bluuebunny/crossref_metadata_2025 huggingface dataset:
Tyrosine phosphorylation regulates the partitioning of STAT1 between different dimer conformations
Authors: Wenta Nikola , Strauss Holger , Meyer Stefanie , Vinkemeier Uwe
Published: July 2008

The activation/inactivation cycle of STAT transcription factors entails their transition between different dimer conformations. Unphosphorylated STATs can dimerize in an antiparallel conformation via extended interfaces of the globular N-domains, whereas STAT activation triggers a parallel dimer conformation with mutual phosphortyrosine:SH2 domain interactions, resulting in DNA-binding and nuclear retention. However, despite the crucial role of STAT tyrosine phosphorylation in cytokine signaling, it has not been determined how this modification affects the stability and the conformational flexibility of STAT dimers. Here, we use analytical ultracentrifugation and electrophoretic mobility shift assay (EMSA) to study the association of STAT1 in solution before and after tyrosine phosphorylation. It is revealed that STAT1 formed high-affinity dimers (
K
d
of ≈50 nM) with estimated half-lives of 20–40 min irrespective of the phosphorylation status. Our results demonstrate that parallel and antiparallel conformations of STAT1 were present simultaneously, supported by mutually exclusive interfaces; and the transition between conformations occurred through affinity-driven dissociation/association reactions. Therefore, tyrosine phosphorylation was dispensable for DNA binding, but the phosphorylation enforced preformed SH2 domain-mediated dimers, thus enhancing the DNA-binding activity of STAT1 >200-fold. Moreover, upon STAT1 activation the N-domains adopted an open conformation and engaged in interdimer interactions, as demonstrated by their participation in tetramerization instead of dimerization. Yet, homotypic N-domain interactions are not conserved in the STAT family, because the N-domain dissociation constants of STAT1, STAT3, and STAT4 differed by more than three orders of magnitude. In conclusion, STAT1 constantly oscillated between different dimer conformations, whereby the abundance of the dimerization interfaces was determined by tyrosine phosphorylation.
A unique life history among tetrapods: An annual chameleon living mostly as an egg
Authors: Karsten Kristopher B. , Andriamandimbiarisoa Laza N. , Fox Stanley F. , Raxworthy Christopher J.
Published: July 2008

The ≈28,300 species of tetrapods (four-limbed vertebrates) almost exclusively have perennial life spans. Here, we report the discovery of a remarkable annual tetrapod from the arid southwest of Madagascar: the chameleon
Furcifer labordi
, with a posthatching life span of just 4–5 months. At the start of the active season (November), an age cohort of hatchlings emerges; larger juveniles or adults are not present. These hatchlings grow rapidly, reach sexual maturity in less than 2 months, and reproduce in January–February. After reproduction, senescence appears, and the active season concludes with population-wide adult death. Consequently, during the dry season, the entire population is represented by developing eggs that incubate for 8–9 months before synchronously hatching at the onset of the following rainy season. Remarkably, this chameleon spends more of its short annual life cycle inside the egg than outside of it. Our review of tetrapod longevity (>1,700 species) finds no others with such a short life span. These findings suggest that the notorious rapid death of chameleons in captivity may, for some species, actually represent the natural adult life span. Consequently, a new appraisal may be warranted concerning the viability of chameleon breeding programs, which could have special significance for species of conservation concern. Additionally, because
F. labordi
is closely related to other perennial species, this chameleon group may prove also to be especially well suited for comparative studies that focus on life history evolution and the ecological, genetic, and/or hormonal determinants of aging, longevity, and senescence.
Spatiotemporal object continuity in human ventral visual cortex
Authors: Yi Do-Joon , Turk-Browne Nicholas B. , Flombaum Jonathan I. , Kim Min-Shik , Scholl Brian J. , Chun Marvin M.
Published: July 2008

Coherent visual experience requires that objects be represented as the same persisting individuals over time and motion. Cognitive science research has identified a powerful principle that guides such processing: Objects must trace continuous paths through space and time. Little is known, however, about how neural representations of objects, typically defined by visual features, are influenced by spatiotemporal continuity. Here, we report the consequences of spatiotemporally continuous vs. discontinuous motion on perceptual representations in human ventral visual cortex. In experiments using both dynamic occlusion and apparent motion, face-selective cortical regions exhibited significantly less activation when faces were repeated in continuous vs. discontinuous trajectories, suggesting that discontinuity caused featurally identical objects to be represented as different individuals. These results indicate that spatiotemporal continuity modulates neural representations of object identity, influencing judgments of object persistence even in the most staunchly “featural” areas of ventral visual cortex.
A stringent test for hydrophobicity scales: Two proteins with 88% sequence identity but different structure and function
Authors: Kister Alexander E. , Phillips James C.
Published: July 2008

Protein–protein interactions (protein functionalities) are mediated by water, which compacts individual proteins and promotes close and temporarily stable large-area protein–protein interfaces. In their classic article, Kyte and Doolittle (KD) concluded that the “simplicity and graphic nature of hydrophobicity scales make them very useful tools for the evaluation of protein structures.” In practice, however, attempts to develop hydrophobicity scales (for example, compatible with classical force fields (CFF) in calculating the energetics of protein folding) have encountered many difficulties. Here, we suggest an entirely different approach based on the idea that proteins are self-organized networks, subject to evolving finite-scale criticality (like some network glasses). We test this proposal against two small proteins that are delicately balanced between α and α/β structures, with different functions encoded with only 12% of their amino acids. This example explains why protein structure prediction is so challenging, and it provides a severe test for the accuracy and content of hydrophobicity scales. This method confirms KD's evaluation and at the same time suggests that protein structure, dynamics, and function can be best discussed without using CFF.
PIF1 directly and indirectly regulates chlorophyll biosynthesis to optimize the greening process in
Arabidopsis
Authors: Moon Jennifer , Zhu Ling , Shen Hui , Huq Enamul
Published: July 2008

