Phenolic acids from beer are absorbed and extensively metabolized in humans

In spite of the wide literature describing the biological effects of phenolic compounds, scarce data are available on their absorption from diet. In the present work, we studied the absorption in humans of phenolic acids from beer, a common beverage rich in different phenolic acids with related chemical structures. Beer was analyzed for free and total (free+bound) phenolic acids. Ferulic, caffeic and sinapic acids were present in beer mainly as bound forms, while 4-hydroxyphenylacetic acid and p-coumaric acid were present mainly as free forms. Vanillic acid was present equally in the free and bound forms. Plasma samples were collected before and 30 and 60 min after beer administration and analyzed for free and conjugated phenolic acid content. A significant two- to fourfold increase in plasma levels of phenolic acids was detected with peak concentrations at 30 min after beer ingestion. 4-Hydroxyphenylacetic acid was present in plasma mainly as nonconjugated forms while p-coumaric acid was present equally as nonconjugated and conjugated forms. Ferulic, vanillic and caffeic acids were present in plasma predominantly as conjugated forms, with a slight prevalence of sulfates with respect to glucuronates. Our results indicate that phenolic acids from beer are absorbed from the gastrointestinal tract and are present in blood after being largely metabolized to the form of glucuronide and sulfate conjugates. The extent of conjugation is related to the chemical structure of phenolic acids: the monohydroxy derivatives showing the lowest conjugation degree and the dihydroxy derivatives showing the highest one.     

Presence and histopathological effects of the Parvatrema sp. (Digenea, Gymnophallidae) in the stout razor clam Tagelus plebeius (Bivalvia, Psammobiidae)

The stout razor clam Tagelus plebeius (Bivalvia, Psammobiidae) has a wide geographic distribution range, including the Brazilian coasts from the northeast (Alagoas) to the south (Santa Catarina). In March 2008, an episode of mass T. plebeius mortality (70%) occurred in an intertidal bed at The Pontal da Daniela, State of Santa Catarina, Brazil. We report here high prevalences (to 100%) of the trematode parasite Parvatrema sp. Cable, 1953 (Digenea, Gymnophallidae) infecting T. plebeius at high intensities. We describe the gymnophalid, echinostomatid and unidentified metacercariae parasites infecting the clam and the host reactions elicited by them. The use of special diagnostic techniques such as Ray’s fluid thioglycollate medium (RFTM) and PCR assays to detect Perkinsus sp. pathogens, hemolymph cytology, and histopathological examinations did not show Perkinsus sp. infections, microcell infections, or neoplastic conditions. However, neither infections or pathology caused by trematode parasites; nor any other pathological condition could be uniquely correlated with the mortality event. A coincident flash flood might have contributed to cause the mortality episode. This is the first report of the Parvatrema sp. metacercariae larvae infecting the stout razor clam T. plebeius from Brazil.     

Genome-Wide Scoring of Positive and Negative Epistasis through Decomposition of Quantitative Genetic Interaction Fitness Matrices

Recent technological developments in genetic screening approaches have offered the means to start exploring quantitative genotype-phenotype relationships on a large-scale. What remains unclear is the extent to which the quantitative genetic interaction datasets can distinguish the broad spectrum of interaction classes, as compared to existing information on mutation pairs associated with both positive and negative interactions, and whether the scoring of varying degrees of such epistatic effects could be improved by computational means. To address these questions, we introduce here a computational approach for improving the quantitative discrimination power encoded in the genetic interaction screening data. Our matrix approximation model decomposes the original double-mutant fitness matrix into separate components, representing variability across the array and query mutants, which can be utilized for estimating and correcting the single-mutant fitness effects, respectively. When applied to three large-scale quantitative interaction datasets in yeast, we could improve the accuracy of scoring various interaction classes beyond that obtained with the original fitness data, especially in synthetic genetic array (SGA) and in genetic interaction mapping (GIM) datasets. In addition to the known pairs of interactions used in the evaluation of the computational approach, a number of novel interaction pairs were also predicted, along with underlying biological mechanisms, which remained undetected by the original datasets. It was shown that the optimal choice of the scoring function depends heavily on the screening approach and on the interaction class under analysis. Moreover, a simple preprocessing of the fitness matrix could further enhance the discrimination power of the epistatic miniarray profiling (E-MAP) dataset. These systematic evaluation results provide in-depth information on the optimal analysis of the future, large-scale screening experiments. In general, the modeling framework, enabling accurate identification and classification of genetic interactions, provides a solid basis for completing and mining the genetic interaction networks in yeast and other organisms.     

