PNAS

Cerebellar-dependent motor learning is based on pruning a Purkinje cell population response.

Publication Date: 2008 May 13 PMID: 18477700
Authors: Catz, N. - Dicke, P. W. - Thier, P.
Journal: Proc Natl Acad Sci U S A

The improvement of motor behavior, based on experience, is a form of learning that is critically dependent on the cerebellum. A well studied example of cerebellar motor learning is short-term saccadic adaptation (STSA). In STSA, information on saccadic errors is used to improve future saccades. The information optimizing saccade metrics is conveyed by Purkinje cells simple spikes (PC-SS) because they are the critical input to the premotor circuits for saccades. We recorded PC-SS of monkeys undergoing STSA to reveal the code used for improving behavior. We found that the discharge of individual PC-SS was unable to account for the behavioral changes. The PC-SS population burst (PB), however, exhibited changes that closely paralleled the qualitatively different changes of saccade kinematics associated with gain-increase and gain-decrease STSA, respectively. Gain-increase STSA, characterized by an increase in saccade duration, replicates the relationship between saccade duration and the end of the PB valid for unadapted saccades. In contrast, gain-decrease STSA, which sports normal saccade duration but reduced saccadic velocity, is characterized by a PB that ends well before the adapted saccade. This suggests that the duration of normal as well as gain-increased saccades is determined by appropriately setting the end of PB end. However, the duration of gain-decreased saccades is apparently not modified by the cerebellum because the PB signals ends too early to determine saccade end. In summary, STSA, and most probably cerebellar-dependent learning in general, is based on optimizing the shape of a PC-SS population response.

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EB1 promotes Aurora-B kinase activity through blocking its inactivation by protein phosphatase 2A.

Publication Date: 2008 May 13 PMID: 18477699
Authors: Sun, L. - Gao, J. - Dong, X. - Liu, M. - Li, D. - Shi, X. - Dong, J. T. - Lu, X. - Liu, C. - Zhou, J.
Journal: Proc Natl Acad Sci U S A

EB1 (end-binding protein 1) is a key player in the regulation of microtubule dynamics. In concert with its binding partners, adenomatous polyposis coli and p150(glued), EB1 plays a crucial role in a variety of microtubule-based cellular processes. In this study we have identified in a yeast two-hybrid screen the mitotic kinase and chromosome passenger protein Aurora-B as a binding partner of EB1. GST pull-down and immunoprecipitation experiments reveal a specific interaction between Aurora-B and EB1 both in cells and in vitro. Immunofluorescence microscopy shows that these two proteins colocalize on the central spindle in anaphase and in the midbody during cytokinesis. Kinase assays using both immunoprecipitated and purified Aurora-B demonstrate that EB1 is not a substrate of Aurora-B. Rather, EB1 positively regulates Aurora-B kinase activity. EB1 overexpression remarkably enhances Aurora-B activity and knockdown of its expression impairs Aurora-B activity. Our data further show that EB1 is able to protect Aurora-B from dephosphorylation/inactivation by protein phosphatase 2A (PP2A) by blocking PP2A binding to Aurora-B. These findings establish Aurora-B as an EB1-interacting protein and suggest that EB1 stimulates Aurora-B activity through antagonizing its dephosphorylation/inactivation by PP2A.

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Electrical microstimulation thresholds for behavioral detection and saccades in monkey frontal eye fields.

Publication Date: 2008 May 13 PMID: 18477698
Authors: Murphey, D. K. - Maunsell, J. H.
Journal: Proc Natl Acad Sci U S A

The frontal eye field (FEF) is involved in the transformation of visual signals into saccadic eye movements. Although it is often considered an oculomotor structure, several lines of evidence suggest that the FEF also contributes to visual perception and attention. To better understand the range of behaviors to which the FEF can contribute, we tested whether monkeys could detect activation of their FEF by electrical microstimulation with currents below those that cause eye movements. We found that stimulation of FEF neurons could almost always be detected at levels below those needed to generate saccades and that the electrical current needed for detection was highly correlated with that needed to generate a saccade. This relationship between detection and saccade thresholds can be explained if FEF neurons represent preparation to make particular saccades and subjects can be aware of such preparations without acting on them when the representation is not strong.

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Prediction of membrane-protein topology from first principles.

Publication Date: 2008 May 13 PMID: 18477697
Authors: Bernsel, A. - Viklund, H. - Falk, J. - Lindahl, E. - Heijne, G. V. - Elofsson, A.
Journal: Proc Natl Acad Sci U S A

The current best membrane-protein topology-prediction methods are typically based on sequence statistics and contain hundreds of parameters that are optimized on known topologies of membrane proteins. However, because the insertion of transmembrane helices into the membrane is the outcome of molecular interactions among protein, lipids and water, it should be possible to predict topology by methods based directly on physical data, as proposed >20 years ago by Kyte and Doolittle. Here, we present two simple topology-prediction methods using a recently published experimental scale of position-specific amino acid contributions to the free energy of membrane insertion that perform on a par with the current best statistics-based topology predictors. This result suggests that prediction of membrane-protein topology and structure directly from first principles is an attainable goal, given the recently improved understanding of peptide recognition by the translocon.

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A rationally engineered misacylating aminoacyl-tRNA synthetase.

Publication Date: 2008 May 13 PMID: 18477696
Authors: Bullock, T. L. - Rodriguez-Hernandez, A. - Corigliano, E. M. - Perona, J. J.
Journal: Proc Natl Acad Sci U S A

Information transfer from nucleic acid to protein is mediated by aminoacyl-tRNA synthetases, which catalyze the specific pairings of amino acids with transfer RNAs. Despite copious sequence and structural information on the 22 tRNA synthetase families, little is known of the enzyme signatures that specify amino acid selectivities. Here, we show that transplanting a conserved arginine residue from glutamyl-tRNA synthetase (GluRS) to glutaminyl-tRNA synthetase (GlnRS) improves the K(M) of GlnRS for noncognate glutamate. Two crystal structures of this C229R GlnRS mutant reveal that a conserved twin-arginine GluRS amino acid identity signature cannot be incorporated into GlnRS without disrupting surrounding protein structural elements that interact with the tRNA. Consistent with these findings, we show that cumulative replacement of other primary binding site residues in GlnRS, with those of GluRS, only slightly improves the ability of the GlnRS active site to accommodate glutamate. However, introduction of 22 amino acid replacements and one deletion, including substitution of the entire primary binding site and two surface loops adjacent to the region disrupted in C229R, improves the capacity of Escherichia coli GlnRS to synthesize misacylated Glu-tRNA(Gln) by 16,000-fold. This hybrid enzyme recapitulates the function of misacylating GluRS enzymes found in organisms that synthesize Gln-tRNA(Gln) by an alternative pathway. These findings implicate the RNA component of the contemporary GlnRS-tRNA(Gln) complex in mediating amino acid specificity. This role for tRNA may persist as a relic of primordial cells in which the evolution of the genetic code was driven by RNA-catalyzed amino acid-RNA pairing.

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Hypoxia and the HIF-1 transcriptional pathway reorganize a neuronal circuit for oxygen-dependent behavior in Caenorhabditis elegans.

Publication Date: 2008 May 13 PMID: 18477695
Authors: Chang, A. J. - Bargmann, C. I.
Journal: Proc Natl Acad Sci U S A

Rapid behavioral responses to oxygen are generated by specialized sensory neurons that sense hypoxia and hyperoxia. On a slower time scale, many cells respond to oxygen through the activity of the hypoxia-inducible transcription factor HIF-1. Here, we show that in the nematode Caenorhabditis elegans, prolonged growth in hypoxia alters the neuronal circuit for oxygen preference by activating the hif-1 pathway. Activation of hif-1 by hypoxia or by mutations in its negative regulator egl-9/prolyl hydroxylase shifts behavioral oxygen preferences to lower concentrations and eliminates a regulatory input from food. At a neuronal level, hif-1 activation transforms a distributed, regulated neuronal network for oxygen preference into a smaller, fixed network that is constitutively active. The hif-1 pathway acts both in neurons and in gonadal endocrine cells to regulate oxygen preference. These results suggest that physiological detection of hypoxia by multiple tissues provides adaptive information to neuronal circuits to modify behavior.

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Atomic interactions of neonicotinoid agonists with AChBP: Molecular recognition of the distinctive electronegative pharmacophore.

Publication Date: 2008 May 13 PMID: 18477694
Authors: Talley, T. T. - Harel, M. - Hibbs, R. E. - Radic, Z. - Tomizawa, M. - Casida, J. E. - Taylor, P.
Journal: Proc Natl Acad Sci U S A

Acetylcholine-binding proteins (AChBPs) from mollusks are suitable structural and functional surrogates of the nicotinic acetylcholine receptors when combined with transmembrane spans of the nicotinic receptor. These proteins assemble as a pentamer with identical ACh binding sites at the subunit interfaces and show ligand specificities resembling those of the nicotinic receptor for agonists and antagonists. A subset of ligands, termed the neonicotinoids, exhibit specificity for insect nicotinic receptors and selective toxicity as insecticides. AChBPs are of neither mammalian nor insect origin and exhibit a distinctive pattern of selectivity for the neonicotinoid ligands. We define here the binding orientation and determinants of differential molecular recognition for the neonicotinoids and classical nicotinoids by estimates of kinetic and equilibrium binding parameters and crystallographic analysis. Neonicotinoid complex formation is rapid and accompanied by quenching of the AChBP tryptophan fluorescence. Comparisons of the neonicotinoids imidacloprid and thiacloprid in the binding site from Aplysia californica AChBP at 2.48 and 1.94 A in resolution reveal a single conformation of the bound ligands with four of the five sites occupied in the pentameric crystal structure. The neonicotinoid electronegative pharmacophore is nestled in an inverted direction compared with the nicotinoid cationic functionality at the subunit interfacial binding pocket. Characteristic of several agonists, loop C largely envelops the ligand, positioning aromatic side chains to interact optimally with conjugated and hydrophobic regions of the neonicotinoid. This template defines the association of interacting amino acids and their energetic contributions to the distinctive interactions of neonicotinoids.

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Common SNP in pre-miR-146a decreases mature miR expression and predisposes to papillary thyroid carcinoma.

Publication Date: 2008 May 12 PMID: 18474871
Authors: Jazdzewski, K. - Murray, E. L. - Franssila, K. - Jarzab, B. - Schoenberg, D. R. - de la Chapelle, A.
Journal: Proc Natl Acad Sci U S A

Although papillary thyroid carcinoma (PTC) displays strong heritability, no predisposing germ-line mutations have been found. We show that a common G/C polymorphism (rs2910164) within the pre-miR-146a sequence reduced the amount of pre- and mature miR-146a from the C allele 1.9- and 1.8-fold, respectively, compared with the G allele. This is matched by a similar decrease in the amount of each pre-miR generated from the corresponding pri-miR-146a in an in vitro processing reaction. The C allele also interfered with the binding of a nuclear factor to pre-miR-146a. The reduction in miR-146a led to less efficient inhibition of target genes involved in the Toll-like receptor and cytokine signaling pathway (TRAF6, IRAK1), and PTC1 (also known as CCDC6 or H4), a gene frequently rearranged with RET proto-oncogene in PTC. In an association study of 608 PTC patients and 901 controls, we found marked differences in genotype distribution of rs2910164 (P = 0.000002), the GC heterozygous state being associated with an increased risk of acquiring PTC (odds ratio = 1.62, P = 0.000007), and both homozygous states protective with odds ratio = 0.42 for the CC genotype (P = 0.003) and odds ratio = 0.69 for the GG genotype (P = 0.0006). Moreover, 4.7% of tumors had undergone somatic mutations of the SNP sequence. Thus, our data suggest that a common polymorphism in pre-miR-146a affects the miR expression, contributes to the genetic predisposition to PTC, and plays a role in the tumorigenesis through somatic mutation. Preliminary evidence suggests that these effects are mediated through target genes whose expression is affected by the SNP status.

