Brain activity during the encoding, retention, and retrieval of stimulus representations

Studies of delayed nonmatching-to-sample (DNMS) performance following lesions of the monkey cortex have revealed a critical circuit of brain regions involved in forming memories and retaining and retrieving stimulus representations. Using event-related functional magnetic resonance imaging (fMRI), we measured brain activity in 10 healthy human participants during performance of a trial-unique visual DNMS task using novel barcode stimuli. The event-related design enabled the identification of activity during the different phases of the task (encoding, retention, and retrieval). Several brain regions identified by monkey studies as being important for successful DNMS performance showed selective activity during the different phases, including the mediodorsal thalamic nucleus (encoding), ventrolateral prefrontal cortex (retention), and perirhinal cortex (retrieval). Regions showing sustained activity within trials included the ventromedial and dorsal prefrontal cortices and occipital cortex. The present study shows the utility of investigating performance on tasks derived from animal models to assist in the identification of brain regions involved in human recognition memory.

Assessment of the ability to model proteins with leucine-rich repeats in light of the latest structural information

The three-dimensional structures of leucine-rich repeat (LRR) -containing proteins from five different families were previously predicted based on the crystal structure of the ribonuclease inhibitor. using an approach that combined homology-based modeling, structure-based sequence alignment of LRRs, and several rational assumptions. The structural models have been produced based on very limited sequence similarity, which, in general. cannot yield trustworthy predictions. Recently, the protein structures from three of these five families have been determined. In this report we estimate the quality of the modeling approach by comparing the models with the experimentally determined structures. The comparison suggests that the general architecture, curvature, interior/exterior orientations of side chains. and backbone conformation of the LRR structures can be predicted correctly. On the other hand. the analysis revealed that, in some cases. it is difficult to predict correctly the twist of the overall super-helical structure. Taking into consideration the conclusions from these comparisons, we identified a new family of bacterial LRR proteins and present its structural model. The reliability of the LRR protein modeling suggests that it would be informative to apply similar modeling approaches to other classes of solenoid proteins.

Structural characterization of the N-terminal autoregulatory sequence of phenylalanine hydroxylase

Phenylalanine hydroxylase (PAH) is activated by its substrate phenylalanine, and through phosphorylation by cAMP-dependent protein kinase at Ser 16 in the N-terminal autoregulatory sequence of the enzyme. The crystal structures of phosphorylated and unphosphorylated forms of the enzyme showed that, in the absence of phenylalanine, in both cases the N-terminal 18 residues including the phosphorylation site contained no interpretable electron density. We used nuclear magnetic resonance (NMR) spectroscopy to characterize this N-terminal region of the molecule in different stages of the regulatory pathway. A number of sharp resonances are observed in PAH with an intact N-terminal region, but no sharp resonances are present in a truncation mutant lacking the N-terminal 29 residues. The N-terminal sequence therefore represents a mobile flexible region of the molecule. The resonances become weaker after the addition of phenylalanine, indicating a loss of mobility. The peptides corresponding to residues 2-20 of PAH have different structural characteristics in the phosphorylated and unphosphorylated forms, with the former showing increased secondary structure. Our results support the model whereby upon phenylalanine binding, the mobile N-terminal 18 residues of PAH associate with the folded core of the molecule; phosphorylation may facilitate this interaction.

Crystal structures of free, IMP-, and GMP-bound Escherichia coli hypoxanthine phosphoribosyltransferase

