceae, Pseudomonas spp, Listeria monocytogenes, moulds and yeasts. Among the tested biopreservatives only Carnobacterium spp was able to control Pseudomonas spp and Enterobacteriaceae. The maximum reduction in the concentration of Pseudomonas spp and Enterobacteriaceae was respectively 1.93 and 2.66 log10 cfu/g, observed 14 days after production. Therefore, Carnobacterium spp was selected as the culture of choice to conduct a challenge study against Pseudomonas spp.

Prenatal restraint stress: an in vivo microdialysis study on catecholamine release in the rat prefrontal cortex.

There is substantial evidence that prenatal exposure to adverse environmental conditions might lead to the psychiatric disorders that can appear in adolescence or in adulthood; vulnerability to drug addiction may increase as well. It is currently accepted that the alteration of catecholamine transmission in the prefrontal cortex plays a prominent role in the etiology of psychiatric disorders. We assessed basal and stimulated dopamine and noradrenaline extracellular concentration in the medial prefrontal cortex by means of microdialysis in awake male adolescent and young adult offspring of rats exposed to restraint stress in the last week of pregnancy. Catecholamine stimulation was obtained by amphetamine or nicotine. We observed that prenatal stress (PNS) did not change dopamine but decreased noradrenaline basal output in both adolescents and adults. Moreover, it decreased amphetamine stimulated dopamine output and increased amphetamine stimulated noradrenaline output. PNS decreased nicotine stimulated noradrenaline (but not dopamine output) in adults, though not in adolescents. These data show that PNS stress modifies prefrontal cortex catecholamine transmission in a complex and age dependent manner. Our results support the view that prenatal stress may be a contributing factor for the development of psychiatric disorders and that its effect may augment drug addiction vulnerability.

Prepuberal subchronic methylphenidate and atomoxetine induce different long-term effects on adult behaviour and forebrain dopamine, norepinephrine and serotonin in Naples high-excitability rats.

The psychostimulant methylphenidate and the non-stimulant atomoxetine are two approved drugs for attention-deficit hyperactivity disorder (ADHD) therapy. The aim of this study was to investigate the long-term effects of prepuberal subchronic methylphenidate and atomoxetine on adult behaviour and the forebrain neurotransmitter and metabolite content of Naples High-Excitability (NHE) rats, a genetic model for the mesocortical variant of ADHD. Male NHE rats were given a daily intraperitoneal injection (1.0mg/kg) of methylphenidate, atomoxetine or vehicle from postnatal day 29 to 42. At postnatal day 70-75, rats were exposed to spatial novelty in the Làt and radial (Olton) mazes. Behavioural analysis for indices of horizontal, vertical, non-selective (NSA) and selective spatial attention (SSA) indicated that only methylphenidate significantly reduced horizontal activity to a different extent, i.e., 39 and 16% respectively. Moreover methylphenidate increased NSA as assessed by higher leaning duration. The high-performance liquid chromatography (HPLC) tissue content assessment of dopamine, norepinephrine, serotonin and relative metabolites in the prefrontal cortex (PFC), cortical motor area (MC), dorsal striatum (DS), ventral striatum (VS), hippocampus and mesencephalon indicated that methylphenidate decreased (i) dopamine, DOPAC, norepinephrine, MHPG, 5-HT and 5-HIAA in the PFC, (ii) dopamine, DOPAC, HVA, serotonin, 5-HIAA in the DS, (iii) dopamine, DOPAC, HVA and MHPG (but increased norepinephrine) in the VS and (iv) norepinephrine, MHPG, serotonin and 5-HIAA in the hippocampus. Atomoxetine increased dopamine and decreased MHPG in the PFC. Like methylphenidate, atomoxetine decreased dopamine, DOPAC, HVA, serotonin and 5-HIAA in the DS, but decreased MHPG in the VS. These results suggest that methylphenidate determined long-term effects on behavioural and neurochemical parameters, whereas atomoxetine affected only the latter.

Influence of extero- and proprioceptive afferents of the plantar surface in determining subjective visual vertical in patients with unilateral vestibular dysfunction.

