The rockefeller university

Background: Recent animal studies have shown that the level of stress-responsive arginine vasopressin (AVP) gene expression in the amygdala is increased during early withdrawal from long-term heroin or cocaine administration. The selective AVP V1b receptor antagonist SSR149415 (capable of exerting antidepressant-like and anxiolytic effects in animal models) also blocked stress-induced reinstatement of drug-seeking behavior. This study was undertaken to investigate the effects of alcohol and to determine whether (i) there are genetically determined differences in basal AVP mRNA levels in the medial/central amygdala (Me/CeA) and medial hypothalamus (MH) between selectively bred Sardinian alcohol-preferring (sP) and alcohol-nonpreferring (sNP) rats; (ii) the AVP mRNA levels are altered by long-term alcohol drinking in sP rats; and (iii) the V1b receptor antagonist SSR149415 alters alcohol drinking in sP rats. Methods: In Experiment 1, AVP mRNA levels were measured in the Me/CeA and MH of alcohol-naive sP and sNP rats, and sP rats exposed to the standard, homecage 2-bottle "alcohol versus water" choice regimen 24 h/d for 17 days. In Experiment 2, SSR149415 (0, 3, 10, or 30 mg/kg; intraperitoneal) was acutely administered 30 minutes before lights off to alcohol-experienced sP rats. Alcohol, water, and food intake were monitored 6 and 24 hours later. Results: We found higher basal AVP mRNA levels in both Me/CeA and MH of alcohol-naive sP than sNP rats; alcohol consumption decreased AVP mRNA levels in both brain regions of sP rats, suggesting genetically determined differences between the 2 rat lines and in the effects of alcohol drinking in sP rats. Acute treatment with SSR149415 significantly reduced alcohol intake of sP rats. Conclusion: The stress-responsive AVP/V1b receptor system is 1 component of the neural circuitry underlying high alcohol drinking in sP rats.

So far, only a few of the interactions between the approximate to 30 nucleoporins comprising the modular structure of the nuclear pore complex have been defined at atomic resolution. Here we report the crystal structure, at 2.6 angstrom resolution, of a heterotrimeric complex, composed of fragments of three cytoplasmically oriented nucleoporins of yeast: Nup82, Nup116, and Nup159. Our data show that the Nup82 fragment, representing more than the N-terminal half of the molecule, folds into an extensively decorated, seven-bladed beta-propeller that forms the centerpiece of this heterotrimeric complex and anchors both a C-terminal fragment of Nup116 and the C-terminal tail of Nup159. Binding between Nup116 and Nup82 is mutually reinforced via two loops, one emanating from the Nup82 beta-propeller and the other one from the beta-sandwich fold of Nup116, each contacting binding pockets in their counterparts. The Nup82-Nup159 interaction occurs through an amphipathic alpha-helix of Nup159, which is cradled in a large hydrophobic groove that is generated from several large surface decorations of the Nup82 beta-propeller. Although Nup159 and Nup116 fragments bind to the Nup82 beta-propeller in close vicinity, there are no direct contacts between them, consistent with the noncooperative binding that was detected biochemically. Extensive mutagenesis delineated hot-spot residues for these interactions. We also showed that the Nup82 beta-propeller binds to other yeast Nup116 family members, Nup145N, Nup100 and to the mammalian homolog, Nup98. Notably, each of the three nucleoporins contains additional nuclear pore complex binding sites, distinct from those that were defined here in the heterotrimeric Nup82.Nup159.Nup116 complex.

A central question in Alzheimer's disease (AD) research is what role beta-amyloid peptide (A beta) plays in synaptic dysfunction. Synaptic activity increases A beta secretion, potentially inhibiting synapses, but also decreases intraneuronal A beta, protecting synapses. We now show that levels of secreted A beta fall with time in culture in neurons of AD-transgenic mice, but not wild-type mice. Moreover, the ability of synaptic activity to elevate secreted A beta and reduce intraneuronal A beta becomes impaired in AD-transgenic but not wild-type neurons with time in culture. We demonstrate that synaptic activity promotes an increase in the A beta-degrading protease neprilysin at the cell surface and a concomitant increase in colocalization with A beta 42. Remarkably, AD-transgenic but not wild-type neurons show reduced levels of neprilysin with time in culture. This impaired ability to secrete A beta and reduce intraneuronal A beta has important implications for the pathogenesis and treatment of AD.