Crystal structures have been determined for free Escherichia coli hypoxanthine phosphoribosyltransferase (HPRT) (2.9 Angstrom resolution) and for the enzyme in complex with the reaction products, inosine 5'-monophosphate (IMP) and guanosine 5-monophosphate (GMP) (2.8 Angstrom resolution). Of the known 6-oxopurine phosphoribosyltransferase (PRTase) structures, E. coli HPRT is most similar in structure to that of Tritrichomonas foetus HGXPRT, with a rmsd for 150 Calpha atoms of 1.0 Angstrom. Comparison of the free and product bound structures shows that the side chain of Phe156 and the polypeptide backbone in this vicinity move to bind IMP or GMP. A nonproline cis peptide bond, also found in some other 6-oxopurine PRTases, is observed between Leu46 and Arg47 in both the free and complexed structures. For catalysis to occur, the 6-oxopurine PRTases have a requirement for divalent metal ion, Usually Mg2+ in vivo. In the free structure, a Mg2+, is coordinated to the side chains of Glu103 and Asp104. This interaction may be important for stabilization of the enzyme before catalysis. E. coli HPRT is unique among the known 6-oxopurine PRTases in that it exhibits a marked preference for hypoxanthine as substrate over both xanthine and guanine. The structures suggest that its substrate specificity is due to the modes of binding of the bases. In E. coli HPRT, the carbonyl oxygen of Asp 163 would likely form a hydrogen bond with the 2-exocyclic nitrogen of guanine (in the HPRT-guanine-PRib-PP-Mg2+ complex). However, hypoxanthine does not have a 2-exocyclic atom and the HPRT-IMP structure suggests that hypoxanthine is likely to occupy a different position in the purine-binding pocket.

Mutations in the human ortholog of Aristaless cause X-linked mental retardation and epilepsy

Mental retardation and epilepsy often occur together. They are both heterogeneous conditions with acquired and genetic causes. Where causes are primarily genetic, major advances have been made in unraveling their molecular basis. The human X chromosome alone is estimated to harbor more than 100 genes that, when mutated, cause mental retardation(1). At least eight autosomal genes involved in idiopathic epilepsy have been identified(2), and many more have been implicated in conditions where epilepsy is a feature. We have identified mutations in an X chromosome-linked, Aristaless-related, homeobox gene (ARX), in nine families with mental retardation (syndromic and nonspecific), various forms of epilepsy, including infantile spasms and myoclonic seizures, and dystonia. Two recurrent mutations, present in seven families, result in expansion of polyalanine tracts of the ARX protein. These probably cause protein aggregation, similar to other polyalanine(3) and polyglutamine(4) disorders. In addition, we have identified a missense mutation within the ARX homeodomain and a truncation mutation. Thus, it would seem that mutation of ARX is a major contributor to X-linked mental retardation and epilepsy.

Effects of molecular crowding by saccharides on alpha-chymotrypsin dimerization

Given the importance of protein complexes as therapeutic targets, it is necessary to understand the physical chemistry of these interactions under the crowded conditions that exist in cells. We have used sedimentation equilibrium to quantify the enhancement of the reversible homodimerization of alpha-chymotrypsin by high concentrations of the osmolytes glucose, sucrose, and raffinose. In an attempt to rationalize the ostuolyte-mediated stabilization of the a-chymotrypsin homodimer, we have used models based on binding interactions (transfer-free energy analysis) and steric interactions (excluded volume theory) to predict the stabilization. Although transfer-free energy analysis predicts reasonably well the relatively small stabilization observed for complex formation between cytochrome c and cytochrome c peroxidase, as well as that between bobtail quail lysozyme and a monoclonal Fab fragment, it underestimates the sugar-mediated stabilization of the alpha-chymotrypsin dimer. Although predictions based on excluded volume theory overestimate the stabilization, it would seem that a major determinant in the observed stabilization of the a-chymotrypsin homodimer is the thermodynamic nonideality arising from molecular crowding by the three small sugars.

MAX4 and RMS1 are orthologous dioxygenase-like genes that regulate shoot branching in Arabidopsis and pea

Shoot branching is inhibited by auxin transported down the stem from the shoot apex. Auxin does not accumulate in inhibited buds and so must act indirectly. We show that mutations in the MAX4 gene of Arabidopsis result in increased and auxin-resistant bud growth. Increased branching in max4 shoots is restored to wild type by grafting to wild-type rootstocks, suggesting that MAX4 is required to produce a mobile branch-inhibiting signal, acting downstream of auxin. A similar role has been proposed for the pea gene, RMS1. Accordingly, MAX4 and RMS1 were found to encode orthologous, auxin-inducible members of the polyene dioxygenase family.