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Cuticular hydrocarbon analysis of an awake behaving fly using direct analysis in real-time time-of-flight mass spectrometry.

Publication Date: 2008 May 12 PMID: 18474870
Authors: Yew, J. Y. - Cody, R. B. - Kravitz, E. A.
Journal: Proc Natl Acad Sci U S A

In mammals and insects, pheromones strongly influence social behaviors such as aggression and mate recognition. In Drosophila melanogaster, pheromones in the form of cuticular hydrocarbons play prominent roles in courtship. GC/MS is the primary analytical tool currently used to study Drosophila cuticular hydrocarbons. Although GC/MS is highly reproducible and sensitive, it requires that the fly be placed in a lethal solution of organic solvent, thereby impeding further behavioral studies. We present a technique for the analysis of hydrocarbons and other surface molecules from live animals by using direct analysis in real-time (DART) MS. Cuticular hydrocarbons were sampled from the surface of a restrained, awake behaving fly by using several brief, carefully controlled depressions of the abdomen with a small steel probe. DART mass spectral analysis of the probe detected ions with mass-to-charge ratio (m/z) of the protonated molecule corresponding to many of the previously identified unsaturated hydrocarbons. Six additional cuticular hydrocarbons also were identified. Consistent with previous GC/MS studies, male and female differences in chemical composition were evident. Spatial differences in the expression profile also were observed on males. Sampling from an individual female first as a virgin and then 45 and 90 min after successful copulation showed that mass signals likely to correspond to cis-vaccenyl acetate, tricosene, and pentacosene increased in relative intensity after courtship. This method provides near-instantaneous analysis of an individual animal's chemical profile in parallel with behavioral studies and could be extended to other models of pheromone-mediated behavior.

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Evidence for the aldo-keto reductase pathway of polycyclic aromatic trans-dihydrodiol activation in human lung A549 cells.

Publication Date: 2008 May 13 PMID: 18474869
Authors: Park, J. H. - Mangal, D. - Tacka, K. A. - Quinn, A. M. - Harvey, R. G. - Blair, I. A. - Penning, T. M.
Journal: Proc Natl Acad Sci U S A

Polycyclic aromatic hydrocarbons (PAHs) are tobacco carcinogens implicated in the causation of human lung cancer. Metabolic activation is a key prerequisite for PAHs to cause their deleterious effects. Using human lung adenocarcinoma (A549) cells, we provide evidence for the metabolic activation of (+/-)-trans-7,8dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-trans-dihydrodiol) by aldo-keto reductases (AKRs) to yield benzo[a]pyrene-7,8-dione (B[a]P-7,8-dione), a redox-active o-quinone. We show that B[a]P-7,8-trans-dihydrodiol (AKR substrate) and B[a]P-7,8-dione (AKR product) lead to the production of intracellular reactive oxygen species (ROS) (measured as an increase in dichlorofluorescin diacetate fluores-cence) and that similar changes were not observed with the regioisomer (+/-)-trans-4,5-dihydroxy-4,5-dihydrobenzo[a]pyrene or the diol-epoxide, (+/-)-anti-7,8-dihydroxy-9alpha,10beta-epoxy-7,8,9,10-tetrahydro-B[a]P. B[a]P-7,8-trans-dihydrodiol and B[a]P-7,8-dione also caused a decrease in glutathione levels and an increase in NADP(+)/NADPH ratios, with a concomitant increase in single-strand breaks (as measured by the comet assay) and 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dGuo). The specificity of the comet assay was validated by coupling it to human 8-oxo-guanine glycosylase (hOGG1), which excises 8-oxo-Gua to yield single-strand breaks. The levels of 8-oxo-dGuo observed were confirmed by an immunoaffinity purification stable isotope dilution ([(15)N(5)]-8-oxo-dGuo) liquid chromatography-electrospray ionization/multiple reaction monitoring/mass spectrometry (LC-ESI/MRM/MS) assay. B[a]P-7,8-trans-dihydrodiol produced DNA strand breaks in the hOGG1-coupled comet assay as well as 8-oxo-dGuo (as measured by LC-ESI/MRM/MS) and was enhanced by a catechol O-methyl transferase (COMT) inhibitor, suggesting that COMT protects against o-quinone-mediated redox cycling. We conclude that activation of PAH-trans-dihydrodiols by AKRs in lung cells leads to ROS-mediated genotoxicity and contributes to lung carcinogenesis.

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A VAST staging area for regulatory proteins.

Publication Date: 2008 May 12 PMID: 18474868
Authors: Mitchell, A. P.
Journal: Proc Natl Acad Sci U S A

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Role of GSK3{beta} in behavioral abnormalities induced by serotonin deficiency.

Publication Date: 2008 May 12 PMID: 18474867
Authors: Belmaker, R. H. - Agam, G. - Bersudsky, Y.
Journal: Proc Natl Acad Sci U S A

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Comprehensive screening for antigens overexpressed on carcinomas via isolation of human mAbs that may be therapeutic.

Publication Date: 2008 May 12 PMID: 18474866
Authors: Kurosawa, G. - Akahori, Y. - Morita, M. - Sumitomo, M. - Sato, N. - Muramatsu, C. - Eguchi, K. - Matsuda, K. - Takasaki, A. - Tanaka, M. - Iba, Y. - Hamada-Tsutsumi, S. - Ukai, Y. - Shiraishi, M. - Suzuki, K. - Kurosawa, M. - Fujiyama, S. - Takahashi, N. - Kato, R. - Mizoguchi, Y. - Shamoto, M. - Tsuda, H. - Sugiura, M. - Hattori, Y. - Miyakawa, S. - Shiroki, R. - Hoshinaga, K. - Hayashi, N. - Sugioka, A. - Kurosawa, Y.
Journal: Proc Natl Acad Sci U S A

Although several murine mAbs that have been humanized became useful therapeutic agents against a few malignancies, therapeutic Abs are not yet available for the majority of the human cancers because of our lack of knowledge of which antigens (Ags) can become useful targets. In the present study we established a procedure for comprehensive identification of such Ags through the extensive isolation of human mAbs that may become therapeutic. Using the phage-display Ab library we isolated a large number of human mAbs that bind to the surface of tumor cells. They were individually screened by immunostaining, and clones that preferentially and strongly stained the malignant cells were chosen. The Ags recognized by those clones were isolated by immunoprecipitation and identified by MS. We isolated 2,114 mAbs with unique sequences and identified 21 distinct Ags highly expressed on several carcinomas. Of those 2,114 mAbs 356 bound specifically to one of the 21 Ags. After preparing complete IgG(1) Abs the in vitro assay for Ab-dependent cell-mediated cytotoxicity (ADCC) and the in vivo assay in cancer-bearing athymic mice were performed to examine antitumor activity. The mAbs converted to IgG(1) revealed effective ADCC as well as antitumor activity in vivo. Because half of the 21 Ags showed distinct tumor-specific expression pattern and the mAbs isolated showed various characteristics with strong affinity to the Ag, it is likely that some of the Ags detected will become useful targets for the corresponding carcinoma therapy and that several mAbs will become therapeutic agents.

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A truer measure of our ignorance.

Publication Date: 2008 May 13 PMID: 18474865
Authors: Amaral, L. A.
Journal: Proc Natl Acad Sci U S A

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Long-distance splicing.

Publication Date: 2008 May 13 PMID: 18474864
Authors: Anderson, A. M. - Staley, J. P.
Journal: Proc Natl Acad Sci U S A

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Increasing fluid intelligence is possible after all.

Publication Date: 2008 May 13 PMID: 18474863
Authors: Sternberg, R. J.
Journal: Proc Natl Acad Sci U S A

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Colloquium Paper: A DNA-based nanomechanical device with three robust states.

Publication Date: 2008 May 12 PMID: 18474862
Authors: Chakraborty, B. - Sha, R. - Seeman, N. C.
Journal: Proc Natl Acad Sci U S A

DNA has been used to build a variety of devices, ranging from those that are controlled by DNA structural transitions to those that are controlled by the addition of specific DNA strands. These sequence-dependent devices fulfill the promise of DNA in nanotechnology because a variety of devices in the same physical environment can be controlled individually. Many such devices have been reported, but most of them contain one or two structurally robust end states, in addition to a floppy intermediate or even a floppy end state. We describe a system in which three different structurally robust end states can be obtained, all resulting from the addition of different set strands to a single floppy intermediate. This system is an extension of the PX-JX(2) DNA device. The three states are related to each other by three different motions, a twofold rotation, a translation of approximately 2.1-2.5 nm, and a twofold screw rotation, which combines these two motions. We demonstrate the transitions by gel electrophoresis, by fluorescence resonance energy transfer, and by atomic force microscopy. The control of this system by DNA strands opens the door to trinary logic and to systems containing N devices that are able to attain 3(N) structural states.

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Estimating the size of the human interactome.

Publication Date: 2008 May 13 PMID: 18474861
Authors: Stumpf, M. P. - Thorne, T. - de Silva, E. - Stewart, R. - An, H. J. - Lappe, M. - Wiuf, C.
Journal: Proc Natl Acad Sci U S A

After the completion of the human and other genome projects it emerged that the number of genes in organisms as diverse as fruit flies, nematodes, and humans does not reflect our perception of their relative complexity. Here, we provide reliable evidence that the size of protein interaction networks in different organisms appears to correlate much better with their apparent biological complexity. We develop a stable and powerful, yet simple, statistical procedure to estimate the size of the whole network from subnet data. This approach is then applied to a range of eukaryotic organisms for which extensive protein interaction data have been collected and we estimate the number of interactions in humans to be approximately 650,000. We find that the human interaction network is one order of magnitude bigger than the Drosophila melanogaster interactome and approximately 3 times bigger than in Caenorhabditis elegans.

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Quorum decision-making facilitates information transfer in fish shoals.