Plants depend on light signals to modulate many aspects of their development and optimize their photosynthetic capacity. Phytochromes (phys), a family of photoreceptors, initiate a signal transduction pathway that alters expression of a large number of genes to induce these responses. Recently, phyA and phyB were shown to bind members of a basic helix–loop–helix family of transcription factors called phy-interacting factors (PIFs). PIF1 negatively regulates chlorophyll biosynthesis and seed germination in the dark, and light-induced degradation of PIF1 relieves this negative regulation to promote photomorphogenesis. Here, we report that PIF1 regulates expression of a discrete set of genes in the dark, including
protochlorophyllide oxidoreductase
(
POR
),
ferrochelatase
(
FeChII
), and
heme oxygenase
(
HO3
), which are involved in controlling the chlorophyll biosynthetic pathway. Using ChIP and DNA gel-shift assays, we demonstrate that PIF1 directly binds to a G-box (CACGTG) DNA sequence element present in the
PORC
promoter. Moreover, in transient assays, PIF1 activates transcription of
PORC
in a G-box-dependent manner. These data strongly suggest that PIF1 directly and indirectly regulates key genes involved in chlorophyll biosynthesis to optimize the greening process in
Arabidopsis
.
Synaptojanin 1-linked phosphoinositide dyshomeostasis and cognitive deficits in mouse models of Down's syndrome
Authors: Voronov Sergey V. , Frere Samuel G. , Giovedi Silvia , Pollina Elizabeth A. , Borel Christelle , Zhang Hong , Schmidt Cecilia , Akeson Ellen C. , Wenk Markus R. , Cimasoni Laurent , Arancio Ottavio , Davisson Muriel T. , Antonarakis Stylianos E. , Gardiner Katheleen , De Camilli Pietro , Di Paolo Gilbert
Published: July 2008

Phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P
2
] is a signaling phospholipid implicated in a wide variety of cellular functions. At synapses, where normal PtdIns(4,5)P
2
balance is required for proper neurotransmission, the phosphoinositide phosphatase synaptojanin 1 is a key regulator of its metabolism. The underlying gene,
SYNJ1
, maps to human chromosome 21 and is thus a candidate for involvement in Down's syndrome (DS), a complex disorder resulting from the overexpression of trisomic genes. Here, we show that PtdIns(4,5)P
2
metabolism is altered in the brain of Ts65Dn mice, the most commonly used model of DS. This defect is rescued by restoring
Synj1
to disomy in Ts65Dn mice and is recapitulated in transgenic mice overexpressing
Synj1
from BAC constructs. These transgenic mice also exhibit deficits in performance of the Morris water maze task, suggesting that PtdIns(4,5)P
2
dyshomeostasis caused by gene dosage imbalance for
Synj1
may contribute to brain dysfunction and cognitive disabilities in DS.
Four-color DNA sequencing with 3′-
O
-modified nucleotide reversible terminators and chemically cleavable fluorescent dideoxynucleotides
Authors: Guo Jia , Xu Ning , Li Zengmin , Zhang Shenglong , Wu Jian , Kim Dae Hyun , Sano Marma Mong , Meng Qinglin , Cao Huanyan , Li Xiaoxu , Shi Shundi , Yu Lin , Kalachikov Sergey , Russo James J. , Turro Nicholas J. , Ju Jingyue
Published: July 2008

DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction can decipher many sequences in parallel. We report here a DNA sequencing method that is a hybrid between the Sanger dideoxynucleotide terminating reaction and SBS. In this approach, four nucleotides, modified as reversible terminators by capping the 3′-OH with a small reversible moiety so that they are still recognized by DNA polymerase as substrates, are combined with four cleavable fluorescent dideoxynucleotides to perform SBS. The ratio of the two sets of nucleotides is adjusted as the extension cycles proceed. Sequences are determined by the unique fluorescence emission of each fluorophore on the DNA products terminated by ddNTPs. On removing the 3′-OH capping group from the DNA products generated by incorporating the 3′-
O
-modified dNTPs and the fluorophore from the DNA products terminated with the ddNTPs, the polymerase reaction reinitiates to continue the sequence determination. By using an azidomethyl group as a chemically reversible capping moiety in the 3′-
O
-modified dNTPs, and an azido-based cleavable linker to attach the fluorophores to the ddNTPs, we synthesized four 3′-
O
-azidomethyl-dNTPs and four ddNTP-azidolinker-fluorophores for the hybrid SBS. After sequence determination by fluorescence imaging, the 3′-
O
-azidomethyl group and the fluorophore attached to the DNA extension product via the azidolinker are efficiently removed by using Tris(2-carboxyethyl)phosphine in aqueous solution that is compatible with DNA. Various DNA templates, including those with homopolymer regions, were accurately sequenced with a read length of >30 bases by using this hybrid SBS method on a chip and a four-color fluorescence scanner.
Humid tropical forest clearing from 2000 to 2005 quantified by using multitemporal and multiresolution remotely sensed data
Authors: Hansen Matthew C. , Stehman Stephen V. , Potapov Peter V. , Loveland Thomas R. , Townshend John R. G. , DeFries Ruth S. , Pittman Kyle W. , Arunarwati Belinda , Stolle Fred , Steininger Marc K. , Carroll Mark , DiMiceli Charlene
Published: July 2008

