Crystal structures of adenine phosphoribosyltransferase from Leishmania donovani.

The enzyme adenine phosphoribosyltransferase (APRT) functions to salvage adenine by converting it to adenosine-5-monophosphate (AMP). APRT deficiency in humans is a well characterized inborn error of metabolism, and APRT may contribute to the indispensable nutritional role of purine salvage in protozoan parasites, all of which lack de novo purine biosynthesis. We determined crystal structures for APRT from Leishmania donovani in complex with the substrate adenine, the product AMP, and sulfate and citrate ions that appear to mimic the binding of phosphate moieties. Overall, these structures are very similar to each other, although the adenine and AMP complexes show different patterns of hydrogen-bonding to the base, and the active site pocket opens slightly to accommodate the larger AMP ligand. Whereas AMP adopts a single conformation, adenine binds in two mutually exclusive orientations: one orientation providing adenine-specific hydrogen bonds and the other apparently positioning adenine for the enzymatic reaction. The core of APRT is similar to that of other phosphoribosyltransferases, although the adenine-binding domain is quite different. A C-terminal extension, unique to Leishmania APRTs, extends an extensive dimer interface by wrapping around the partner molecule. The active site involves residues from both subunits of the dimer, indicating that dimerization is essential for catalysis.     

A neurocognitive perspective on language: the declarative/procedural model

What are the psychological, computational and neural underpinnings of language? Are these neurocognitive correlates dedicated to language? Do different parts of language depend on distinct neurocognitive systems? Here I address these and other issues that are crucial for our understanding of two fundamental language capacities: the memorization of words in the mental lexicon, and the rule-governed combination of words by the mental grammar. According to the declarative/procedural model, the mental lexicon depends on declarative memory and is rooted in the temporal lobe, whereas the mental grammar involves procedural memory and is rooted in the frontal cortex and basal ganglia. I argue that the declarative/procedural model provides a new framework for the study of lexicon and grammar.     

Domain-specific antibodies reveal multiple-site topology of Nup153 within the nuclear pore complex

Nup153, one of the best characterized nuclear pore complex proteins (nucleoporins), plays a critical role in the import of proteins into the nucleus as well as in the export of RNAs and proteins from the nucleus. Initially an epitope of Nup153 was found to reside at the distal ring of the NPC, whereas more recently another epitope was localized to the nuclear ring moiety of the NPC. In an effort to more definitively determine the location of Nup153 within the 3-D architecture of the NPC we have generated domain-specific antibodies against distinct domains of Xenopus Nup153. With this approach we have found that the N-terminal domain is exposed at the nuclear ring of the NPC, whereas the zinc-finger domain of Nup153 is exposed at the distal ring of the NPC. In contrast, the C-terminal domain of Nup153 is not restricted to one particular subdomain of the NPC but rather appears to be highly flexible. Exogenous epitope-tagged hNup153 incorporated into Xenopus oocyte NPCs further underscored these findings. Our data illustrate that multiple domain-specific antibodies are essential to understanding the topology of a nucleoporin within the context of the NPC. Moreover, this approach has revealed new clues to the mechanisms by which Nup153 may contribute to nucleocytoplasmic transport.     

Peroxisomal targeting signal-1 receptor protein PEX5 from Leishmania donovani. Molecular, biochemical, and immunocytochemical characterization