Publication Date: 2008 May 13 PMID: 18474860
Authors: Ward, A. J. - Sumpter, D. J. - Couzin, I. D. - Hart, P. J. - Krause, J.
Journal: Proc Natl Acad Sci U S A

Despite the growing interest in collective phenomena such as "swarm intelligence" and "wisdom of the crowds," little is known about the mechanisms underlying decision-making in vertebrate animal groups. How do animals use the behavior of others to make more accurate decisions, especially when it is not possible to identify which individuals possess pertinent information? One plausible answer is that individuals respond only when they see a threshold number of individuals perform a particular behavior. Here, we investigate the role of such "quorum responses" in the movement decisions of fish (three-spine stickleback, Gasterosteus aculeatus). We show that a quorum response to conspecifics can explain how sticklebacks make collective movement decisions, both in the absence and presence of a potential predation risk. Importantly our experimental work shows that a quorum response can reduce the likelihood of amplification of nonadaptive following behavior. Whereas the traveling direction of solitary fish was strongly influenced by a single replica conspecific, the replica was largely ignored by larger groups of four or eight sticklebacks under risk, and the addition of a second replica was required to exert influence on the movement decisions of such groups. Model simulations further predict that quorum responses by fish improve the accuracy and speed of their decision-making over that of independent decision-makers or those using a weak linear response. This study shows that effective and accurate information transfer in groups may be gained only through nonlinear responses of group members to each other, thus highlighting the importance of quorum decision-making.

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Sildenafil and cardiomyocyte-specific cGMP signaling prevent cardiomyopathic changes associated with dystrophin deficiency.

Publication Date: 2008 May 13 PMID: 18474859
Authors: Khairallah, M. - Khairallah, R. J. - Young, M. E. - Allen, B. G. - Gillis, M. A. - Danialou, G. - Deschepper, C. F. - Petrof, B. J. - Des Rosiers, C.
Journal: Proc Natl Acad Sci U S A

We recently demonstrated early metabolic alterations in the dystrophin-deficient mdx heart that precede overt cardiomyopathy and may represent an early "subclinical" signature of a defective nitric oxide (NO)/cGMP pathway. In this study, we used genetic and pharmacological approaches to test the hypothesis that enhancing cGMP, downstream of NO formation, improves the contractile function, energy metabolism, and sarcolemmal integrity of the mdx heart. We first generated mdx mice overexpressing, in a cardiomyocyte-specific manner, guanylyl cyclase (GC) (mdx/GC(+/0)). When perfused ex vivo in the working mode, 12- and 20-week-old hearts maintained their contractile performance, as opposed to the severe deterioration observed in age-matched mdx hearts, which also displayed two to three times more lactate dehydrogenase release than mdx/GC(+/0). At the metabolic level, mdx/GC(+/0) displayed a pattern of substrate selection for energy production that was similar to that of their mdx counterparts, but levels of citric acid cycle intermediates were significantly higher (36 +/- 8%), suggesting improved mitochondrial function. Finally, the ability of dystrophin-deficient hearts to resist sarcolemmal damage induced in vivo by increasing the cardiac workload acutely with isoproterenol was enhanced by the presence of the transgene and even more so by inhibiting cGMP breakdown using the phosphodiesterase inhibitor sildenafil (44.4 +/- 1.0% reduction in cardiomyocyte damage). Overall, these findings demonstrate that enhancing cGMP signaling, specifically downstream and independent of NO formation, in the dystrophin-deficient heart improves contractile performance, myocardial metabolic status, and sarcolemmal integrity and thus constitutes a potential clinical avenue for the treatment of the dystrophin-related cardiomyopathies.

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Prediction of the tissue-specificity of selective estrogen receptor modulators by using a single biochemical method.

Publication Date: 2008 May 12 PMID: 18474858
Authors: Dai, S. Y. - Chalmers, M. J. - Bruning, J. - Bramlett, K. S. - Osborne, H. E. - Montrose-Rafizadeh, C. - Barr, R. J. - Wang, Y. - Wang, M. - Burris, T. P. - Dodge, J. A. - Griffin, P. R.
Journal: Proc Natl Acad Sci U S A

Here, we demonstrate that a single biochemical assay is able to predict the tissue-selective pharmacology of an array of selective estrogen receptor modulators (SERMs). We describe an approach to classify estrogen receptor (ER) modulators based on dynamics of the receptor-ligand complex as probed with hydrogen/deuterium exchange (HDX) mass spectrometry. Differential HDX mapping coupled with cluster and discriminate analysis effectively predicted tissue-selective function in most, but not all, cases tested. We demonstrate that analysis of dynamics of the receptor-ligand complex facilitates binning of ER modulators into distinct groups based on structural dynamics. Importantly, we were able to differentiate small structural changes within ER ligands of the same chemotype. In addition, HDX revealed differentially stabilized regions within the ligand-binding pocket that may contribute to the different pharmacology phenotypes of the compounds independent of helix 12 positioning. In summary, HDX provides a sensitive and rapid approach to classify modulators of the estrogen receptor that correlates with their pharmacological profile.

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Expression of an estrogen receptor agonist in differentiating osteoblast cultures.

Publication Date: 2008 May 13 PMID: 18474857
Authors: McCarthy, T. L. - Clough, M. E. - Gundberg, C. M. - Centrella, M.
Journal: Proc Natl Acad Sci U S A

Osteoblasts respond in direct and indirect ways to estrogens, and age-dependent changes in hormone levels and bone health can be limited by focused hormone replacement therapy. In this study, we report the release and isolation of an estrogen receptor agonist from osteoblast cultures. This entity reprises many aspects of estradiol activity in isolated osteoblasts, but differs from authentic estradiol by several biochemical and physical criteria. At levels that occur in conditioned medium from differentiating osteoblast cultures, the agonist directly drives gene expression through estrogen-sensitive response elements, activates the obligate osteoblast transcription factor Runx2, and potently enhances Smad-dependent gene expression in response to TGF-beta, but exhibits relatively lesser suppressive effects on gene expression through C/EBP and AP-1-binding protein transcription factors. Estrogen receptor agonist activity is resistant to heating at 100 degrees C and separable from the bulk of the remaining alcohol- and hexane-soluble molecules by C18 chromatography. MS and molecular fragmentation analyses predict a M(r) of 415.2 to 437.2. Therefore, in addition to earlier studies showing that osteoblasts readily respond to and metabolize various sex steroid-like substrates, we find that they also generate a potent estrogen receptor agonist during differentiation in vitro. Changes in the availability of a molecule like this within bone may relate to differences in skeletal integrity with aging or metabolic disease.

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Hyperpolarization-activated graded persistent activity in the prefrontal cortex.

Publication Date: 2008 May 12 PMID: 18474856
Authors: Winograd, M. - Destexhe, A. - Sanchez-Vives, M. V.
Journal: Proc Natl Acad Sci U S A

We describe a phenomenon of hyperpolarization-activated graded persistent activity (HAGPA) in prefrontal cortex neurons. Successive hyperpolarizing pulses induced increasingly higher rates of tonic firing that remained stable for tens of seconds, allowing the neuron to retain a memory of the previous history of stimulation. This phenomenon occurred at the cellular level and in the absence of neuromodulators. Neurons with HAGPA had a sag during hyperpolarization, and blocking h-current eliminated the sag and prevented HAGPA, suggesting that the activation of this hyperpolarization-activated cationic current was necessary for the occurrence of the phenomenon. A single-neuron biophysical model including h-current modulation by intracellular calcium was able to display HAGPA. This form of neuronal memory not only allows the transformation of inhibition into an increase of firing rate, but also endows neurons with a mechanism to compute the properties of successive inputs into persistent activity, thus solving a difficult computational problem.

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Fecal transmission of AA amyloidosis in the cheetah contributes to high incidence of disease.

Publication Date: 2008 May 12 PMID: 18474855
Authors: Zhang, B. - Une, Y. - Fu, X. - Yan, J. - Ge, F. - Yao, J. - Sawashita, J. - Mori, M. - Tomozawa, H. - Kametani, F. - Higuchi, K.
Journal: Proc Natl Acad Sci U S A

AA amyloidosis is one of the principal causes of morbidity and mortality in captive cheetahs (Acinonyx jubatus), which are in danger of extinction, but little is known about the underlying mechanisms. Given the transmissible characteristics of AA amyloidosis, transmission between captive cheetahs may be a possible mechanism involved in the high incidence of AA amyloidosis. In this study of animals with AA amyloidosis, we found that cheetah feces contained AA amyloid fibrils that were different from those of the liver with regard to molecular weight and shape and had greater transmissibility. The infectious activity of fecal AA amyloid fibrils was reduced or abolished by the protein denaturants 6 M guanidine.HCl and formic acid or by AA immunodepletion. Thus, we propose that feces are a vehicle of transmission that may accelerate AA amyloidosis in captive cheetah populations. These results provide a pathogenesis for AA amyloidosis and suggest possible measures for rescuing cheetahs from extinction.

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Reversal of opiate-induced apoptosis by human recombinant growth hormone in murine foetus primary hippocampal neuronal cell cultures.

Publication Date: 2008 May 12 PMID: 18474854
Authors: Svensson, A. L. - Bucht, N. - Hallberg, M. - Nyberg, F.
Journal: Proc Natl Acad Sci U S A

Previous studies have shown that chronic opiates may inhibit cell growth and trigger apoptosis leading to impaired cognitive capabilities in both humans and other mammals. In contrast, growth hormone (GH) has been demonstrated to stimulate cell growth and counteract apoptosis. GH has also been shown to improve learning and memory in both human and rodents. In this work, we demonstrate that GH may reverse opiate-induced apoptosis in cells derived from prenatal mouse hippocampus. Primary hippocampal cell cultures derived from 16-day-old fetal mouse neurons were treated with morphine for 7 days during growth in the absence or presence of recombinant human GH (rhGH). The release of lactate dehydrogenase (LDH) into the culture media and the level of cleaved caspase-3 were measured. Results indicate that morphine (15 muM) decreased the cell content in a concentration-dependent manner and increased LDH release and caspase-3 activity. Thus, fetal mouse neurons treated with morphine showed less viability compared with controls. Interestingly, the addition of rhGH (1 muM) counteracted the morphine-induced effect on the cell density. Furthermore, the hormone attenuated the effects on LHD release and caspase-3 activity elicited by morphine. These results suggest that the hormone is capable of preventing or even repairing morphine-induced damage to hippocampal cells.

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Involvement of testicular growth factors in fetal Leydig cell aggregation after exposure to phthalate in utero.

Publication Date: 2008 May 9 PMID: 18469139
Authors: Lin, H. - Ge, R. S. - Chen, G. R. - Hu, G. X. - Dong, L. - Lian, Q. Q. - Hardy, D. O. - Sottas, C. M. - Li, X. K. - Hardy, M. P.
Journal: Proc Natl Acad Sci U S A

Exposures to di-(2-ethylhexyl) phthalate (DEHP) have been shown to be associated with decreased adult testosterone (T) levels and increased Leydig cell numbers. As yet, little is known about DEHP effects in utero on fetal Leydig cells (FLC). The present study investigated effects of DEHP on FLC function. Pregnant Long-Evans female rats received vehicle (corn oil) or DEHP at 10, 100, or 750 mg/kg by oral gavage from gestational day (GD)2-20. At GD21, T production, FLC numbers and distribution, and testicular gene expression were examined. The percentage of FLC clusters containing 6-30 cells increased in all treatment groups, with 29 +/- 2% in control vs. 37 +/- 3, 35 +/- 3, and 56 +/- 4% in rats receiving 10, 100, and 750 mg/kg DEHP, respectively. In contrast, FLC numbers were 33% and 39% lower than control after exposures to 100 and 750 mg/kg DEHP, respectively. At these doses, mRNA levels of leukemia inhibitory factor (LIF) increased. LIF was found to induce cell aggregation in FLCs in vitro, consistent with the hypothesis that DEHP induced FLC aggregation. Testicular T levels were doubled by the 10 mg/kg dose and halved at 750 mg/kg. The mRNA levels of IGF-1 and c-Kit ligand (KITL) were induced by 10 mg/kg DEHP. These results, taken together, indicate that fetal exposures to DEHP have effects on FLC number, distribution, and most importantly, steroidogenic capacity and suggest that abnormal expressions of IGF1, KITL, and LIF genes may contribute to the reproductive toxicity of phthalates.