Forest cover is an important input variable for assessing changes to carbon stocks, climate and hydrological systems, biodiversity richness, and other sustainability science disciplines. Despite incremental improvements in our ability to quantify rates of forest clearing, there is still no definitive understanding on global trends. Without timely and accurate forest monitoring methods, policy responses will be uninformed concerning the most basic facts of forest cover change. Results of a feasible and cost-effective monitoring strategy are presented that enable timely, precise, and internally consistent estimates of forest clearing within the humid tropics. A probability-based sampling approach that synergistically employs low and high spatial resolution satellite datasets was used to quantify humid tropical forest clearing from 2000 to 2005. Forest clearing is estimated to be 1.39% (SE 0.084%) of the total biome area. This translates to an estimated forest area cleared of 27.2 million hectares (SE 2.28 million hectares), and represents a 2.36% reduction in area of humid tropical forest. Fifty-five percent of total biome clearing occurs within only 6% of the biome area, emphasizing the presence of forest clearing “hotspots.” Forest loss in Brazil accounts for 47.8% of total biome clearing, nearly four times that of the next highest country, Indonesia, which accounts for 12.8%. Over three-fifths of clearing occurs in Latin America and over one-third in Asia. Africa contributes 5.4% to the estimated loss of humid tropical forest cover, reflecting the absence of current agro-industrial scale clearing in humid tropical Africa.
Two different mitotic checkpoint inhibitors of the anaphase-promoting complex/cyclosome antagonize the action of the activator Cdc20
Authors: Eytan Esther , Braunstein Ilana , Ganoth Dvora , Teichner Adar , Hittle James C. , Yen Tim J. , Hershko Avram
Published: July 2008

The mitotic checkpoint system ensures the fidelity of chromosome segregation by preventing the completion of mitosis in the presence of any misaligned chromosome. When activated, it blocks the initiation of anaphase by inhibiting the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C). Little is known about the biochemical mechanisms by which this system inhibits APC/C, except for the existence of a mitotic checkpoint complex (MCC) inhibitor of APC/C composed of the APC/C activator Cdc20 associated with the checkpoint proteins Mad2, BubR1, and Bub3. We have been studying the mechanisms of the mitotic checkpoint system in extracts that reproduce its downstream events. We found that inhibitory factors are associated with APC/C in the checkpoint-arrested state, which can be recovered from immunoprecipitates. Only a part of the inhibitory activity was caused by MCC [Braunstein I, Miniowitz S, Moshe Y, Hershko A (2007)
Proc Natl Acad Sci USA
104:4870–4875]. Here, we show that during exit from checkpoint, rapid disassembly of MCC takes place while APC/C is still inactive. This observation suggested the possible involvement of multiple factors in the regulation of APC/C by the mitotic checkpoint. We have separated a previously unknown inhibitor of APC/C from MCC. This inhibitor, called mitotic checkpoint factor 2 (MCF2), is associated with APC/C only in the checkpoint-arrested state. The inhibition of APC/C by both MCF2 and MCC was decreased at high concentrations of Cdc20. We propose that both MCF2 and MCC inhibit APC/C by antagonizing Cdc20, possibly by interaction with the Cdc20-binding site of APC/C.
Clearance of influenza virus from the lung depends on migratory langerin+CD11b− but not plasmacytoid dendritic cells
Authors: GeurtsvanKessel Corine H. , Willart Monique A.M. , van Rijt Leonie S. , Muskens Femke , Kool Mirjam , Baas Chantal , Thielemans Kris , Bennett Clare , Clausen Björn E. , Hoogsteden Henk C. , Osterhaus Albert D.M.E. , Rimmelzwaan Guus F. , Lambrecht Bart N.
Published: July 2008

Although dendritic cells (DCs) play an important role in mediating protection against influenza virus, the precise role of lung DC subsets, such as CD11b− and CD11b+ conventional DCs or plasmacytoid DCs (pDCs), in different lung compartments is currently unknown. Early after intranasal infection, tracheal CD11b−CD11chi DCs migrated to the mediastinal lymph nodes (MLNs), acquiring co-stimulatory molecules in the process. This emigration from the lung was followed by an accumulation of CD11b+CD11chi DCs in the trachea and lung interstitium. In the MLNs, the CD11b+ DCs contained abundant viral nucleoprotein (NP), but these cells failed to present antigen to CD4 or CD8 T cells, whereas resident CD11b−CD8α+ DCs presented to CD8 cells, and migratory CD11b−CD8α− DCs presented to CD4 and CD8 T cells. When lung CD11chi DCs and macrophages or langerin+CD11b−CD11chi DCs were depleted using either CD11c–diphtheria toxin receptor (DTR) or langerin-DTR mice, the development of virus-specific CD8+ T cells was severely delayed, which correlated with increased clinical severity and a delayed viral clearance. 120G8+ CD11cint pDCs also accumulated in the lung and LNs carrying viral NP, but in their absence, there was no effect on viral clearance or clinical severity. Rather, in pDC-depleted mice, there was a reduction in antiviral antibody production after lung clearance of the virus. This suggests that multiple DCs are endowed with different tasks in mediating protection against influenza virus.
A rush to judgment on Th17
Authors: Steinman Lawrence
Published: July 2008