The human pathogens of the Leishmania and Trypanosoma genera compartmentalize glycolytic and other key metabolic pathways in unique subcellular microbodies called glycosomes, organelles related to the peroxisomes of mammals and yeast. The molecular machinery that carries out the specific targeting of glycosomal proteins to the organelle has not been characterized, although the bulk of glycosomal proteins contain the COOH-terminal tripeptide glycosomal peroxisomal targeting signal-1 (PTS-1) similar to the mammalian and fungal peroxisomal targeting signal. To characterize the mechanisms of glycosomal targeting, the gene encoding PEX5, designated LdPEX5, has been isolated from Leishmania donovani. LdPEX5 encodes a 625-amino acid protein with a molecular mass of 69.7 kDa. Like its homologs in yeast and humans, LdPEX5 predicts a protein with seven copies of a tetratricopeptide repeat in its COOH-terminal half proposed to mediate PTS-1 binding and three copies of a WXXX(Y/F) motif in its NH(2) terminus conjectured to be essential for protein translocation into the organelle. LdPEX5 was overexpressed in Escherichia coli and purified to homogeneity for binding experiments and generation of antibodies. Recombinant LdPEX5 bound xanthine phosphoribosyltransferase (XPRT), a PTS-1 containing glycosomal protein with a K(D) of 4.2 nm, but did not bind an XPRT in which the PTS-1 had been deleted. Moreover, binding studies with the COOH-terminal half of the LdPEX5 confirmed that this portion of the PEX5 protein was capable of binding the XPRT PTS-1 with an affinity of 17.3 nm. Confocal microsocopy revealed that LdPEX5 was predominantly in the cytosolic milieu, and genetic analysis implied that LdPEX5 was an essential gene.     

The optical flow of planar surfaces

The human visual system can recover the 3-D shape of moving objects on the basis of motion information alone. Computational studies of this capacity have considered primarily non-planar rigid objects. With respect to moving planar surfaces, previous studies have shown that the planar velocity field has in general a two-fold ambiguity: there are two different planes engaged in different motions that can induce the same velocity field. The current analysis extends the analysis of the planar velocity field in four directions: (1) the use of flow parameters of the type suggested by Koenderink and van Doorn (Optica Acta, 1975, 22, 773-791), (2) the exclusion of confusable non-planar solutions, (3) a new proof and a new method for computing the 3-D motion and surface orientation (4) a comparison with the information available in orthographic velocity fields, which is important for determining the stability of the 3-D recovery process.     

An effect of parasite-encoded arginase on the outcome of murine cutaneous leishmaniasis

Classical activation of macrophages infected with Leishmania species results in expression and activation of inducible NO synthase (iNOS) leading to intracellular parasite killing. Macrophages can contrastingly undergo alternative activation with increased arginase activity, metabolism of arginine along the polyamine pathway, and consequent parasite survival. An active role for parasite-encoded arginase in host microbicidal responses has not previously been documented. To test the hypothesis that parasite-encoded arginase can influence macrophage responses to intracellular Leishmania, a comparative genetic approach featuring arginase-deficient mutants of L. mexicana lacking both alleles of the gene encoding arginase (Deltaarg), as well as wild-type and complemented Deltaarg controls (Deltaarg[pArg]), was implemented. The studies showed: 1) the absence of parasite arginase resulted in a significantly attenuated infection of mice (p<0.05); 2) poorer survival of Deltaarg in mouse macrophages than controls correlated with greater NO generation; 3) the difference between Deltaarg or control intracellular survival was abrogated in iNOS-deficient macrophages, suggesting iNOS activity was responsible for increased Deltaarg killing; 4) consistently, immunohistochemistry showed enhanced nitrotyrosine modifications in tissues of mice infected with Deltaarg compared with control parasites. Furthermore, 5) in the face of decreased parasite survival, lymph node cells draining cutaneous lesions of Deltaarg parasites produced more IFN-gamma and less IL-4 and IL-10 than controls. These data intimate that parasite-encoded arginase of Leishmania mexicana subverts macrophage microbicidal activity by diverting arginine away from iNOS.     