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On the coevolution of Ediacaran oceans and animals.

Publication Date: 2008 May 9 PMID: 18469138
Authors: Shen, Y. - Zhang, T. - Hoffman, P. F.
Journal: Proc Natl Acad Sci U S A

Fe speciation and S-isotope of pyrite data from the terminal Proterozoic Sheepbed Formation in Canada and Doushantuo Formation in China reveal that ocean deep waters were anoxic after the global glaciations (snowball Earth) ending 635 million years ago, but that marine sulfate concentrations and inferentially atmospheric oxygen levels were higher than before the glaciations. This supports a long-postulated link between oxygen levels and the emergence of eumetazoa. Subsequent ventilation of the deep ocean, inferred from shifts in Fe speciation in Newfoundland (previously published data) and western Canada (this report), paved the way for Ediacaran macrobiota to colonize the deep seafloors.

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Proteasomal adaptation to environmental stress links resistance to proteotoxicity with longevity in Caenorhabditis elegans.

Publication Date: 2008 May 13 PMID: 18467495
Authors: Yun, C. - Stanhill, A. - Yang, Y. - Zhang, Y. - Haynes, C. M. - Xu, C. F. - Neubert, T. A. - Mor, A. - Philips, M. R. - Ron, D.
Journal: Proc Natl Acad Sci U S A

The burden of protein misfolding is believed to contribute to aging. However, the links between adaptations to conditions associated with protein misfolding and resistance to the time-dependent attrition of cellular function remain poorly understood. We report that worms lacking aip-1, a homologue of mammalian AIRAP (arsenic-inducible proteasomal 19S regulatory particle-associated protein), are not only impaired in their ability to resist exposure to arsenite but also exhibit shortened lifespan and hypersensitivity to misfolding-prone proteins under normal laboratory conditions. Mammals have a second, constitutively expressed AIRAP-like gene (AIRAPL) that also encodes a proteasome-interacting protein, which shares with AIRAP the property of enhancing peptide accessibility to the proteasome's active site. Genetic rescue experiments suggest that features common to the constitutively expressed worm AIP-1 and mammalian AIRAPL (but missing in the smaller, arsenite-inducible AIRAP) are important to lifespan extension. In worms, a single AIRAP-related protein links proteasomal adaptation to environmental stress with resistance to both proteotoxic insults and maintenance of animal life span under normal conditions.

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Interferon signaling and treatment outcome in chronic hepatitis C.

Publication Date: 2008 May 13 PMID: 18467494
Authors: Sarasin-Filipowicz, M. - Oakeley, E. J. - Duong, F. H. - Christen, V. - Terracciano, L. - Filipowicz, W. - Heim, M. H.
Journal: Proc Natl Acad Sci U S A

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. The current standard therapy for chronic hepatitis C (CHC) consists of a combination of pegylated IFN alpha (pegIFNalpha) and ribavirin. It achieves a sustained viral clearance in only 50-60% of patients. To learn more about molecular mechanisms underlying treatment failure, we investigated IFN-induced signaling in paired liver biopsies collected from CHC patients before and after administration of pegIFNalpha. In patients with a rapid virological response to treatment, pegIFNalpha induced a strong up-regulation of IFN-stimulated genes (ISGs). As shown previously, nonresponders had high expression levels of ISGs before therapy. Analysis of posttreatment biopsies of these patients revealed that pegIFNalpha did not induce expression of ISGs above the pretreatment levels. In accordance with ISG expression data, phosphorylation, DNA binding, and nuclear localization of STAT1 indicated that the IFN signaling pathway in nonresponsive patients is preactivated and refractory to further stimulation. Some features characteristic of nonresponders were more accentuated in patients infected with HCV genotypes 1 and 4 compared with genotypes 2 and 3, providing a possible explanation for the poor response of the former group to therapy. Taken together with previous findings, our data support the concept that activation of the endogenous IFN system in CHC not only is ineffective in clearing the infection but also may impede the response to therapy, most likely by inducing a refractory state of the IFN signaling pathway.

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Diverse syntrophic partnerships from deep-sea methane vents revealed by direct cell capture and metagenomics.

Publication Date: 2008 May 13 PMID: 18467493
Authors: Pernthaler, A. - Dekas, A. E. - Brown, C. T. - Goffredi, S. K. - Embaye, T. - Orphan, V. J.
Journal: Proc Natl Acad Sci U S A

Microorganisms play a fundamental role in the cycling of nutrients and energy on our planet. A common strategy for many microorganisms mediating biogeochemical cycles in anoxic environments is syntrophy, frequently necessitating close spatial proximity between microbial partners. We are only now beginning to fully appreciate the diversity and pervasiveness of microbial partnerships in nature, the majority of which cannot be replicated in the laboratory. One notable example of such cooperation is the interspecies association between anaerobic methane oxidizing archaea (ANME) and sulfate-reducing bacteria. These consortia are globally distributed in the environment and provide a significant sink for methane by substantially reducing the export of this potent greenhouse gas into the atmosphere. The interdependence of these currently uncultured microbes renders them difficult to study, and our knowledge of their physiological capabilities in nature is limited. Here, we have developed a method to capture select microorganisms directly from the environment, using combined fluorescence in situ hybridization and immunomagnetic cell capture. We used this method to purify syntrophic anaerobic methane oxidizing ANME-2c archaea and physically associated microorganisms directly from deep-sea marine sediment. Metagenomics, PCR, and microscopy of these purified consortia revealed unexpected diversity of associated bacteria, including Betaproteobacteria and a second sulfate-reducing Deltaproteobacterial partner. The detection of nitrogenase genes within the metagenome and subsequent demonstration of (15)N(2) incorporation in the biomass of these methane-oxidizing consortia suggest a possible role in new nitrogen inputs by these syntrophic assemblages.

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Endochondral bone tissue engineering using embryonic stem cells.

Publication Date: 2008 May 13 PMID: 18467492
Authors: Jukes, J. M. - Both, S. K. - Leusink, A. - Sterk, L. M. - van Blitterswijk, C. A. - de Boer, J.
Journal: Proc Natl Acad Sci U S A

Embryonic stem cells can provide an unlimited supply of pluripotent cells for tissue engineering applications. Bone tissue engineering by directly differentiating ES cells (ESCs) into osteoblasts has been unsuccessful so far. Therefore, we investigated an alternative approach, based on the process of endochondral ossification. A cartilage matrix was formed in vitro by mouse ESCs seeded on a scaffold. When these cartilage tissue-engineered constructs (CTECs) were implanted s.c., the cartilage matured, became hypertrophic, calcified, and was ultimately replaced by bone tissue in the course of 21 days. Bone aligning hypertrophic cartilage was observed frequently. Using various chondrogenic differentiation periods in vitro, we demonstrated that a cartilage matrix is required for bone formation by ESCs. Chondrogenic differentiation of mesenchymal stem cells and articular chondrocytes showed that a cartilage matrix alone was not sufficient to drive endochondral bone formation. Moreover, when CTECs were implanted orthotopically into critical-size cranial defects in rats, efficient bone formation was observed. We report previously undescribed ESC-based bone tissue engineering under controlled reproducible conditions. Furthermore, our data indicate that ESCs can also be used as a model system to study endochondral bone formation.

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Dachshund inhibits oncogene-induced breast cancer cellular migration and invasion through suppression of interleukin-8.

Publication Date: 2008 May 13 PMID: 18467491
Authors: Wu, K. - Katiyar, S. - Li, A. - Liu, M. - Ju, X. - Popov, V. M. - Jiao, X. - Lisanti, M. P. - Casola, A. - Pestell, R. G.
Journal: Proc Natl Acad Sci U S A

Oncogene-mediated signaling to the host environment induces a subset of cytokines and chemokines. The Drosophila Dac gene promotes migration of the morphogenetic furrow during eye development. Expression of the cell-fate determination factor Dachshund (DACH1) was lost in poor prognosis invasive breast cancer. Mouse embryo fibroblasts derived from Dach1(-/-) mice demonstrated endogenous Dach1 constitutively represses cellular migration. DACH1 inhibited cellular migration and invasion of oncogene (Ras, Myc, ErbB2, c-Raf)-transformed human breast epithelial cells. An unbiased proteomic analysis identified and immunoneutralizing antibody and reconstitution experiments demonstrated IL-8 is a critical target of DACH1 mediating breast cancer cellular migration and metastasis in vivo. DACH1 bound the endogenous IL-8 promoter in ChIP assays and repressed the IL-8 promoter through the AP-1 and NF-kappaB binding sites. Collectively, our data identify a pathway by which an endogenous cell-fate determination factor blocks oncogene-dependent tumor metastasis via a key heterotypic mediator.

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Modulation of brassinosteroid-regulated gene expression by jumonji domain-containing proteins ELF6 and REF6 in Arabidopsis.

Publication Date: 2008 May 8 PMID: 18467490
Authors: Yu, X. - Li, L. - Li, L. - Guo, M. - Chory, J. - Yin, Y.
Journal: Proc Natl Acad Sci U S A

Plant steroid hormones, brassinosteroids (BRs), are of great importance for plant growth and development. BRs signal through a cell surface receptor kinase, BRI1, and a GSK3-like kinase, BIN2, to regulate the BES1/BZR1 family of transcription factors, which directly bind to target gene promoters to activate or repress gene expression and mediate BR responses. To understand how BES1 regulates target gene expression, we identified two BES1-interacting proteins, ELF6 (early flowering 6) and its homolog REF6 (relative of early flowering 6), both of which are Jumonji N/C (JmjN/C) domain-containing proteins and were previously found to regulate flowering time. The interactions between BES1 and ELF6/REF6 were confirmed by GST pull-down and BiFC (bimolecular fluorescence complementation) experiments. Mutations in ELF6 or REF6 genes in Arabidopsis lead to BR-related phenotypes, including impaired cell elongation and reduced expression of BR target genes. Chromatin immunoprecipitation (ChIP) experiments indicated that histone 3 lysine 9 (H3K9) methylation status was changed in elf6 and ref6 mutants, consistent with recent findings that many Jmj proteins are histone demethylases. Our results demonstrate that BES1 recruits other transcriptional regulators such as ELF6 and REF6 to regulate target gene expression and coordinate BR responses with other developmental processes such as control of flowering time. Jmj domain-containing histone demethylases are involved in gene expression in many developmental processes and diseases, but how these proteins affect specific pathways is not well understood. Thus, our study establishes an important mechanism by which Jmj domain proteins modulate specific gene expression by interacting with pathway-specific transcription factors such as BES1.