Some immunologists have characterized T helper (Th)17 T cells as the master mediators of tissue damage in a variety of pathological conditions. New data now demonstrate that Th1 and Th17 T cells are independently capable of inducing disease in two established models of autoimmunity. Thus, the role of Th17 cytokines as the central mediators of pathological tissue damage seems to require clarification.
Length-dependent recognition of double-stranded ribonucleic acids by retinoic acid–inducible gene-I and melanoma differentiation–associated gene 5
Authors: Kato Hiroki , Takeuchi Osamu , Mikamo-Satoh Eriko , Hirai Reiko , Kawai Tomoji , Matsushita Kazufumi , Hiiragi Akane , Dermody Terence S. , Fujita Takashi , Akira Shizuo
Published: July 2008

The ribonucleic acid (RNA) helicases retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation–associated gene 5 (MDA5) recognize distinct viral and synthetic RNAs, leading to the production of interferons. Although 5′-triphosphate single-stranded RNA is a RIG-I ligand, the role of RIG-I and MDA5 in double-stranded (ds) RNA recognition remains to be characterized. In this study, we show that the length of dsRNA is important for differential recognition by RIG-I and MDA5. The MDA5 ligand, polyinosinic-polycytidylic acid, was converted to a RIG-I ligand after shortening of the dsRNA length. In addition, viral dsRNAs differentially activated RIG-I and MDA5, depending on their length. Vesicular stomatitis virus infection generated dsRNA, which is responsible for RIG-I–mediated recognition. Collectively, RIG-I detects dsRNAs without a 5′-triphosphate end, and RIG-I and MDA5 selectively recognize short and long dsRNAs, respectively.
Deficiency of Th17 cells in hyper IgE syndrome due to mutations in STAT3
Authors: Ma Cindy S. , Chew Gary Y.J. , Simpson Nicholas , Priyadarshi Archana , Wong Melanie , Grimbacher Bodo , Fulcher David A. , Tangye Stuart G. , Cook Matthew C.
Published: July 2008

Hyper–immunoglobulin E syndrome (HIES) is a primary immune deficiency characterized by abnormal and devastating susceptibility to a narrow spectrum of infections, most commonly Staphylococcus aureus and Candida albicans. Recent investigations have identified mutations in STAT3 in the majority of HIES patients studied. Despite the identification of the genetic cause of HIES, the mechanisms underlying the pathological features of this disease remain to be elucidated. Here, we demonstrate a failure of CD4+ T cells harboring heterozygous STAT3 mutations to generate interleukin 17–secreting (i.e., T helper [Th]17) cells in vivo and in vitro due to a failure to express sufficient levels of the Th17-specific transcriptional regulator retinoid-related orphan receptor γt. Because Th17 cells are enriched for cells with specificities against fungal antigens, our results may explain the pattern of infection susceptibility characteristic of patients with HIES. Furthermore, they underscore the importance of Th17 responses in normal host defense against the common pathogens S. aureus and C. albicans.
Mutations in STAT3 and IL12RB1 impair the development of human IL-17–producing T cells
Authors: de Beaucoudrey Ludovic , Puel Anne , Filipe-Santos Orchidée , Cobat Aurélie , Ghandil Pegah , Chrabieh Maya , Feinberg Jacqueline , von Bernuth Horst , Samarina Arina , Jannière Lucile , Fieschi Claire , Stéphan Jean-Louis , Boileau Catherine , Lyonnet Stanislas , Jondeau Guillaume , Cormier-Daire Valérie , Le Merrer Martine , Hoarau Cyrille , Lebranchu Yvon , Lortholary Olivier , Chandesris Marie-Olivia , Tron François , Gambineri Eleonora , Bianchi Lucia , Rodriguez-Gallego Carlos , Zitnik Simona E. , Vasconcelos Julia , Guedes Margarida , Vitor Artur Bonito , Marodi Laszlo , Chapel Helen , Reid Brenda , Roifman Chaim , Nadal David , Reichenbach Janine , Caragol Isabel , Garty Ben-Zion , Dogu Figen , Camcioglu Yildiz , Gülle Sanyie , Sanal Ozden , Fischer Alain , Abel Laurent , Stockinger Birgitta , Picard Capucine , Casanova Jean-Laurent
Published: July 2008

The cytokines controlling the development of human interleukin (IL) 17–producing T helper cells in vitro have been difficult to identify. We addressed the question of the development of human IL-17–producing T helper cells in vivo by quantifying the production and secretion of IL-17 by fresh T cells ex vivo, and by T cell blasts expanded in vitro from patients with particular genetic traits affecting transforming growth factor (TGF) β, IL-1, IL-6, or IL-23 responses. Activating mutations in TGFB1, TGFBR1, and TGFBR2 (Camurati-Engelmann disease and Marfan-like syndromes) and loss-of-function mutations in IRAK4 and MYD88 (Mendelian predisposition to pyogenic bacterial infections) had no detectable impact. In contrast, dominant-negative mutations in STAT3 (autosomal-dominant hyperimmunoglobulin E syndrome) and, to a lesser extent, null mutations in IL12B and IL12RB1 (Mendelian susceptibility to mycobacterial diseases) impaired the development of IL-17–producing T cells. These data suggest that IL-12Rβ1– and STAT-3–dependent signals play a key role in the differentiation and/or expansion of human IL-17–producing T cell populations in vivo.
Differential recognition of double-stranded RNA by RIG-I–like receptors in antiviral immunity
Authors: Saito Takeshi , Gale Michael
Published: July 2008