Adenovirus E4 ORF3 protein inhibits the interferon-mediated antiviral response

The PML oncogenic domain (POD/ND10/PML body) is a common target of DNA viruses, which replicate their genomes in proximity to this nuclear structure. The adenovirus early protein E4 ORF3 is both necessary and sufficient to rearrange PODs from punctate bodies into track-like structures. Although multiple hypotheses exist, the precise reason for this activity has not yet been elucidated. PML, the protein responsible for nucleating PODs, is an interferon (IFN)-stimulated gene, implicating the participation of this nuclear body in an innate antiviral response. Here, we demonstrate that E4 ORF3 is critical to the replicative success of adenovirus during the IFN-induced antiviral state. When cells are pretreated with either IFN-alpha or IFN-gamma, a mutant virus that does not express E4 ORF3 is severely compromised for replication. This result suggests the functional significance of ORF3 track formation is the inhibition of a POD-mediated, antiviral mechanism. Replication of the E4 ORF3 mutant virus can be rescued following the introduction of E4 ORF3 from evolutionarily divergent adenoviruses, suggesting a conserved function for E4 ORF3 inhibition of the IFN-induced antiviral state. Furthermore, E4 ORF3 inhibition of an IFN-induced response is unrelated to the inhibition of adenovirus replication by the Mre11-Rad50-Nbs1 DNA repair complex. We propose that the evolutionarily conserved function of the adenovirus E4 ORF3 protein is the inhibition of a host interferon response to viral infection via disruption of the PML oncogenic domain.     

Genetic selection for a highly functional cysteine-less membrane protein using site saturation mutagenesis

We describe an efficient method for generating highly functional membrane proteins with variant amino acids at defined positions that couples a modified site saturation strategy with functional genetic selection. We applied this method to the production of a cysteine-less variant of the Crithidia fasciculata inosine-guanosine permease CfNT2 to facilitate biochemical studies using thiol-specific modifying reagents. Of 10 endogenous cysteine residues in CfNT2, two cannot be replaced with serine or alanine without loss of function. High-quality single- and double-mutant libraries were produced by combining a previously reported site saturation mutagenesis scheme based on the Stratagene Quikchange method with a novel gel purification step that effectively eliminated template DNA from the products. Following selection for functional complementation in Saccharomyces cerevisiae cells auxotrophic for purines, several highly functional noncysteine substitutions were efficiently identified at each desired position, allowing the construction of cysteine-less variants of CfNT2 that retained wild-type affinity for inosine. This combination of an improved site saturation mutagenesis technique and positive genetic selection provides a simple and efficient means to identify functional and perhaps unexpected amino acid variants at a desired position.     

The national DBS brain tissue network pilot study: need for more tissue and more standardization

Over 70,000 DBS devices have been implanted worldwide; however, there remains a paucity of well-characterized post-mortem DBS brains available to researchers. We propose that the overall understanding of DBS can be improved through the establishment of a Deep Brain Stimulation-Brain Tissue Network (DBS-BTN), which will further our understanding of DBS and brain function. The objectives of the tissue bank are twofold: (a) to provide a complete (clinical, imaging and pathological) database for DBS brain tissue samples, and (b) to make available DBS tissue samples to researchers, which will help our understanding of disease and underlying brain circuitry. Standard operating procedures for processing DBS brains were developed as part of the pilot project. Complete data files were created for individual patients and included demographic information, clinical information, imaging data, pathology, and DBS lead locations/settings. 19 DBS brains were collected from 11 geographically dispersed centers from across the U.S. The average age at the time of death was 69.3 years (51?C92, with a standard deviation or SD of 10.13). The male:female ratio was almost 3:1. Average post-mortem interval from death to brain collection was 10.6 h (SD of 7.17). The DBS targets included: subthalamic nucleus, globus pallidus interna, and ventralis intermedius nucleus of the thalamus. In 16.7% of cases the clinical diagnosis failed to match the pathological diagnosis. We provide neuropathological findings from the cohort, and perilead responses to DBS. One of the most important observations made in this pilot study was the missing data, which was approximately 25% of all available data fields. Preliminary results demonstrated the feasibility and utility of creating a National DBS-BTN resource for the scientific community. We plan to improve our techniques to remedy omitted clinical/research data, and expand the Network to include a larger donor pool. We will enhance sample preparation to facilitate advanced molecular studies and progenitor cell retrieval.