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Cross-polarized excitons in carbon nanotubes.

Publication Date: 2008 May 13 PMID: 18463293
Authors: Kilina, S. - Tretiak, S. - Doorn, S. K. - Luo, Z. - Papadimitrakopoulos, F. - Piryatinski, A. - Saxena, A. - Bishop, A. R.
Journal: Proc Natl Acad Sci U S A

Polarization of low-lying excitonic bands in finite-size semiconducting single-walled carbon nanotubes (SWNTs) is studied by using quantum-chemical methodologies. Our calculations elucidate properties of cross-polarized excitons, which lead to the transverse optical absorption of nanotubes and presumably couple to intermediate-frequency modes recently observed in resonance Raman excitation spectroscopy. We identify up to 12 distinct excitonic transitions below the second fundamental band associated with the E(22) van Hove singularity. Calculations for several chiral SWNTs distinguish the optically active "bright" excitonic band polarized parallel to the tube axis and several optically "weak" cross-polarized excitons. The rest are optically (near) forbidden "dark" transitions. An analysis of the transition density matrices related to excitonic bands provides detailed information about delocalization of excitonic wavefunction along the tube. Utilization of the natural helical coordinate system accounting for the tube chirality allows one to disentangle longitudinal and circumferential components. The distribution of the transition density matrix along a tube axis is similar for all excitons. However, four parallel-polarized excitons associated with the E(11) transition are more localized along the circumference of a tube, compared with others related to the E(12) and E(21) cross-polarized transitions. Calculated splitting between optically active parallel- and cross-polarized transitions increases with tube diameter, which compares well with experimental spectroscopic data.

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Characterization of selective binding of alkali cations with carboxylate by x-ray absorption spectroscopy of liquid microjets.

Publication Date: 2008 May 13 PMID: 18463292
Authors: Uejio, J. S. - Schwartz, C. P. - Duffin, A. M. - Drisdell, W. S. - Cohen, R. C. - Saykally, R. J.
Journal: Proc Natl Acad Sci U S A

We describe an approach for characterizing selective binding between oppositely charged ionic functional groups under biologically relevant conditions. Relative shifts in K-shell x-ray absorption spectra of aqueous cations and carboxylate anions indicate the corresponding binding strengths via perturbations of carbonyl antibonding orbitals. XAS spectra measured for aqueous formate and acetate solutions containing lithium, sodium, and potassium cations reveal monotonically stronger binding of the lighter metals, supporting recent results from simulations and other experiments. The carbon K-edge spectra of the acetate carbonyl feature centered near 290 eV clearly indicate a preferential interaction of sodium versus potassium, which was less apparent with formate. These results are in accord with the Law of Matching Water Affinities, relating relative hydration strengths of ions to their respective tendencies to form contact ion pairs. Density functional theory calculations of K-shell spectra support the experimental findings.

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Platinum-based inhibitors of amyloid-beta as therapeutic agents for Alzheimer's disease.

Publication Date: 2008 May 13 PMID: 18463291
Authors: Barnham, K. J. - Kenche, V. B. - Ciccotosto, G. D. - Smith, D. P. - Tew, D. J. - Liu, X. - Perez, K. - Cranston, G. A. - Johanssen, T. J. - Volitakis, I. - Bush, A. I. - Masters, C. L. - White, A. R. - Smith, J. P. - Cherny, R. A. - Cappai, R.
Journal: Proc Natl Acad Sci U S A

Amelyoid-beta peptide (Abeta) is a major causative agent responsible for Alzheimer's disease (AD). Abeta contains a high affinity metal binding site that modulates peptide aggregation and toxicity. Therefore, identifying molecules targeting this site represents a valid therapeutic strategy. To test this hypothesis, a range of L-PtCl(2) (L = 1,10-phenanthroline derivatives) complexes were examined and shown to bind to Abeta, inhibit neurotoxicity and rescue Abeta-induced synaptotoxicity in mouse hippocampal slices. Coordination of the complexes to Abeta altered the chemical properties of the peptide inhibiting amyloid formation and the generation of reactive oxygen species. In comparison, the classic anticancer drug cisplatin did not affect any of the biochemical and cellular effects of Abeta. This implies that the planar aromatic 1,10-phenanthroline ligands L confer some specificity for Abeta onto the platinum complexes. The potent effect of the L-PtCl(2) complexes identifies this class of compounds as therapeutic agents for AD.

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Human biliverdin reductase is an ERK activator; hBVR is an ERK nuclear transporter and is required for MAPK signaling.

Publication Date: 2008 May 13 PMID: 18463290
Authors: Lerner-Marmarosh, N. - Miralem, T. - Gibbs, P. E. - Maines, M. D.
Journal: Proc Natl Acad Sci U S A

Activation of the MEK/ERK/Elk-signaling cascade is a mechanism for relaying mitogenic and stress stimuli for gene activation. MEK1 is the proximate kinase for activation of ERK1/2, and nuclear targeting of ERK1/2 is obligatory for Elk1 transcriptional activity. Human biliverdin reductase (hBVR) is a recently described Ser/Thr/Tyr kinase in the MAPK insulin/insulin-like growth factor 1 (IGF1)-signaling cascade. Using 293A cells and in vitro experiments, we detail the formation of a ternary complex of MEK/ERK/hBVR, activation of MEK1 and ERK1/2 kinase activities by hBVR, and phosphorylation of hBVR by ERK1/2. hBVR is nearly as effective as IGF1 in activating ERK; intact hBVR ATP-binding domain is necessary for Elk1 activation, whereas protein-protein interaction is the basis for hBVR activation of MEK1 and ERK. The two MAPK docking consensus sequences present in hBVR, F(162)GFP and K(275)KRILHCLGL (C- and D-box, respectively), are ERK interactive sites; interaction at each site is critical for ERK/Elk1 activation. Transfection with mutant hBVR-P(165) or peptides corresponding to the C- or D-box blocked activation of ERK by IGF1. Transfection with D-box mutant hBVR prevented the activation of ERK by wild-type protein and dramatically decreased Elk1 transcriptional activity. hBVR is a nuclear transporter of ERK; experiments with hBVR nuclear export signal (NES) and nuclear localization signal (NLS) mutants demonstrated its critical role in the nuclear localization of IGF-stimulated ERK for Elk1 activation. These findings, together with observations that si-hBVR blocked activation of ERK and Elk1 by IGF1 and prevented formation of ternary complex between MEK/ERK/hBVR, define the critical role of hBVR in ERK signaling and nuclear functions of the kinase.

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Force-induced growth of adhesion domains is controlled by receptor mobility.

Publication Date: 2008 May 13 PMID: 18463289
Authors: Smith, A. S. - Sengupta, K. - Goennenwein, S. - Seifert, U. - Sackmann, E.
Journal: Proc Natl Acad Sci U S A

In living cells, adhesion structures have the astonishing ability to grow and strengthen under force. Despite the rising evidence of the importance of this phenomenon, little is known about the underlying mechanism. Here, we show that force-induced adhesion-strengthening can occur purely because of the thermodynamic response to the elastic deformation of the membrane, even in the absence of the actively regulated cytoskeleton of the cell, which was hitherto deemed necessary. We impose pN-forces on two fluid membranes, locally pre-adhered by RGD-integrin binding. One of the binding partners is always mobile whereas the mobility of the other can be switched on or off. Immediate passive strengthening of adhesion structures occurs in both cases. When both binding partners are mobile, strengthening is aided by lateral movement of intact bonds as a transient response to force-induced membrane-deformation. By extending our microinterferometric technique to the suboptical regime, we show that the adhesion, as well as the resistance to force-induced de-adhesion, is greatly enhanced when both, rather than only one, of the binding partners are mobile. We formulate a theory that explains our observations by linking the macroscopic shape deformation with the microscopic formation of bonds, which further elucidates the importance of receptor mobility. We propose this fast passive response to be the first-recognition that triggers signaling events leading to mechanosensing in living cells.

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Electronic duality in strongly correlated matter.

Publication Date: 2008 May 13 PMID: 18463288
Authors: Park, T. - Graf, M. J. - Boulaevskii, L. - Sarrao, J. L. - Thompson, J. D.
Journal: Proc Natl Acad Sci U S A

Superconductivity develops from an attractive interaction between itinerant electrons that creates electron pairs, which condense into a macroscopic quantum state-the superconducting state. On the other hand, magnetic order in a metal arises from electrons localized close to the ionic core and whose interaction is mediated by itinerant electrons. The dichotomy between local moment magnetic order and superconductivity raises the question of whether these two states can coexist and involve the same electrons. Here, we show that the single 4f electron of cerium in CeRhIn(5) simultaneously produces magnetism, characteristic of localization, and superconductivity that requires itinerancy. The dual nature of the 4f-electron allows microscopic coexistence of antiferromagnetic order and superconductivity whose competition is tuned by small changes in pressure and magnetic field. Electronic duality contrasts with conventional interpretations of coexisting spin-density magnetism and superconductivity and offers a new avenue for understanding complex states in classes of materials.

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Identification of innate immunity genes and pathways using a comparative genomics approach.

Publication Date: 2008 May 13 PMID: 18463287
Authors: Alper, S. - Laws, R. - Lackford, B. - Boyd, W. A. - Dunlap, P. - Freedman, J. H. - Schwartz, D. A.
Journal: Proc Natl Acad Sci U S A

To reveal regulators of innate immunity, we used RNAi assays to monitor the immune response when genes are inhibited in Caenorhabditis elegans and mouse macrophages. Genes that altered innate immune responsiveness in C. elegans were validated in murine macrophages, resulting in the discovery of 11 genes that regulate the innate immune response in both systems and the subsequent identification of a protein interaction network with a conserved role in innate immunity regulation. We confirmed the role of four of these 11 genes in antimicrobial gene regulation using available mutants in C. elegans. Several of these genes (acy-1, tub-2, and tbc-1) also regulate susceptibility to the pathogen Pseudomonas aeruginosa. These genes may prove critical to understanding host defense and represent potential therapeutic targets for infectious and immunological diseases.

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Alternative splicing: A missing piece in the puzzle of intron gain.

Publication Date: 2008 May 7 PMID: 18463286
Authors: Tarrio, R. - Ayala, F. J. - Rodriguez-Trelles, F.
Journal: Proc Natl Acad Sci U S A

Spliceosomal introns, a hallmark of eukaryotic gene organization, were an unexpected discovery. After three decades, crucial issues such as when and how introns first appeared in evolution remain unsettled. An issue yet to be answered is how intron positions arise de novo. Phylogenetic investigations concur that intron positions continue to emerge, at least in some lineages. Yet genomic scans for the sources of introns occupying new positions have been fruitless. Two alternative solutions to this paradox are: (i) formation of new intron positions halted before the recent past and (ii) it continues to occur, but through processes different from those generally assumed. One process generally dismissed is intron sliding-the relocation of a preexisting intron over short distances-because of supposed associated deleterious effects. The puzzle of intron gain arises owing to a pervasive operational definition of introns, which sees them as precisely demarcated segments of the genome separated from the neighboring nonintronic DNA by unmovable limits. Intron homology is defined as position homology. Recent studies of pre-mRNA processing indicate that this assumption needs to be revised. We incorporate recent advances on the evolutionarily frequent process of alternative splicing, by which exons of primary transcripts are spliced in different patterns, into a new model of intron sliding that accounts for the diversity of intron positions. We posit that intron positional diversity is driven by two overlapping processes: (i) background process of continuous relocation of preexisting introns by sliding and (ii) spurts of extensive gain/loss of new intron sequences.