Mouse proteome analysis

A general overview of the protein sequence set for the mouse transcriptome produced during the FANTOM2 sequencing project is presented here. We applied different algorithms to characterize protein sequences derived from a nonredundant representative protein set (RPS) and a variant protein set (VPS) of the mouse transcriptome. The functional characterization and assignment of Gene Ontology terms was done by analysis of the proteome using InterPro. The Superfamily database analyses gave a detailed structural classification according to SCOP and provide additional evidence for the functional characterization of the proteome data. The MDS database analysis revealed new domains which are not presented in existing protein domain databases. Thus the transcriptome gives us a unique source of data for the detection of new functional groups. The data obtained for the RPS and VPS sets facilitated the comparison of different patterns of protein expression. A comparison of other existing mouse and human protein sequence sets (e.g., the International Protein Index) demonstrates the common patterns in mammalian proteornes. The analysis of the membrane organization within the transcriptome of multiple eukaryotes provides valuable statistics about the distribution of secretory and transmembrane proteins

Analysis of the mouse transcriptome for genes involved in the function of the nervous system

We analyzed the mouse Representative Transcript and Protein Set for molecules involved in brain function. We found full-length cDNAs of many known brain genes and discovered new members of known brain gene families, including Family 3 G-protein coupled receptors, voltage-gated channels, and connexins. We also identified previously unknown candidates for secreted neuroactive molecules. The existence of a large number of unique brain ESTs suggests an additional molecular complexity that remains to be explored. A list of genes containing CAG stretches in the coding region represents a first step in the potential identification of candidates for hereditary neurological disorders.

The mouse secretome: functional classification of the proteins secreted into the extracellular environment

We have developed a computational strategy to identify the set of soluble proteins secreted into the extracellular environment of a cell. Within the protein sequences predominantly derived from the RIKEN representative transcript and protein set, we identified 2033 unique soluble proteins that are potentially secreted from the cell. These proteins contain a signal peptide required for entry into the secretory pathway and lack any transmembrane domains or intracellular localization signals. This class of proteins, which we have termed the mouse secretome, included >500 novel proteins and 92 proteins

Uma cidade portuária: a Horta entre 1880 e 1926. Sociedade e cultura com a política em fundo

Tese de Doutoramento, História (História Contemporânea), 29 de Novembro de 2013, Universidade dos Açores.

Routine screening of harmful microorganisms in beach sands: implications to public health

Beaches worldwide provide recreational opportunities to hundreds of millions of people and serve as important components of coastal economies. Beach water is often monitored for microbiological quality to detect the presence of indicators of human sewage contamination so as to prevent public health outbreaks associated with water contact. However, growing evidence suggests that beach sand can harbor microbes harmful to human health, often in concentrations greater than the beach water. Currently, there are no standards for monitoring, sampling, analyzing, or managing beach sand quality. In addition to indicator microbes, growing evidence has identified pathogenic bacteria, viruses, and fungi in a variety of beach sands worldwide. The public health threat associated with these populations through direct and indirect contact is unknown because so little research has been conducted relating to health outcomes associated with sand quality. In this manuscript, we present the consensus findings of a workshop of experts convened in Lisbon, Portugal to discuss the current state of knowledge on beach sand microbiological quality and to develop suggestions for standardizing the evaluation of sand at coastal beaches. The expert group at the "Microareias 2012" workshop recommends that 1) beach sand should be screened for a variety of pathogens harmful to human health, and sand monitoring should then be initiated alongside regular water monitoring; 2) sampling and analysis protocols should be standardized to allow proper comparisons among beach locations; and 3) further studies are needed to estimate human health risk with exposure to contaminated beach sand. Much of the manuscript is focused on research specific to Portugal, but similar results have been found elsewhere, and the findings have worldwide implications.

Zinc-substituted desulfovibrio gigas desulforedoxins

Protein Science (2002), 11:2464–2470

Children and sand play: screening of potential harmful microorganisms in sandboxes, parks, and beaches

Sand serves as a reservoir for potentially pathogenic microorganisms. Children, a high-risk group, can acquire infections from sand in sandboxes, recreational areas, and beaches. This paper reviews the microbes in sands, with an emphasis on fungi. Recreational areas and beach sands have been found to harbor many types of fungi and microbes. A newly emerging group of fungi of concern include the black yeast-like fungi. After establishing that sand is a reservoir for fungi, clinical manifestations of fungal infections are described with an emphasis on ocular and ear infections. Overall, we recommend environmental studies to develop monitoring strategies for sand and studies to evaluate the link between fungi exposure in sand and human health impacts.