Retinoic acid–inducible gene (RIG)-I–like receptors (RLRs) are cytosolic RNA helicases that sense viral RNA and trigger signaling pathways that induce the production of type I interferons (IFNs) and proinflammatory cytokines. RLRs recognize distinct and overlapping sets of viruses, but the mechanisms that dictate this specificity were unknown. A new study now provides evidence for size-based discrimination of double-stranded RNA (dsRNA) by RLRs and suggests how host cells recognize a variety of RNA viruses.
Predicting protein function from domain content
Authors: Forslund Kristoffer , Sonnhammer Erik L. L.
Published: July 2008

Motivation: Computational assignment of protein function may be the single most vital application of bioinformatics in the post-genome era. These assignments are made based on various protein features, where one is the presence of identifiable domains. The relationship between protein domain content and function is important to investigate, to understand how domain combinations encode complex functions.

Results: Two different models are presented on how protein domain combinations yield specific functions: one rule-based and one probabilistic. We demonstrate how these are useful for Gene Ontology annotation transfer. The first is an intuitive generalization of the Pfam2GO mapping, and detects cases of strict functional implications of sets of domains. The second uses a probabilistic model to represent the relationship between domain content and annotation terms, and was found to be better suited for incomplete training sets. We implemented these models as predictors of Gene Ontology functional annotation terms. Both predictors were more accurate than conventional best BLAST-hit annotation transfer and more sensitive than a single-domain model on a large-scale dataset. We present a number of cases where combinations of Pfam-A protein domains predict functional terms that do not follow from the individual domains.

Availability: Scripts and documentation are available for download at http://sonnhammer.sbc.su.se/multipfam2go_source_docs.tar

Contact:  Kristoffer.Forslund@sbc.su.se

Supplementary information:  Supplementary data are available at Bioinformatics online.
Nuclear HuR accumulation through phosphorylation by Cdk1
Authors: Kim Hyeon Ho , Abdelmohsen Kotb , Lal Ashish , Pullmann Rudolf , Yang Xiaoling , Galban Stefanie , Srikantan Subramanya , Martindale Jennifer L. , Blethrow Justin , Shokat Kevan M. , Gorospe Myriam
Published: July 2008

A predominantly nuclear RNA-binding protein, HuR translocates to the cytoplasm in response to stress and proliferative signals, where it stabilizes or modulates the translation of target mRNAs. Here, we present evidence that HuR phosphorylation at S202 by the G2-phase kinase Cdk1 influences its subcellular distribution. HuR was specifically phosphorylated in synchronous G2-phase cultures; its cytoplasmic levels increased by Cdk1-inhibitory interventions and declined in response to Cdk1-activating interventions. In keeping with the prominently cytoplasmic location of the nonphosphorylatable point mutant HuR(S202A), phospho-HuR(S202) was shown to be predominantly nuclear using a novel anti-phospho-HuR(S202) antibody. The enhanced cytoplasmic presence of unphosphorylated HuR was linked to its decreased association with 14–3–3 and to its heightened binding to target mRNAs. Our findings suggest that Cdk1 phosphorylates HuR during G2, thereby helping to retain it in the nucleus in association with 14–3–3 and hindering its post-transcriptional function and anti-apoptotic influence.
Distinct activities of the DExD/H-box splicing factor hUAP56 facilitate stepwise assembly of the spliceosome
Authors: Shen Haihong , Zheng Xuexiu , Shen Jingping , Zhang Lingdi , Zhao Rui , Green Michael R.
Published: July 2008

The essential splicing factor human UAP56 (hUAP56) is a DExD/H-box protein known to promote prespliceosome assembly. Here, using a series of hUAP56 mutants that are defective for ATP-binding, ATP hydrolysis, or dsRNA unwindase/helicase activity, we assess the relative contributions of these biochemical functions to pre-mRNA splicing. We show that prespliceosome assembly requires hUAP56’s ATP-binding and ATPase activities, which, unexpectedly, are required for hUAP56 to interact with U2AF65 and be recruited into splicing complexes. Surprisingly, we find that hUAP56 is also required for mature spliceosome assembly, which requires, in addition to the ATP-binding and ATPase activities, hUAP56’s dsRNA unwindase/helicase activity. We demonstrate that hUAP56 directly contacts U4 and U6 snRNAs and can promote unwinding of the U4/U6 duplex, and that both these activities are dependent on U2AF65. Our results indicate that hUAP56 first interacts with U2AF65 in an ATP-dependent manner, and subsequently with U4/U6 snRNAs to facilitate stepwise assembly of the spliceosome.
The phospho-occupancy of an atypical SLIMB-binding site on PERIOD that is phosphorylated by DOUBLETIME controls the pace of the clock
Authors: Chiu Joanna C. , Vanselow Jens T. , Kramer Achim , Edery Isaac
Published: July 2008