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A soluble activin type IIA receptor induces bone formation and improves skeletal integrity.

Publication Date: 2008 May 13 PMID: 18460605
Authors: Pearsall, R. S. - Canalis, E. - Cornwall-Brady, M. - Underwood, K. W. - Haigis, B. - Ucran, J. - Kumar, R. - Pobre, E. - Grinberg, A. - Werner, E. D. - Glatt, V. - Stadmeyer, L. - Smith, D. - Seehra, J. - Bouxsein, M. L.
Journal: Proc Natl Acad Sci U S A

Diseases that affect the regulation of bone turnover can lead to skeletal fragility and increased fracture risk. Members of the TGF-beta superfamily have been shown to be involved in the regulation of bone mass. Activin A, a TGF-beta signaling ligand, is present at high levels in bone and may play a role in the regulation of bone metabolism. Here we demonstrate that pharmacological blockade of ligand signaling through the high affinity receptor for activin, type II activin receptor (ActRIIA), by administration of the soluble extracellular domain of ActRIIA fused to a murine IgG2a-Fc, increases bone formation, bone mass, and bone strength in normal mice and in ovariectomized mice with established bone loss. These observations support the development of this pharmacological strategy for the treatment of diseases with skeletal fragility.

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The evolution of modularity in bacterial metabolic networks.

Publication Date: 2008 May 13 PMID: 18460604
Authors: Kreimer, A. - Borenstein, E. - Gophna, U. - Ruppin, E.
Journal: Proc Natl Acad Sci U S A

Deciphering the modular organization of metabolic networks and understanding how modularity evolves have attracted tremendous interest in recent years. Here, we present a comprehensive large scale characterization of modularity across the bacterial tree of life, systematically quantifying the modularity of the metabolic networks of >300 bacterial species. Three main determinants of metabolic network modularity are identified. First, network size is an important topological determinant of network modularity. Second, several environmental factors influence network modularity, with endosymbionts and mammal-specific pathogens having lower modularity scores than bacterial species that occupy a wider range of niches. Moreover, even among the pathogens, those that alternate between two distinct niches, such as insect and mammal, tend to have relatively high metabolic network modularity. Third, horizontal gene transfer is an important force that contributes significantly to metabolic modularity. We additionally reconstruct the metabolic network of ancestral bacterial species and examine the evolution of modularity across the tree of life. This reveals a trend of modularity decrease from ancestors to descendants that is likely the outcome of niche specialization and the incorporation of peripheral metabolic reactions.

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Gene regulation logic in retinal ganglion cell development: Isl1 defines a critical branch distinct from but overlapping with Pou4f2.

Publication Date: 2008 May 13 PMID: 18460603
Authors: Mu, X. - Fu, X. - Beremand, P. D. - Thomas, T. L. - Klein, W. H.
Journal: Proc Natl Acad Sci U S A

Understanding gene regulatory networks (GRNs) that control neuronal differentiation will provide systems-level perspectives on neurogenesis. We have previously constructed a model for a GRN in retinal ganglion cell (RGC) differentiation in which four hierarchical tiers of transcription factors ultimately control the expression of downstream terminal genes. Math5 occupies a central node in the hierarchy because it is essential for the formation of RGCs and the expression of the immediate downstream factor Pou4f2. Based on its expression, we also proposed that Isl1, a LIM-homeodomain factor, functions in parallel with Pou4f2 and downstream of Math5 in the RGC GRN. To determine whether this was the case, a conditional Isl1 allele was generated and deleted specifically in the developing retina. Although RGCs formed in Isl1-deleted retinas, most underwent apoptosis, and few remained at later stages. By microarray analysis, we identified a distinct set of genes whose expression depended on Isl1. These genes are all downstream of Math5, and some of them, but not all, also depend on Pou4f2. Additionally, Isl1 was required for the sustained expression of Pou4f2, suggesting that Isl1 positively regulates Pou4f2 after Math5 levels are diminished. The results demonstrate an essential role for Isl1 in RGC development and reveal two distinct but intersecting branches of the RGC GRN downstream of Math5, one directed by Pou4f2 and the other by Isl1. They also reveal that identical RGC expression patterns are achieved by different combinations of divergent inputs from upstream transcription factors.

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Different assemblies of the DAM1 complex follow shortening microtubules by distinct mechanisms.

Publication Date: 2008 May 13 PMID: 18460602
Authors: Grishchuk, E. L. - Spiridonov, I. S. - Volkov, V. A. - Efremov, A. - Westermann, S. - Drubin, D. - Barnes, G. - Ataullakhanov, F. I. - McIntosh, J. R.
Journal: Proc Natl Acad Sci U S A

Mitotic chromosomes segregate at the ends of shortening spindle microtubules (MTs). In budding yeast, the Dam1 multiprotein complex supports this dynamic attachment, thereby contributing to accurate chromosome segregation. Purified Dam1 will track the end of a depolymerizing MT and can couple it to microbead transport in vitro. The processivity of such motions has been thought to depend on rings that the Dam1 complex can form around MTs, but the possibility that alternative coupling geometries contribute to these motilities has not been considered. Here, we demonstrate that both rings and nonencircling Dam1 oligomers can track MT ends and enable processive cargo movement in vitro. The coupling properties of these two assemblies are, however, quite different, so each may make a distinct contribution to chromosome motility.

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Reply to Boudreau: Morphologic expression of ferromanganese nodules.

Publication Date: 2008 May 13 PMID: 18460601
Authors: Asikainen, C. A.
Journal: Proc Natl Acad Sci U S A

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Nodule morphology and growth model.

Publication Date: 2008 May 13 PMID: 18460600
Authors: Boudreau, B. P.
Journal: Proc Natl Acad Sci U S A

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What's the point of the type III secretion system needle?

Publication Date: 2008 May 6 PMID: 18458349
Authors: Blocker, A. J. - Deane, J. E. - Veenendaal, A. K. - Roversi, P. - Hodgkinson, J. L. - Johnson, S. - Lea, S. M.
Journal: Proc Natl Acad Sci U S A

Recent work by several groups has significantly expanded our knowledge of the structure, regulation of assembly, and function of components of the extracellular portion of the type III secretion system (T3SS) of Gram-negative bacteria. This perspective presents a structure-informed analysis of functional data and discusses three nonmutually exclusive models of how a key aspect of T3SS biology, the sensing of host cells, may be performed.

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Impacts of climate warming on terrestrial ectotherms across latitude.

Publication Date: 2008 May 6 PMID: 18458348
Authors: Deutsch, C. A. - Tewksbury, J. J. - Huey, R. B. - Sheldon, K. S. - Ghalambor, C. K. - Haak, D. C. - Martin, P. R.
Journal: Proc Natl Acad Sci U S A

The impact of anthropogenic climate change on terrestrial organisms is often predicted to increase with latitude, in parallel with the rate of warming. Yet the biological impact of rising temperatures also depends on the physiological sensitivity of organisms to temperature change. We integrate empirical fitness curves describing the thermal tolerance of terrestrial insects from around the world with the projected geographic distribution of climate change for the next century to estimate the direct impact of warming on insect fitness across latitude. The results show that warming in the tropics, although relatively small in magnitude, is likely to have the most deleterious consequences because tropical insects are relatively sensitive to temperature change and are currently living very close to their optimal temperature. In contrast, species at higher latitudes have broader thermal tolerance and are living in climates that are currently cooler than their physiological optima, so that warming may even enhance their fitness. Available thermal tolerance data for several vertebrate taxa exhibit similar patterns, suggesting that these results are general for terrestrial ectotherms. Our analyses imply that, in the absence of ameliorating factors such as migration and adaptation, the greatest extinction risks from global warming may be in the tropics, where biological diversity is also greatest.

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Dendritic cell-mediated NK cell activation is controlled by Jagged2-Notch interaction.

Publication Date: 2008 May 13 PMID: 18458347
Authors: Kijima, M. - Yamaguchi, T. - Ishifune, C. - Maekawa, Y. - Koyanagi, A. - Yagita, H. - Chiba, S. - Kishihara, K. - Shimada, M. - Yasutomo, K.
Journal: Proc Natl Acad Sci U S A

Natural killer (NK) cells regulate various immune responses by exerting cytotoxic activity or secreting cytokines. The interaction of NK cells with dendritic cells (DC) contributes to NK cell-mediated antitumor or antimicrobial responses. However, the cellular and molecular mechanisms for controlling this interaction are largely unknown. Here, we show an involvement of Jagged2-Notch interaction in augmenting NK cell cytotoxicity mediated by DC. Enforced expression of Jagged2 on A20 cells (Jag2-A20 cells) suppressed their growth in vivo, which was abrogated by depleting NK cells. Moreover, Jag2-A20 cells exerted a suppression on the growth of nonmanipulated A20 cells in SCID mice in an NK-dependent manner. Consistently, coinoculation of A20 cells with DC overexpressing Jagged2 (Jag2-DC) suppressed the growth of A20 cells in mice. Stimulation of NK cells with Jagged2 directly enhanced their cytotoxicity, IFN-gamma production, and proliferation. Ligation of Notch2 on NK cells enhanced their cytotoxic activity, and Jag2-DC or CpG-treated DC-mediated NK cell cytotoxicity was suppressed by a gamma-secretase inhibitor. These results indicate that the Jagged2-Notch axis plays a crucial role in DC-mediated NK cell cytotoxicity. Furthermore, manipulation of this interaction may provide an approach to induce potent tumor immunity or to inhibit certain autoimmune diseases caused by NK cell activation.

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Iodide accumulation provides kelp with an inorganic antioxidant impacting atmospheric chemistry.

Publication Date: 2008 May 13 PMID: 18458346
Authors: Kupper, F. C. - Carpenter, L. J. - McFiggans, G. B. - Palmer, C. J. - Waite, T. J. - Boneberg, E. M. - Woitsch, S. - Weiller, M. - Abela, R. - Grolimund, D. - Potin, P. - Butler, A. - Luther, G. W. 3rd - Kroneck, P. M. - Meyer-Klaucke, W. - Feiters, M. C.
Journal: Proc Natl Acad Sci U S A

Brown algae of the Laminariales (kelps) are the strongest accumulators of iodine among living organisms. They represent a major pump in the global biogeochemical cycle of iodine and, in particular, the major source of iodocarbons in the coastal atmosphere. Nevertheless, the chemical state and biological significance of accumulated iodine have remained unknown to this date. Using x-ray absorption spectroscopy, we show that the accumulated form is iodide, which readily scavenges a variety of reactive oxygen species (ROS). We propose here that its biological role is that of an inorganic antioxidant, the first to be described in a living system. Upon oxidative stress, iodide is effluxed. On the thallus surface and in the apoplast, iodide detoxifies both aqueous oxidants and ozone, the latter resulting in the release of high levels of molecular iodine and the consequent formation of hygroscopic iodine oxides leading to particles, which are precursors to cloud condensation nuclei. In a complementary set of experiments using a heterologous system, iodide was found to effectively scavenge ROS in human blood cells.