Subjective visual vertical refers to an individual's ability to indicate what, in his or her opinion, is a perfectly vertical line in specific experimental conditions. Although the otolith organs play a key role in the perception of verticality, the contribution of other sensory systems, e.g. the visual and proprioceptive systems, cannot be overlooked. The aim of this study was to test the hypothesis that extero- and proprioceptive afferent signals, particularly from the plantar surface of the foot, can influence the temporal evolution of altered subjective visual vertical following unilateral acute vestibular dysfunction. Subjective visual vertical was studied in 40 consecutive patients: 19 females and 21 males (mean age 46.4 years). It was first measured at diagnosis (1-2 days after onset of symptoms). For this measurement, a baseline test was performed (patient standing in direct contact with the floor), followed by a provocation test with a soft support between the patient's feet and the floor. Based on a comparison between the baseline and provocation tests, the patients were divided into three groups: Group A--patients showing a significant increase (p < 0.05) in subjective visual vertical(0 )values in the provocation test compared to baseline values; Group B--patients showing a significant decrease (p < 0.05) in subjective visual vertical(0 )values in the provocation test compared to baseline values; Group C--patients showing no significant changes (p < 0.05) in subjective visual vertical(0 )values in the provocation test compared to baseline values. The baseline test was repeated at 30, 90 and 180 days. At the end of the follow-up, a persistent change in subjective visual vertical was noted in 87% of the patients from Group B, 31% of the patients from Group C but none of the patients from Group A, all of whom were able to correct the perception error during the second examination. The study demonstrates that normalisation of subjective visual vertical in subjects with unilateral vestibular lesions seems to be influenced by the possibility of exploiting extra-vestibular sensory information, particularly extero- and proprioceptive information from the plantar surface.

The ubiquitin-proteasome system regulates p53-mediated transcription at p21waf1 promoter.

The ubiquitin (Ub)-proteasome system (UPS) promotes the proteasomal degradation of target proteins by decorating them with Ub labels. Emerging evidence indicates a role of UPS in regulating gene transcription. In this study, we provided evidence for the involvement of UPS in the transcriptional activation function of tumor suppressor p53. We showed that both ubiquitylation and proteasomal functions are required for efficient transcription mediated by p53. Disruption of transcription by actinomycin D, 5,6-dichloro-1-beta-D-ribofuranosyl-benzimadazole or alpha-amanitin leads to accumulation of cellular p53 protein. Proteasome inhibition by MG132 increases the occupancy of p53 protein at p53-responsive p21(waf1) promoter. In addition, the Sug-1 component of 19S proteasome physically interacts with p53 in vitro and in vivo. Moreover, in response to ultraviolet-induced DNA damage, both the 19S proteasomal components, Sug1 and S1, are recruited to p21(waf1) promoter region in a kinetic pattern similar to that of p53. These results suggested that UPS positively regulates p53-mediated transcription at p21(waf1) promoter.

Genomics and the evolution of aminoacyl-tRNA synthesis.

Translation is the process by which ribosomes direct protein synthesis using the genetic information contained in messenger RNA (mRNA). Transfer RNAs (tRNAs) are charged with an amino acid and brought to the ribosome, where they are paired with the corresponding trinucleotide codon in mRNA. The amino acid is attached to the nascent polypeptide and the ribosome moves on to the next codon. Thus, the sequential pairing of codons in mRNA with tRNA anticodons determines the order of amino acids in a protein. It is therefore imperative for accurate translation that tRNAs are only coupled to amino acids corresponding to the RNA anticodon. This is mostly, but not exclusively, achieved by the direct attachment of the appropriate amino acid to the 3'-end of the corresponding tRNA by the aminoacyl-tRNA synthetases. To ensure the accurate translation of genetic information, the aminoacyl-tRNA synthetases must display an extremely high level of substrate specificity. Despite this highly conserved function, recent studies arising from the analysis of whole genomes have shown a significant degree of evolutionary diversity in aminoacyl-tRNA synthesis. For example, non-canonical routes have been identified for the synthesis of Asn-tRNA, Cys-tRNA, Gln-tRNA and Lys-tRNA. Characterization of non-canonical aminoacyl-tRNA synthesis has revealed an unexpected level of evolutionary divergence and has also provided new insights into the possible precursors of contemporary aminoacyl-tRNA synthetases.

Post-transcriptional modification in archaeal tRNAs: identities and phylogenetic relations of nucleotides from mesophilic and hyperthermophilic Methanococcales.