A common feature of animal circadian clocks is the progressive phosphorylation of PERIOD (PER) proteins, which is highly dependent on casein kinase Iδ/ε (CKIδ/ε; termed DOUBLETIME [DBT] in Drosophila) and ultimately leads to the rapid degradation of hyperphosphorylated isoforms via a mechanism involving the F-box protein, β-TrCP (SLIMB in Drosophila). Here we use the Drosophila melanogaster model system, and show that a key step in controlling the speed of the clock is phosphorylation of an N-terminal Ser (S47) by DBT, which collaborates with other nearby phosphorylated residues to generate a high-affinity atypical SLIMB-binding site on PER. DBT-dependent increases in the phospho-occupancy of S47 are temporally gated, dependent on the centrally located DBT docking site on PER and partially counterbalanced by protein phosphatase activity. We propose that the gradual DBT-mediated phosphorylation of a nonconsensus SLIMB-binding site establishes a temporal threshold for when in a daily cycle the majority of PER proteins are tagged for rapid degradation. Surprisingly, most of the hyperphosphorylation is unrelated to direct effects on PER stability. We also use mass spectrometry to map phosphorylation sites on PER, leading to the identification of a number of “phospho-clusters” that explain several of the classic per mutants.
SpoIIIE strips proteins off the DNA during chromosome translocation
Authors: Marquis Kathleen A. , Burton Briana M. , Nollmann Marcelo , Ptacin Jerod L. , Bustamante Carlos , Ben-Yehuda Sigal , Rudner David Z.
Published: July 2008

The FtsK/SpoIIIE family of DNA transporters are responsible for translocating missegregated chromosomes after the completion of cell division. An extreme example of this post-cytokinetic DNA segregation occurs during spore formation in the bacterium Bacillus subtilis, where SpoIIIE pumps three-quarters of the chromosome (>3 megabases) into one of the two daughter cells. Here, we investigate the fate of the proteins associated with the translocated DNA. Taking advantage of several unique features of Bacillus sporulation, we demonstrate that RNA polymerase, transcription factors, and chromosome remodeling proteins are stripped off the DNA during translocation of the chromosome into the forespore compartment. Furthermore, we show that in vitro the soluble ATPase domain of SpoIIIE can displace RNA polymerase bound to DNA, suggesting that SpoIIIE alone is capable of this wire-stripping activity. Our data suggest that the bulk of the forespore chromosome is translocated naked into the forespore compartment. We propose that the translocation-stripping activity of SpoIIIE plays a key role in reprogramming developmental gene expression in the forespore.
Fibrinogen drives dystrophic muscle fibrosis via a TGFβ/alternative macrophage activation pathway
Authors: Vidal Berta , Serrano Antonio L. , Tjwa Marc , Suelves Mònica , Ardite Esther , De Mori Roberta , Baeza-Raja Bernat , Martínez de Lagrán María , Lafuste Peggy , Ruiz-Bonilla Vanessa , Jardí Mercè , Gherardi Romain , Christov Christo , Dierssen Mara , Carmeliet Peter , Degen Jay L. , Dewerchin Mieke , Muñoz-Cánoves Pura
Published: July 2008

In the fatal degenerative Duchenne muscular dystrophy (DMD), skeletal muscle is progressively replaced by fibrotic tissue. Here, we show that fibrinogen accumulates in dystrophic muscles of DMD patients andmdxmice. Genetic loss or pharmacological depletion of fibrinogen in these mice reduced fibrosis and dystrophy progression. Our results demonstrate that fibrinogen–Mac-1 receptor binding, through induction of IL-1β, drives the synthesis of transforming growth factor-β (TGFβ) bymdxmacrophages, which in turn induces collagen production inmdxfibroblasts. Fibrinogen-produced TGFβ further amplifies collagen accumulation through activation of profibrotic alternatively activated macrophages. Fibrinogen, by engaging its αvβ3 receptor on fibroblasts, also directly promotes collagen synthesis. These data unveil a profibrotic role of fibrinogen deposition in muscle dystrophy.
Identification of Differential Genes by Suppression Subtractive Hybridization: II. Subtractive Hybridization
Authors: Rebrikov Denis V.
Published: July 2008

INTRODUCTION

Suppression subtractive hybridization (SSH) is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. This technique can be used to compare two mRNA populations and obtain cDNAs representing genes that are either overexpressed or exclusively expressed in one population as compared to another. It can also be used for comparison of genomic DNA populations. This protocol describes a method for subtractive hybridization using adapter-ligated tester and RsaI-digested driver DNA samples.
Generating HapMap Data Text Reports Using the Genome Browser
Authors: Smith Albert Vernon
Published: July 2008

INTRODUCTION

The primary goal of the International Haplotype Map Project has been to develop a haplotype map of the human genome that describes the common patterns of genetic variation, in order to accelerate the search for the genetic causes of human disease. Within the project, ~3.9 million distinct single-nucleotide polymorphisms (SNPs) have been genotyped in 270 individuals from four worldwide populations. The project data are available for unrestricted public use at the HapMap website. This site, which is the primary portal to genotype data produced by the project, offers bulk downloads of the data set, as well as interactive data browsing and analysis tools that are not available elsewhere. In many cases, a researcher will be interested in downloading HapMap data from a region of interest for local analysis. This protocol describes the direct download of genotype, frequency, tag-SNPs, and other reports from the project website, using the genome browser.
Manipulating HapMap Data Using HaploView
Authors: Smith Albert Vernon
Published: July 2008