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Inhibition of Wnt signaling by the osteoblast-specific transcription factor Osterix.

Publication Date: 2008 May 13 PMID: 18458345
Authors: Zhang, C. - Cho, K. - Huang, Y. - Lyons, J. P. - Zhou, X. - Sinha, K. - McCrea, P. D. - de Crombrugghe, B.
Journal: Proc Natl Acad Sci U S A

The recent identification of the genes responsible for several human genetic diseases affecting bone homeostasis and the characterization of mouse models for these diseases indicated that canonical Wnt signaling plays a critical role in the control of bone mass. Here, we report that the osteoblast-specific transcription factor Osterix (Osx), which is required for osteoblast differentiation, inhibits Wnt pathway activity. First, in calvarial cells of embryonic day (E)18.5 Osx-null embryos, expression of the Wnt antagonist Dkk1 was abolished, and that of Wnt target genes c-Myc and cyclin D1 was increased. Moreover, our studies demonstrated that Osx bound to and activated the Dkk1 promoter. In addition, Osx inhibited beta-catenin-induced Topflash reporter activity and beta-catenin-induced secondary axis formation in Xenopus embryos. Importantly, in calvaria of E18.5 Osx-null embryos harboring the TOPGAL reporter transgene, beta-galactosidase activity was increased, suggesting that Osx inhibited the Wnt pathway in osteoblasts in vivo. Our data further showed that Osx disrupted binding of Tcf to DNA, providing a likely mechanism for the inhibition by Osx of beta-catenin transcriptional activity. We also showed that Osx decreased osteoblast proliferation. Indeed, E18.5 Osx-null calvaria showed greater BrdU incorporation than wild-type calvaria and that Osx overexpression in C2C12 mesenchymal cells inhibited cell growth. Because Wnt signaling has a major role in stimulating osteoblast proliferation, we speculate that Osx-mediated inhibition of osteoblast proliferation is a consequence of the Osx-mediated control of Wnt/beta-catenin activity. Our results add a layer of control to Wnt/beta-catenin signaling in bone.

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Direct phylogenetic evidence for lateral transfer of elongation factor-like gene.

Publication Date: 2008 May 13 PMID: 18458344
Authors: Kamikawa, R. - Inagaki, Y. - Sako, Y.
Journal: Proc Natl Acad Sci U S A

Genes encoding elongation factor-like (EFL) proteins, which show high similarity to elongation factor-1alpha (EF-1alpha), have been found in phylogenetically distantly related eukaryotes. The sporadic distribution of "EFL-containing" lineages within "EF-1alpha-containing" lineages indirectly, but strongly, suggests lateral gene transfer as the principal driving force in EFL evolution. However, one of the most critical aspects in the above hypothesis, the donor lineages in any putative cases of lateral EFL gene transfer, remained unclear. In this study, we provide direct evidence for lateral transfer of an EFL gene through the analyses of 10 diatom EFL genes. All diatom EFL homologues tightly clustered in phylogenetic analyses, suggesting acquisition of the exogenous EFL gene early in diatom evolution. Our survey additionally identified Thalassiosira pseudonana as a eukaryote bearing EF-1alpha and EFL genes and secondary EFL gene loss in Phaeodactylum tricornutum, the complete genome of which encodes only the EF-1alpha gene. Most importantly, the EFL phylogeny recovered a robust grouping of homologues from diatoms, the cercozoan Bigelowiella natans, and the foraminifer Planoglabratella opecularis, with the diatoms nested within the Bigelowiella plus Planoglabratella (Rhizaria) grouping. The particular relationships recovered are further consistent with two characteristic sequence motifs. The best explanation of our data analyses is an EFL gene transfer from a foraminifer to a diatom, the first case in which the donor-recipient relationship was clarified. Finally, based on a reverse transcriptase quantitative PCR assay and the genome information of Thalassiosira and Phaeodactylum, we propose the loss of elongation factor function in Thalassiosira EF-1alpha.

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Unlocking evidence of early diet from tooth enamel.

Publication Date: 2008 May 13 PMID: 18458343
Authors: Humphrey, L. T. - Dean, M. C. - Jeffries, T. E. - Penn, M.
Journal: Proc Natl Acad Sci U S A

Recent developments in microspatial analysis of enamel chemistry provide the resolution needed to reconstruct detailed chronological records of an individual's early life history. Evidence of nutritional history, residential mobility, and exposure to heavy metals can potentially be retrieved from archaeological and even fossil teeth. Understanding the pattern and timing of incorporation of each trace element or stable isotope into enamel is crucial to the interpretation of the primary data. Here, we use laser ablation inductively coupled plasma mass spectrometry and ArcGIS software to map variation in calcium-normalized strontium intensities across thin sections of enamel from exfoliated deciduous teeth. Differences in calcium-normalized strontium intensities across each tooth reflect variation in tooth mineralization, implying that sampling location must be taken into account in interpreting results. Chronologically consistent shifts in calcium-normalized strontium intensities in teeth from children with known nursing histories reflect the onset and duration of breastfeeding and the introduction of nonmaternal sources of food. This tool is likely to be valuable for studying weaning and nursing behavior in the past. The distribution of normalized strontium intensities presented here is consistent with a model for the differential incorporation of strontium and calcium into enamel during the secretory and maturational phases of formation.

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In vitro reconstitution and crystal structure of p-aminobenzoate N-oxygenase (AurF) involved in aureothin biosynthesis.

Publication Date: 2008 May 13 PMID: 18458342
Authors: Choi, Y. S. - Zhang, H. - Brunzelle, J. S. - Nair, S. K. - Zhao, H.
Journal: Proc Natl Acad Sci U S A

p-Aminobenzoate N-oxygenase (AurF) from Streptomyces thioluteus catalyzes the formation of unusual polyketide synthase starter unit p-nitrobenzoic acid (pNBA) from p-aminobenzoic acid (pABA) in the biosynthesis of antibiotic aureothin. AurF is a metalloenzyme, but its native enzymatic activity has not been demonstrated in vitro, and its catalytic mechanism is unclear. In addition, the nature of the cofactor remains a controversy. Here, we report the in vitro reconstitution of the AurF enzyme activity, the crystal structure of AurF in the oxidized state, and the cocrystal structure of AurF with its product pNBA. Our combined biochemical and structural analysis unequivocally indicates that AurF is a non-heme di-iron monooxygenase that catalyzes sequential oxidation of aminoarenes to nitroarenes via hydroxylamine and nitroso intermediates.

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Arbovirus evolution in vivo is constrained by host alternation.

Publication Date: 2008 May 13 PMID: 18458341
Authors: Coffey, L. L. - Vasilakis, N. - Brault, A. C. - Powers, A. M. - Tripet, F. - Weaver, S. C.
Journal: Proc Natl Acad Sci U S A

The intrinsic plasticity of RNA viruses can facilitate host range changes that lead to epidemics. However, evolutionary processes promoting cross-species transfers are poorly defined, especially for arthropod-borne viruses (arboviruses). In theory, cross species transfers by arboviruses may be constrained by their alternating infection of disparate hosts, where optimal replication in one host involves a fitness tradeoff for the other. Accordingly, freeing arboviruses from alternate replication via specialization in a single host should accelerate adaptation. This hypothesis has been tested by using cell culture model systems with inconclusive results. Therefore, we tested it using an in vivo system with Venezuelan equine encephalitis virus (VEEV), an emerging alphavirus of the Americas. VEEV serially passaged in mosquitoes exhibited increased mosquito infectivity and vertebrate-specialized strains produced higher viremias. Conversely, alternately passaged VEEV experienced no detectable fitness gains in either host. These results suggest that arbovirus adaptation and evolution is limited by obligate host alternation and predict that arboviral emergence via host range changes may be less frequent than that of single host animal RNA viruses.

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Glucose sensing by MondoA:Mlx complexes: a role for hexokinases and direct regulation of thioredoxin-interacting protein expression.

Publication Date: 2008 May 13 PMID: 18458340
Authors: Stoltzman, C. A. - Peterson, C. W. - Breen, K. T. - Muoio, D. M. - Billin, A. N. - Ayer, D. E.
Journal: Proc Natl Acad Sci U S A

Glucose is a fundamental metabolite, yet how cells sense and respond to changes in extracellular glucose concentration is not completely understood. We recently reported that the MondoA:Mlx dimeric transcription factor directly regulates glycolysis. In this article, we consider whether MondoA:Mlx complexes have a broader role in sensing and responding to glucose status. In their latent state, MondoA:Mlx complexes localize to the outer mitochondrial membrane, yet shuttle between the mitochondria and the nucleus. We show that MondoA:Mlx complexes accumulate in the nucleus in response to glucose and 2-deoxyglucose (2-DG). Furthermore, nuclear localization of MondoA:Mlx depends on the enzymatic activity of hexokinases. These enzymes catalyze conversion of glucose to glucose-6-phosphate (G6P), which is the first step in the glycolytic pathway. Together, these findings suggest that MondoA:Mlx monitors intracellular G6P concentration and translocates to the nucleus when levels of this key metabolite increase. Transcriptional profiling experiments demonstrate that MondoA is required for >75% of the 2-DG-induced transcription signature. We identify thioredoxin-interacting protein (TXNIP) as a direct and glucose-regulated MondoA:Mlx transcriptional target. Furthermore, MondoA:Mlx complexes, via their regulation of TXNIP, are potent negative regulators of glucose uptake. These studies suggest a key role for MondoA:Mlx complexes in the adaptive transcriptional response to changes in extracellular glucose concentration and peripheral glucose uptake.

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Mitochondrial copper(I) transfer from Cox17 to Sco1 is coupled to electron transfer.

Publication Date: 2008 May 13 PMID: 18458339
Authors: Banci, L. - Bertini, I. - Ciofi-Baffoni, S. - Hadjiloi, T. - Martinelli, M. - Palumaa, P.
Journal: Proc Natl Acad Sci U S A

The human protein Cox17 contains three pairs of cysteines. In the mitochondrial intermembrane space (IMS) it exists in a partially oxidized form with two S-S bonds and two reduced cysteines (HCox17(2S-S)). HCox17(2S-S) is involved in copper transfer to the human cochaperones Sco1 and Cox11, which are implicated in the assembly of cytochrome c oxidase. We show here that Cu(I)HCox17(2S-S), i.e., the copper-loaded form of the protein, can transfer simultaneously copper(I) and two electrons to the human cochaperone Sco1 (HSco1) in the oxidized state, i.e., with its metal-binding cysteines forming a disulfide bond. The result is Cu(I)HSco1 and the fully oxidized apoHCox17(3S-S), which can be then reduced by glutathione to apoHCox17(2S-S). The HSco1/HCox17(2S-S) redox reaction is thermodynamically driven by copper transfer. These reactions may occur in vivo because HSco1 can be found in the partially oxidized state within the IMS, consistent with the variable redox properties of the latter compartment. The electron transfer-coupled metallation of HSco1 can be a mechanism within the IMS for an efficient specific transfer of the metal to proteins, where metal-binding thiols are oxidized. The same reaction of copper-electron-coupled transfer does not occur with the human homolog of Sco1, HSco2, for kinetic reasons that may be ascribed to the lack of a specific metal-bridged protein-protein complex, which is instead observed in the Cu(I)HCox17(2S-S)/HSco1 interaction.