Tryptophan 243 affects interprotein contacts, cofactor binding and stability in D-amino acid oxidase from Rhodotorula gracilis

The flavoenzyme d-amino acid oxidase from Rhodotorula gracilis is a homodimeric protein whose dimeric state has been proposed to occur as a result of (a) the electrostatic interactions between positively charged residues of the betaF5-betaF6 loop of one monomer and negatively charged residues belonging to the alpha-helices I3' and I3' of the other monomer, and (b) the interaction of residues (e.g. Trp243) belonging to the two monomers at the mixed interface region. The role of Trp243 was investigated by substituting it with either tyrosine or isoleucine: both substitutions were nondisruptive, as confirmed by the absence of significant changes in catalytic activity, but altered the tertiary structure (yielding a looser conformation) and decreased the stability towards temperature and denaturants. The change in conformation interferes both with the interaction of the coenzyme to the apoprotein moiety (although the kinetics of the apoprotein-FAD complex reconstitution process are similar between wild-type and mutant D-amino acid oxidases) and with the interaction between monomers. Our results indicate that, in the folded holoenzyme, Trp243 is situated at a position optimal for increasing the interactions between monomers by maximizing van der Waals interactions and by efficiently excluding solvent.     

The mirror neuron system and the consequences of its dysfunction

The discovery of premotor and parietal cells known as mirror neurons in the macaque brain that fire not only when the animal is in action, but also when it observes others carrying out the same actions provides a plausible neurophysiological mechanism for a variety of important social behaviours, from imitation to empathy. Recent data also show that dysfunction of the mirror neuron system in humans might be a core deficit in autism, a socially isolating condition. Here, we review the neurophysiology of the mirror neuron system and its role in social cognition and discuss the clinical implications of mirror neuron dysfunction.     

The interferon-inducible IFI16 gene inhibits tube morphogenesis and proliferation of primary, but not HPV16 E6/E7-immortalized human endothelial cells

Immunohistochemical analysis has demonstrated that the human IFI16 gene, in addition to the hematopoietic tissues, is highly expressed in endothelial cells and squamous stratified epithelia. In this study, we have developed a reliable HSV-derived replication-defective vector (TO-IFI16) to efficiently transduce IFI16 into primary human umbilical vein endothelial cells (HUVEC), which are usually poorly transfectable. HUVEC infection with TO-IFI16 virus suppressed endothelial migration, invasion and formation of capillary-like structures in vitro. In parallel, sustained IFI16 expression inhibited HUVEC cell cycle progression, accompanied by significant induction of p53, p21, and hypophosphorylated pRb. Further support for the involvement of these pathways in IFI16 activity came from the finding that infection with TO-IFI16 virus does not impair the in vitro angiogenic activity and cell cycle progression of HUVEC immortalized by HPV16 E6/E7 oncogenes, which are known to inactivate both p53 and pRb systems. This use of a reliable viral system for gene delivery into primary human endothelial cells assigns a potent angiostatic activity to an IFN-inducible gene, namely IFI16, and thus throws further light on antiangiogenic therapy employing IFNs.     

Human poly(ADP-ribose) glycohydrolase is expressed in alternative splice variants yielding isoforms that localize to different cell compartments

Poly(ADP-ribose) glycohydrolase (PARG) is the only protein known to catalyze hydrolysis of ADP-ribose (ADPR) polymers to free ADP-ribose. While numerous genes encode different poly(ADP-ribose) polymerases (PARPs) that all synthesize ADP-ribose polymer, only a single gene coding for PARG has been detected in mammalian cells. Here, we describe two splice variants of human PARG mRNA, which lead to expression of PARG isoforms of 102 kDa (hPARG102) and 99 kDa (hPARG99) in addition to the full-length PARG protein (hPARG111). These splice variants differ from hPARG111 by the lack of exon 1 (hPARG102) or exons 1 and 2 (hPARG99). They are generated by the utilization of ambiguous splice donor sites in the PARG gene 5' untranslated region. The hPARG111 isoform localizes to the nucleus, whereas hPARG102 and hPARG99 are cytoplasmic proteins. The nuclear targeting of hPARG111 is due to a nuclear localization signal (NLS) in exon 1 that was mapped to the amino acids (aa) (10)CTKRPRW(16). Immunocytochemistry, immunoblotting, and PARG enzyme activity measurements show that the cytoplasmic isoforms of PARG account for most of the PARG activity in cells in the absence and presence of genotoxic stress. The predominantly cytoplasmic location of cellular PARG is intriguing as most known cellular PARPs have a nuclear localization.     