INTRODUCTION

The primary goal of the International Haplotype Map Project has been to develop a haplotype map of the human genome that describes the common patterns of genetic variation, in order to accelerate the search for the genetic causes of human disease. Within the project, ~3.9 million distinct single-nucleotide polymorphisms (SNPs) have been genotyped in 270 individuals from four worldwide populations. The project data are available for unrestricted public use at the HapMap website. This site, which is the primary portal to genotype data produced by the project, offers bulk downloads of the data set, as well as interactive data browsing and analysis tools that are not available elsewhere. Advanced users who wish to exercise fine control over the display of regions of high linkage disequilibrium (LD) or who wish to experiment with new algorithms for tag-SNP picking may wish to analyze HapMap data using the HaploView program. This program works well in combination with the HapMap website genome browser. A big advantage of HaploView over the genome browser is that it displays simultaneous high- and low-power views of regions of LD, and gives immediate feedback during scrolling and zooming operations. This protocol describes the use of HaploView to manipulate HapMap data.
Retrieving HapMap Data Using HapMart: Figure 1.
Authors: Smith Albert Vernon
Published: July 2008

INTRODUCTION

The primary goal of the International Haplotype Map Project has been to develop a haplotype map of the human genome that describes the common patterns of genetic variation, in order to accelerate the search for the genetic causes of human disease. Within the project, ~3.9 million distinct single-nucleotide polymorphisms (SNPs) have been genotyped in 270 individuals from four worldwide populations. The project data are available for unrestricted public use at the HapMap Web site. This site, which is the primary portal to genotype data produced by the project, offers bulk downloads of the data set, as well as interactive data browsing and analysis tools that are not available elsewhere. Because of performance considerations, interactive access to HapMap data via the genome browser on the HapMap Web site is limited to regions no more than 5 Mb wide. Researchers who wish to obtain data for chromosome- or genome-wide data have two choices: Bulk download or HapMart access. HapMart, described in this protocol, allows researchers to select SNPs using diverse criteria and to display just those aspects of the data set that they are interested in.
Generation of Driver and Reporter Constructs for the GAL4 Expression System in Drosophila: Figure 1.
Authors: Southall Tony D. , Brand Andrea H.
Published: July 2008

INTRODUCTION

The GAL4 system is a method for ectopic gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. This protocol describes the generation of driver and reporter lines for use with the GAL4 system in Drosophila. A promoter-GAL4 fusion is constructed using a P-element transformable vector, and a GAL4-responsive target gene is created via generation of an upstream activation sequence (UAS)-reporter construct. An alternative strategy for integration using the phiC31 system is also provided. Transformant lines are generated using standard procedures for microinjection.
High-Throughput Paraffin Section In Situ Hybridization and Dual Immunohistochemistry on Mouse Tissues: Figure 1.
Authors: Rumballe Bree , Georgas Kylie , Little Melissa H.
Published: July 2008

INTRODUCTION

Section in situ hybridization (SISH) is a high-resolution tool used to analyze gene expression patterns. This protocol utilizes the Tecan Freedom EVO150 platform to perform high-throughput SISH on paraffin sections to detect mRNA with a digoxigenin (DIG)-labeled probe. The slide is mounted and imaged before performing immunohistochemistry (IHC) on the same section. The dual reaction enables a marker of protein expression to be localized on the same section as the mRNA and facilitates more accurate annotation of the gene expression.
Identification of Differential Genes by Suppression Subtractive Hybridization: An Overview
Authors: Rebrikov Denis V.
Published: July 2008

INTRODUCTION

Suppression subtractive hybridization (SSH) is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. This technique can be used to compare two mRNA populations and obtain cDNAs representing genes that are either overexpressed or exclusively expressed in one population as compared to another. It can also be used for comparison of genomic DNA populations. We have used SSH in studies of regeneration and development on various types of model organisms (including freshwater planaria regeneration, Xenopus laevis development, and mammalian brain cortex development). We also use SSH for the analysis of strain-specific genes in bacteria with different characteristics. During these studies, a large number of differentially regulated and differentially presented genes have been identified, including transcriptional regulation factors and restriction modification enzymes. This article describes the SSH method and considerations for its use.
The GAL4 System: A Versatile Toolkit for Gene Expression in Drosophila
Authors: Southall Tony D. , Elliott David A. , Brand Andrea H.
Published: July 2008

INTRODUCTION

The generation of gain-of-function phenotypes by ectopic expression of known genes provides a powerful complement to the genetic approach, in which genes are studied or identified through mutations that generally reduce or eliminate gene function. The GAL4 system is a method for ectopic gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. A key advantage of the system is the separation of the GAL4 protein from its target gene in distinct transgenic lines, which ensures that the target gene is silent until the introduction of GAL4. Recent modifications and adaptations of the GAL4 system to make the system inducible have further expanded its scope, enabling greater temporal control over the activity of GAL4. There are now large resources for the community, including thousands of GAL4 lines and a wide selection of reporter lines. Here we present an overview of the GAL4 system, highlighting recent developments and discussing methods for generating and analyzing transgenic flies for GAL4-mediated ectopic expression.
Nuclear Stains
Authors: Kiernan John Alan
Published: July 2008

INTRODUCTION

For the study of microscopic anatomy and of pathological material, it is usual to stain sections of tissue in such a way as to impart a dark color to the nuclei of cells and a lighter, contrasting color to the cytoplasm and extracellular structures. Nuclear stains, including cationic, anionic, and metal complexing dyes, are considered in this article. The rationales of the techniques are discussed, but methods of higher chemical specificity (e.g., for nucleic acids, carbohydrates, and functional groups of proteins) are not covered.
Anionic Counterstains
Authors: Kiernan John Alan
Published: July 2008