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Dynamics of phosphodiester synthesis by DNA ligase.

Publication Date: 2008 May 13 PMID: 18458338
Authors: Crut, A. - Nair, P. A. - Koster, D. A. - Shuman, S. - Dekker, N. H.
Journal: Proc Natl Acad Sci U S A

Ligases are essential actors in DNA replication, recombination, and repair by virtue of their ability to seal breaks in the phosphodiester backbone. Ligation proceeds through a nicked DNA-adenylate intermediate (AppDNA), which must be sealed quickly to avoid creating a potentially toxic lesion. Here, we take advantage of ligase-catalyzed AMP-dependent incision of a single supercoiled DNA molecule to observe the step of phosphodiester synthesis in real time. An exponentially distributed number of supercoils was relaxed per successful incision-resealing event, from which we deduce the torque-dependent ligation probability per DNA swivel. Premature dissociation of ligase from nicked DNA-adenylate accounted for approximately 10% of the observed events. The ability of ligase to form a C-shaped protein clamp around DNA is a key determinant of ligation probability per turn and the stability of the ligase-AppDNA intermediate. The estimated rate of phosphodiester synthesis by DNA ligase (400 s(-1)) is similar to the high rates of phosphodiester synthesis by replicative DNA polymerases.

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Null mutations in human and mouse orthologs frequently result in different phenotypes.

Publication Date: 2008 May 13 PMID: 18458337
Authors: Liao, B. Y. - Zhang, J.
Journal: Proc Natl Acad Sci U S A

One-to-one orthologous genes of relatively closely related species are widely assumed to have similar functions and cause similar phenotypes when deleted from the genome. Although this assumption is the foundation of comparative genomics and the basis for the use of model organisms to study human biology and disease, its validity is known only from anecdotes rather than from systematic examination. Comparing documented phenotypes of null mutations in humans and mice, we find that >20% of human essential genes have nonessential mouse orthologs. These changes of gene essentiality appear to be associated with adaptive evolution at the protein-sequence, but not gene-expression, level. Proteins localized to the vacuole, a cellular compartment for waste management, are highly enriched among essentiality-changing genes. It is probable that the evolution of the prolonged life history in humans required enhanced waste management for proper cellular function until the time of reproduction, which rendered these vacuole proteins essential and generated selective pressures for their improvement. If our gene sample represents the entire genome, our results would mean frequent changes of phenotypic effects of one-to-one orthologous genes even between relatively closely related species, a possibility that should be considered in comparative genomic studies and in making cross-species inferences of gene function and phenotypic effect.

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Microdeletions are a general feature of adult and adolescent acute lymphoblastic leukemia: Unexpected similarities with pediatric disease.

Publication Date: 2008 May 6 PMID: 18458336
Authors: Paulsson, K. - Cazier, J. B. - Macdougall, F. - Stevens, J. - Stasevich, I. - Vrcelj, N. - Chaplin, T. - Lillington, D. M. - Lister, T. A. - Young, B. D.
Journal: Proc Natl Acad Sci U S A

We present here a genome-wide map of abnormalities found in diagnostic samples from 45 adults and adolescents with acute lymphoblastic leukemia (ALL). A 500K SNP array analysis uncovered frequent genetic abnormalities, with cryptic deletions constituting half of the detected changes, implying that microdeletions are a characteristic feature of this malignancy. Importantly, the pattern of deletions resembled that recently reported in pediatric ALL, suggesting that adult, adolescent, and childhood cases may be more similar on the genetic level than previously thought. Thus, 70% of the cases displayed deletion of one or more of the CDKN2A, PAX5, IKZF1, ETV6, RB1, and EBF1 genes. Furthermore, several genes not previously implicated in the pathogenesis of ALL were identified as possible recurrent targets of deletion. In total, the SNP array analysis identified 367 genetic abnormalities not corresponding to known copy number polymorphisms, with all but two cases (96%) displaying at least one cryptic change. The resolution level of this SNP array study is the highest used to date to investigate a malignant hematologic disorder. Our findings provide insights into the leukemogenic process and may be clinically important in adult and adolescent ALL. Most importantly, we report that microdeletions of key genes appear to be a common, characteristic feature of ALL that is shared among different clinical, morphological, and cytogenetic subgroups.

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Cis- and trans-splicing of mRNAs mediated by tRNA sequences in eukaryotic cells.

Publication Date: 2008 May 13 PMID: 18458335
Authors: Di Segni, G. - Gastaldi, S. - Tocchini-Valentini, G. P.
Journal: Proc Natl Acad Sci U S A

The formation of chimeric mRNAs is a strategy used by human cells to increase the complexity of their proteome, as revealed by the ENCODE project. Here, we use Saccharomyces cerevisiae to show a way by which trans-spliced mRNAs can be generated. We demonstrate that a pretRNA inserted into a premRNA context directs the splicing reaction precisely to the sites of the tRNA intron. A suppressor pretRNA gene was inserted, in cis, into the sequence encoding the third cytoplasmic loop of the Ste2 or Ste3 G protein-coupled receptor. The hybrid RNAs are spliced at the specific pretRNA splicing sites, releasing both functional tRNAs that suppress nonsense mutations and translatable mRNAs that activate the signal transduction pathway. The RNA molecules extracted from yeast cells were amplified by RT-PCR, and their sequences were determined, confirming the identity of the splice junctions. We then constructed two fusions between the premRNA sequence (STE2 or STE3) and the 5'- or 3'-pretRNA half, so that the two hybrid RNAs can associate with each other, in trans, through their tRNA halves. Splicing occurs at the predicted pretRNA sites, producing a chimeric STE3-STE2 receptor mRNA. RNA trans-splicing mediated by tRNA sequences, therefore, is a mechanism capable of producing new kinds of RNAs, which could code for novel proteins.

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Human ApoD, an apolipoprotein up-regulated in neurodegenerative diseases, extends lifespan and increases stress resistance in Drosophila.

Publication Date: 2008 May 13 PMID: 18458334
Authors: Muffat, J. - Walker, D. W. - Benzer, S.
Journal: Proc Natl Acad Sci U S A

Apolipoprotein D (ApoD) expression increases in several neurological disorders and in spinal cord injury. We provide a report of a physiological role for human ApoD (hApoD): Flies overexpressing hApoD are long-lived and protected against stress conditions associated with aging and neurodegeneration, including hyperoxia, dietary paraquat, and heat stress. We show that the fly ortholog, Glial Lazarillo, is strongly up-regulated in response to these extrinsic stresses and also can protect in vitro-cultured cells in situations modeling Alzheimer's disease (AD) and Parkinson's disease (PD). In adult flies, hApoD overexpression reduces age-associated lipid peroxide accumulation, suggesting a proximal mechanism of action. Similar data obtained in the mouse [Ganfornina, M.D., et al., (2008) Apolipoprotein D is involved in the mechanisms regulating protection from oxidative stress. Aging Cell 10.1111/j.1474-9726.2008.00395.] as well as in plants (Charron et al., personal communication) suggest that ApoD and its orthologs play an evolutionarily conserved role in response to stress, possibly managing or preventing lipid peroxidation.

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Genomic and epigenetic alterations deregulate microRNA expression in human epithelial ovarian cancer.

Publication Date: 2008 May 13 PMID: 18458333
Authors: Zhang, L. - Volinia, S. - Bonome, T. - Calin, G. A. - Greshock, J. - Yang, N. - Liu, C. G. - Giannakakis, A. - Alexiou, P. - Hasegawa, K. - Johnstone, C. N. - Megraw, M. S. - Adams, S. - Lassus, H. - Huang, J. - Kaur, S. - Liang, S. - Sethupathy, P. - Leminen, A. - Simossis, V. A. - Sandaltzopoulos, R. - Naomoto, Y. - Katsaros, D. - Gimotty, P. A. - DeMichele, A. - Huang, Q. - Butzow, R. - Rustgi, A. K. - Weber, B. L. - Birrer, M. J. - Hatzigeorgiou, A. G. - Croce, C. M. - Coukos, G.
Journal: Proc Natl Acad Sci U S A

MicroRNAs (miRNAs) are an abundant class of small noncoding RNAs that function as negative gene regulators. miRNA deregulation is involved in the initiation and progression of human cancer; however, the underlying mechanism and its contributions to genome-wide transcriptional changes in cancer are still largely unknown. We studied miRNA deregulation in human epithelial ovarian cancer by integrative genomic approach, including miRNA microarray (n = 106), array-based comparative genomic hybridization (n = 109), cDNA microarray (n = 76), and tissue array (n = 504). miRNA expression is markedly down-regulated in malignant transformation and tumor progression. Genomic copy number loss and epigenetic silencing, respectively, may account for the down-regulation of approximately 15% and at least approximately 36% of miRNAs in advanced ovarian tumors and miRNA down-regulation contributes to a genome-wide transcriptional deregulation. Last, eight miRNAs located in the chromosome 14 miRNA cluster (Dlk1-Gtl2 domain) were identified as potential tumor suppressor genes. Therefore, our results suggest that miRNAs may offer new biomarkers and therapeutic targets in epithelial ovarian cancer.

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Centrosomal RNA correlates with intron-poor nuclear genes in Spisula oocytes.

Publication Date: 2008 May 13 PMID: 18458332
Authors: Alliegro, M. C. - Alliegro, M. A.
Journal: Proc Natl Acad Sci U S A

The evolutionary origin of centriole/kinetosomes, centrosomes, and other microtubule organizing centers (MTOCs), whether by direct filiation or symbiogenesis, has been controversial for >50 years. Centrioles, like mitochondria and chloroplasts, duplicate independently of the nucleus and constitute a heritable system independent of chromosomal DNA. Nucleic acids endogenous to the MTOC would support evolutionary origin by symbiogenesis. To date, most reports of centrosome-associated nucleic acids have used generalized reagents such as RNases and nucleic acid dyes. Here, from a library of RNAs extracted from isolated surf clam (Spisula solidissima) centrosomes, we describe a group of centrosome-associated transcripts representing a structurally unique intron-poor collection of nuclear genes skewed toward nucleic acid metabolism. Thus, we resolve the debate over the existence of centrosome-associated RNA (cnRNA). A subset of cnRNAs contain functional domains that are highly conserved across distant taxa, such as nucleotide polymerase motifs. In situ localization of cnRNA65, a molecule with an RNA polymerase domain, showed it is present in the intact oocyte nucleus (germinal vesicle). Its expression, therefore, precedes the appearance of gamma-tubulin-containing centrosomes. At this stage, the in situ signal resembles the nucleolinus, a poorly understood organelle proposed to play a role in spindle formation. After oocyte activation and germinal vesicle breakdown, cnRNA65 persists as a cytoplasmic patch within which gamma-tubulin-stained centrosomes can be seen. These observations provoke the question of whether cnRNAs and the nucleolinus serve as cytological progenitors of the centrosome and may support a symbiogenetic model for its evolution.

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