Immunization of BALB/c mice with irradiated sporozoites (IrSp) of Plasmodium yoelii can lead to sterile immunity. The circumsporozoite protein (CSP) plays a dominant role in protection. Nevertheless after hyper-immunization with IrSp, complete protection is obtained in CSP-transgenic BALB/c mice that are T-cell tolerant to the CSP and cannot produce antibodies [CSP-Tg/JhT(-/-)]. This protection is mediated exclusively by CD8(+) T cells [1]. To identify the non-CSP protective T cell antigens, we studied the properties of 34 P. yoelii sporozoite antigens that are predicted to be secreted and to contain strong Kd-restricted CD8(+) T cell epitopes. The synthetic peptides corresponding to the epitopes were used to screen for the presence of peptide-specific CD8(+) T cells secreting interferon-gamma (IFN-gamma) in splenocytes from CSP-Tg/JhT(-/-) BALB/c mice hyper immunized with IrSp. However, the numbers of IFN-gamma-secreting splenocytes specific for the non-CSP antigen-derived peptides were 20-100 times lower than those specific for the CSP-specific peptide. When mice were immunized with recombinant adenoviruses expressing selected non-CSP antigens, the animals were not protected against challenge with P. yoelii sporozoites although large numbers of CD8(+). specific T cells were generated. (C) 2011 Elsevier Ltd. All rights reserved.

The nuclear pore complex encloses a central channel for nucleocytoplasmic transport, which is thought to consist of three nucleoporins, Nup54, Nup58, and Nup62. However, the structure and composition of the channel are elusive. We determined the crystal structures of the interacting domains between these nucleoporins and pieced together the molecular architecture of the mammalian transport channel. Located in the channel midplane is a flexible Nup54, Nup58 ring that can undergo large rearrangements yielding diameter changes from similar to 20 to similar to 40 nm. Nup62 . Nup54 triple helices project alternately up and down from either side of the midplane ring and form nucleoplasmic and cytoplasmic entries. The channel consists of as many as 224 copies of the three nucleoporins, amounting to a molar mass of 12.3 MDa and contributing 256 phenylalanine-glycine repeat regions. We propose that the occupancy of these repeat regions with transport receptors modulates ring diameter and transport activity.

Natural killer T cells (NKT cells) recognize glycolipid antigens presented by CD1d. These cells express an evolutionarily conserved, invariant T cell antigen receptor (TCR), but the forces that drive TCR conservation have remained uncertain. Here we show that NKT cells recognized diacylglycerol-containing glycolipids from Streptococcus pneumoniae, the leading cause of community-acquired pneumonia, and group B Streptococcus, which causes neonatal sepsis and meningitis. Furthermore, CD1d-dependent responses by NKT cells were required for activation and host protection. The glycolipid response was dependent on vaccenic acid, which is present in low concentrations in mammalian cells. Our results show how microbial lipids position the sugar for recognition by the invariant TCR and, most notably, extend the range of microbes recognized by this conserved TCR to several clinically important bacteria.

Child low-level lead (Pb) exposure is an unresolved public health problem and an unaddressed child health disparity. Particularly in cases of low-level exposure, source removal can be impossible to accomplish, and the only practical strategy for reducing risk may be primary prevention. Genetic biomarkers of increased neurotoxic risk could help to identify small subgroups of children for early intervention. Previous studies have suggested that, by way of a distinct mechanism, delta-aminolevulinic acid dehydratase single nucleotide polymorphism 2 (ALAD(2)) and/or peptide transporter 2*2 haplotype (hPEPT2*2) increase Pb blood burden in children. Studies have not yet examined whether sex mediates the effects of genotype on blood Pb burden. Also, previous studies have not included blood iron (Fe) level in their analyses. Blood and cheek cell samples were obtained from 306 minority children, ages 5.1 to 12.9 years. 208 Pb and 56 Fe levels were determined with inductively coupled plasma-mass spectrometry. General linear model analyses were used to examine differences in Pb blood burden by genotype and sex while controlling for blood Fe level. The sample geometric mean Pb level was 2.75 mu g/dl. Pb blood burden was differentially higher in ALAD2 heterozygous boys and hPEPT2*2 homozygous boys. These results suggest that the effect of ALAD2 and hPEPT2*2 on Pb blood burden may be sexually dimorphic. ALAD2 and hPEPT2*2 may be novel biomarkers of health and mental health risks in male children exposed to low levels of Pb.