Solvent-induced ligand dissociation and conformational states of Cellular Retinol-Binding Protein type I

Cellular Retinol-Binding Protein type I (CRBP) exhibits very high affinity for its ligand, bound within a buried cavity completely shielded from the outside medium. Three-dimensional structure and backbone dynamics in aqueous solution at neutral pH, either in the absence or in the presence of retinol, fail to represent the protein in a state capable of ligand uptake and release. The question was asked whether changes in the composition of the outside medium might facilitate ligand dissociation. Acidic aqueous solutions and water-alcohol mixtures were selected, among the best described denaturing solvents, to investigate their effects on the stability of the carrier-ligand complex and the conformational state of the protein upon ligand release. Circular dichroism (CD) and fluorescence spectroscopy were used to probe protein secondary and tertiary structure, compactness and retinol dissociation. While in purely aqueous media retinol dissociation parallels the acid-induced denaturation of the carrier, in water-alcohol mixtures it occurs in a range of co-solvent content lower than that required for protein denaturation. In light of these results, it is suggested that local solvent properties in vivo might modulate protein conformation and flexibility and thus play a fundamental role in the control of retinol exchange between carrier and membrane-bound donors and acceptors.     

An<Emphasis Type="Italic"> Alu</Emphasis>-mediated rearrangement as cause of exon skipping in Hunter disease

Hunter syndrome (Mucopolysaccharidosis type II), a rare X-linked lysosomal storage disorder, results from deleterious mutations in the iduronate-2-sulfatase ( IDS) gene located on Xq27.3-q28. Partial or complete deletions and large rearrangements have been extensively reported in the IDS gene as the basis of Hunter disease. The present report, however, is the first report on a Hunter patient in which Alu-mediated recombinations are implicated. Our patient showed the skipping of exon 8 at the cDNA level, without any splice-junction defects at the genomic level, where a new large rearrangement was identified instead. This new mutant allele consisted of an extensive deletion of IDS sequence of about 3 kb, as well as an additional inserted sequence of 157 bp. Two different computer programs were necessary to elucidate the nature of the insert. NCBI-BLAST query detected a single match for 126 bp out of 157 of the fragment that aligned exactly with a specific chromosomal region, Xq25-27.1, where an AluSg sequence is adjacent to an L1. Instead, the Repeat Masker program identified only 83 bp out of 157 of the insert, which was confirmed as an AluS. The observed homology between the AluSc sequence in the IDS intron 8 and the inserted AluS element, as well as the closeness of 26 bp Alu core sequence, considered to be a recombination hotspot, made us hypothesise upon the fact that both an Alu retrotransposition and an Alu-mediated deletion underlie the disease-producing rearrangement. We, therefore, now propose a mechanism that led to the large genomic deletion causing the production of the aberrant mRNA splicing.     

CDK4 coexpression with Ras generates malignant human epidermal tumorigenesis

Ras acts with other proteins to induce neoplasia. By itself, however, strong Ras signaling can suppress proliferation of normal cells. In primary epidermal cells, we found that oncogenic Ras transiently decreases cyclin-dependent kinase (CDK) 4 expression in association with cell cycle arrest in G1 phase. CDK4 co-expression circumvents Ras growth suppression and induces invasive human neoplasia resembling squamous cell carcinoma. Tumorigenesis is dependent on CDK4 kinase function, with cyclin D1 required but not sufficient for this process. In facilitating escape from G1 growth restraints, Ras and CDK4 alter the composition of cyclin D and cyclin E complexes and promote resistance to growth inhibition by INK4 cyclin-dependent kinase inhibitors. These data identify a new role for oncogenic Ras in CDK4 regulation and highlight the functional importance of CDK4 suppression in preventing uncontrolled growth.