Post-transcriptional modifications in archaeal RNA are known to be phylogenetically distinct but relatively little is known of tRNA from the Methanococci, a lineage of methanogenic marine euryarchaea that grow over an unusually broad temperature range. Transfer RNAs from Methanococcus vannielii, Methanococcus maripaludis, the thermophile Methanococcus thermolithotrophicus, and hyperthermophiles Methanococcus jannaschii and Methanococcus igneus were studied to determine whether modification patterns reflect the close phylogenetic relationships inferred from small ribosomal subunit RNA sequences, and to examine modification differences associated with temperature of growth. Twenty-four modified nucleosides were characterized, including the complex tricyclic nucleoside wyosine characteristic of position 37 in tRNA(Phe) and known previously only in eukarya, plus two new wye family members of presently unknown structure. The hypermodified nucleoside 5-methylaminomethyl-2-thiouridine, reported previously only in bacterial tRNA at the first position of the anticodon, was identified by liquid chromatography-electrospray ionization mass spectrometry in four of the five organisms. The ribose-methylated nucleosides, 2'-O-methyladenosine, N(2),2'-O-dimethylguanosine and N(2),N(2),2'-O-trimethylguanosine, were found only in hyperthermophile tRNA, consistent with their proposed roles in thermal stabilization of tRNA.

Protein synthesis: twenty three amino acids and counting.

The genetic code can be interpreted during translation as 21 amino acids and three termination signals. Recent advances at the interface of chemistry and molecular biology are extending the genetic code to allow assignment of new amino acids to existing codons, providing new functional groups for protein synthesis.

The renaissance of aminoacyl-tRNA synthesis.

The role of tRNA as the adaptor in protein synthesis has held an enduring fascination for molecular biologists. Over four decades of study, taking in numerous milestones in molecular biology, led to what was widely held to be a fairly complete picture of how tRNAs and amino acids are paired prior to protein synthesis. However, recent developments in genomics and structural biology have revealed an unexpected array of new enzymes, pathways and mechanisms involved in aminoacyl-tRNA synthesis. As a more complete picture of aminoacyl-tRNA synthesis now begins to emerge, the high degree of evolutionary diversity in this universal and essential process is becoming clearer.

Genomics-based identification of targets in pathogenic bacteria for potential therapeutic and diagnostic use.

The availability of numerous complete microbial genome sequences has profoundly altered our understanding of a number of fundamental biological processes. For example the enzymes involved in aminoacyl-tRNA (AA-tRNA) synthesis, the key process responsible for the accuracy of protein synthesis, have been found to be highly species-specific. In particular, a number of pathogens contain certain pathways of AA-tRNA synthesis that are unrelated to those found in their mammalian hosts. Since AA-tRNA synthesis is indispensable for cell viability, the discovery of pathogen-specific pathways and enzymes presents novel therapeutic and diagnostic targets. Here we will review recent advances in the elucidation of AA-tRNA synthesis pathways and discuss the possible pharmaceutical exploitation of these discoveries. In particular, the integration of genomic and biochemical approaches to identify novel targets for the treatment of Chlamydial infections and the diagnosis and treatment of Lyme disease will be presented.

Context-dependent anticodon recognition by class I lysyl-tRNA synthetases.

Lysyl-tRNA synthesis is catalyzed by two unrelated families of aminoacyl-tRNA synthetases. In most bacteria and all eukarya, the known lysyl-tRNA synthetases (LysRSs) are subclass IIb-type aminoacyl-tRNA synthetases, whereas many archaea and a scattering of bacteria contain an unrelated class I-type LysRS. Examination of the recognition of partially modified tRNA(Lys) anticodon variants by a bacterial (from Borrelia burgdorferi) and an archaeal (from Methanococcus maripaludis) class I lysyl-tRNA synthetase revealed differences in the pattern of anticodon recognition between the two enzymes. U35 and U36 were both important for recognition by the B. burgdorferi enzyme, whereas only U36 played a role in recognition by M. maripaludis LysRS. Examination of the phylogenetic distribution of class I LysRSs suggested a correlation between recognition of U35 and U36 and the presence of asparaginyl-tRNA synthetase (AsnRS), which also recognizes U35 and U36 in the anticodon of tRNA(Asn). However, the class II LysRS of Helicobacter pylori, an organism that lacks AsnRS, also recognizes both U35 and U36, indicating that the presence of AsnRS has solely influenced the phylogenetic distribution of class I LysRSs. These data suggest that competition between unrelated aminoacyl-tRNA synthetases for overlapping anticodon sequences is a determinant of the phylogenetic distribution of extant synthetase families. Such patterns of competition also provide a basis for the two separate horizontal gene transfer events hypothesized in the evolution of the class I lysyl-tRNA synthetases.