The dynamic cellular reorganization needed for successful mitosis requires regulatory cues that vary across microns. The chromosomal passenger complex (CPC) is a conserved regulator involved in key mitotic events such as chromosome-microtubule attachment and spindle midzone formation. Recently, spatial phosphorylation gradients have been reported for CPC substrates, raising the possibility that CPC-dependent signaling establishes order on the micron-length scale in dividing cells. However, this hypothesis has not been tested, largely because of incomplete characterization of the CPC-dependent phosphorylation dynamics. Without these data it is difficult to evaluate perturbations of CPC signaling and select one that alters the spatial organization of substrate phosphorylation at a particular stage of mitosis, without changing overall phosphorylation levels. Here we examine the spatiotemporal dynamics of CPC-dependent phosphorylation along microtubules throughout mitosis using a Forster resonance energy transfer-based sensor. We find that a CPC substrate phosphorylation gradient, with highest phosphorylation levels between the two spindle poles, emerges when a cell enters mitosis. Interestingly, this gradient becomes undetectable at metaphase, but can be revealed by partially suppressing CPC activity, suggesting that high substrate phosphorylation levels can mask persistent CPC-dependent spatial patterning. After anaphase onset, the gradient emerges and persists until cell cleavage. Selective mislocalization of the CPC during anaphase suppresses gradient formation, but overall substrate phosphorylation levels remain unchanged. Under these conditions, the spindle midzone fails to organize and function properly. Our findings suggest a model in which the CPC establishes phosphorylation gradients to coordinate the spatiotemporal dynamics needed for error-free cell division.

Clones that encode the biosynthesis of long-chain N-acyl amino acids are frequently recovered from activity-based screens of soil metagenomic libraries. Members of a diverse set of enzymes referred to as N-acyl amino acid synthases are responsible for the production of all metagenome-derived N-acyl amino acids characterized to date. Based on the frequency at which N-acyl amino acid synthase genes have been identified from metagenomic samples, related genes are expected to be common throughout the global bacterial metagenome. Homologs of metagenome-derived N-acyl amino acid synthase genes are scarce, however, within the sequenced genomes of cultured bacterial species. Toward the goal of understanding the role(s) played by N-acyl amino acids in environmental bacteria, we looked for conserved genetic features that are positionally linked to metagenome-derived N-acyl amino acid synthase genes. This analysis revealed that N-acyl amino acid synthase genes are frequently found adjacent to genes predicted to encode PEP-CTERM motif-containing proteins and, in some cases, other conserved elements of the PEP-CTERM/exosortase system. Although relatively little is known about the PEP-CTERM/exosortase system, its core components are believed to represent the putative Gram-negative equivalent of the LPXTG/sortase protein-sorting system of Gram-positive bacteria. During the course of this investigation, we were able to provide evidence that an uncharacterized family of hypothetical acyltransferases, which had previously been linked to the PEP-CTERM/exosortase system by bioinformatics, is a new family of N-acyl amino acid synthases that is widely distributed among the PEP-CTERM/exosortase system-containing Proteobacteria.

A measurement of the top-quark mass is presented using Tevatron data from proton-antiproton collisions at center-of-mass energy root s = 1.96 TeV collected with the CDF II detector. Events are selected from a sample of candidates for production of t (t) over bar pairs that decay into the lepton + jets channel. The top-quark mass is measured with an unbinned maximum likelihood method where the event probability density functions are calculated using signal and background matrix elements, as well as a set of parametrized jet-to-parton transfer functions. The likelihood function is maximized with respect to the top-quark mass, the signal fraction in the sample, and a correction to the jet energy scale (JES) calibration of the calorimeter jets. The simultaneous measurement of the JES correction (Delta(JES)) amounts to an additional in situ jet energy calibration based on the known mass of the hadronically decaying W boson. Using the data sample of 578 lepton + jets candidate events, corresponding to 3.2 fb(-1) of integrated luminosity, the top-quark mass is measured to be m(t) = 172.4 +/- 1.4(stat + Delta(JES)) +/- 1.3(syst) GeV/c(2).