Ancient adaptation of the active site of tryptophanyl-tRNA synthetase for tryptophan binding.

The amino acid binding domains of the tryptophanyl (TrpRS)- and tyrosyl-tRNA synthetases (TyrRS) of Bacillus stearothermophilus are highly homologous. These similarities suggest that conserved residues in TrpRS may be responsible for both determining tryptophan recognition and discrimination against tyrosine. This was investigated by the systematic mutation of TrpRS residues based upon the identity of homologous positions in TyrRS. Of the four residues which interact directly with the aromatic side chain of tryptophan (Phe5, Met129, Asp132, and Val141) replacements of Asp132 led to significant changes in the catalytic efficiency of Trp aminoacylation (200-1250-fold reduction in k(cat)/K(M)) and substitution of Val141 by the larger Glu side chain reduced k(cat)/K(M) by 300-fold. Mutation of Pro127, which determines the position of active-site residues, did not significantly effect Trp binding. Of the mutants tested, D132N TrpRS also showed a significant reduction in discrimination against Tyr, with Tyr acting as a competitive inhibitor but not a substrate. The analogous residue in B. stearothermophilusTyrRS (Asp176) has also been implicated as a determinant of amino acid specificity in earlier studies [de Prat Gay, G., Duckworth, H. W., and Fersht, A. R. (1993) FEBS Lett. 318, 167-171]. This striking similarity in the function of a highly conserved residue found in both TrpRS and TyrRS provides mechanistic support for a common origin of the two enzymes.

Aminoacyl-tRNA synthesis.

Aminoacyl-tRNAs are substrates for translation and are pivotal in determining how the genetic code is interpreted as amino acids. The function of aminoacyl-tRNA synthesis is to precisely match amino acids with tRNAs containing the corresponding anticodon. This is primarily achieved by the direct attachment of an amino acid to the corresponding tRNA by an aminoacyl-tRNA synthetase, although intrinsic proofreading and extrinsic editing are also essential in several cases. Recent studies of aminoacyl-tRNA synthesis, mainly prompted by the advent of whole genome sequencing and the availability of a vast body of structural data, have led to an expanded and more detailed picture of how aminoacyl-tRNAs are synthesized. This article reviews current knowledge of the biochemical, structural, and evolutionary facets of aminoacyl-tRNA synthesis.

[Role of pH as a regulator of vaginal physiological environment]. / Ruolo del pH come modulatore dell'equilibrio fisiopatologico vaginale.

In the vagina there are several microorganisms. Their survival is fundamental to have a physiological environment in the vagina (ecosystem). The same vaginal environment playg a fundamental role to guarantee tho survival of resident microorganisms. Several factors, like changes of temperature and oxygenation, can interfere on vaginal ecosystem, but the resident vaginal microorganisms are the main factors to stabilize the vaginal ecosystem. The Doderlein's lactobacillus is the predominant vaginal microorganism. It is capable of fermenting the glycogen deriving from the decline of the eutrophic vaginal mmucosa, to lactic acid with release of hydrogen ions. The final result of that metabolism is an acid pH with values between 4-4.5. Vaginal pH undergoes physiologically changes from birth to menopause, according to changes of ovarian steroids occurring during woman's life. Adequate levels of estrogens play a fundamental role in the trophism of vaginal mucosa. In fact, estrogens increase the cellular content of glycogen. Exogenous acbvities on vaginal pH can be exerted by several factors, such as sexual activily, oral contraceptives, systemic diseases, vaginal infections (candidosis, thrichomonias, vaginosis), systemic or local therapies. They incrcase vaginal pH by acting through different mechanisms. The increase of vaginal pH is detrimental for the survival of Doderlein's lactobacillus, but not for the pathogenetic microorganisms whose replication, on the contrary, is favored by the absence of contraction exerted by Doderlein's bacillus. It has been showed that local acidifying substances (lactic acid, lactobacillus and substances recently synthesized like alpha-aminovalerianic acid, policarbophil and carbopol 934) are useful in restoring the biological and chemical characteristics of the vaginal ecosystem.