INTRODUCTION

For the study of microscopic anatomy and of pathological material, it is usual to stain sections of tissue in such a way as to impart a dark color to the nuclei of cells and a lighter, contrasting color to the cytoplasm and extracellular structures. In most of the general “oversight” methods used in histology, a blue, purple, or black nuclear stain is followed by a paler, usually pink, counterstain, which colors all the other components of the tissue. Simple counterstaining procedures using anionic dyes are considered in this article. The rationales of the techniques are discussed, but methods of higher chemical specificity (e.g., for nucleic acids, carbohydrates, and functional groups of proteins) are not covered.
Mutation ofArabidopsis BARD1Causes Meristem Defects by Failing to ConfineWUSCHELExpression to the Organizing Center
Authors: Han Pei , Li Qing , Zhu Yu-Xian
Published: July 2008

Stem cell fate in the Arabidopsis thaliana shoot apical meristem (SAM) is controlled by WUSCHEL (WUS) and CLAVATA. Here, we examine BARD1 (for BRCA1-associated RING domain 1), which had previously been implicated in DNA repair functions; we find that it also regulates WUS expression. We observed severe SAM defects in the knockout mutant bard1-3. WUS transcripts accumulated >238-fold in bard1-3 compared with the wild type and were located mainly in the outermost cell layers instead of the usual organizing center. A specific WUS promoter region was recognized by nuclear protein extracts obtained from wild-type plants, and this protein-DNA complex was recognized by antibodies against BARD1. The double mutant (wus-1 bard1-3) showed prematurely terminated SAM structures identical to those of wus-1, indicating that BARD1 functions through regulation of WUS. BARD1 overexpression resulted in reduced WUS transcript levels, giving a wus-1–like phenotype. Either full-length BARD1 or a clone that encoded the C-terminal domain (BARD1:C-ter;bard1-3) was sufficient to complement the bard1-3 phenotype, indicating that BARD1 functions through its C-terminal domain. Our data suggest that BARD1 regulates SAM organization and maintenance by limiting WUS expression to the organizing center.
PDE-Driven Level Sets and Shape Sensitivity for Structural Topology Optimization
Authors: Wang Michael Yu , Wang Xiaoming
Published: July 2008

This paper addresses the problem of structural shape and topology optimization. A level set method is adopted as an alternative approach to the popular homogenization based methods. The paper focuses on four areas of discussion: (1) The level-set model of the structure’s shape is characterized as a region and global representation; the shape boundary is embedded in a higher-dimensional scalar function as its “iso-surface.” Changes of the shape and topology are governed by a partial differential equation (PDE). (2) The velocity vector of the Hamilton-Jacobi PDE is shown to be naturally related to the shape derivative from the classical shape variational analysis. Thus, the level set method provides a natural setting to combine the rigorous shape variations into the optimization process. Finally, the benefit and the advantages of the developed method are illustrated with several 2D examples that have been extensively used in the recent literature of topology optimization, especially in the homogenization based methods.
Numerical and Experimental Study of Shielding Gas Orientation Effects on Particle Stream Concentration Mode in Coaxial Laser Aided Material Deposition Process
Authors: Pan Heng , Thakar Yogesh D. , Liou Frank
Published: July 2008

Laser aided deposition quality largely depends on the powder stream structure below the nozzle. Modeling of the powder concentration distribution rarely relies on the numerical approach partially due to the complex phenomenon involved in the two-phase turbulence flow. In this paper, a numerical model is introduced to predict the particle-gas flow precisely and economically in order to meet the practical requirement for coaxial nozzle design optimizations. This model is able to quantitatively predict the powder stream concentration mode under different outer shielding gas directions and inner/outer gas velocity ratio. The numerical simulation results are compared with the experimental study using prototyped coaxial nozzles. The results are found to match. This study shows that the particle concentration mode is influenced significantly by the outer gas direction and gas flow settings.
A Method to Solve Inverse Kinematics Problems Using Lie Algebra and Its Application to Robot Spray Painting Simulation
Authors: Tokunaga Hitoshi , Okano Takaaki , Matsuki Norio , Tanaka Fumiki , Kishinami Takeshi
Published: July 2008

This paper presents a new method for robot spray painting simulation. First, the authors introduce a method for rigid body motion interpolation proposed by Zefran for generating smooth trajectories of a spray gun. Second, the authors propose a method to solve inverse kinematics problems using Lie algebra for computing the motion of the robot manipulator from the spray gun’s motion. Third, the authors propose a method for calculation of color depth of the workpiece painted with the spray gun. Finally, in order to evaluate the usefulness of the three proposed methods, the authors develop a simulation system for robot spray painting.
Derivation of Design Guidelines for Miniature Machine Tools
Authors: Mishima Nozomu
Published: July 2008

As has been demonstrated the “microfactory”, which is a miniature manufacturing system proposed by the author and his research group, small machine tools that are comparable in size to their target products lead to large reductions in energy consumption and occupied space. They also increase the flexibility of system reconfiguration because of their low weight and small size. Although it had been thought that micro machine tools might not have sufficient metal cutting capability, experiments showed that were capable of micro mechanical fabrication. However, the design of miniature machine tools has not been fully optimized. For example, the design target of the first prototype, a performable miniature machine (“Micro lathe”), was to make the overall size as small as possible. The author proposed a design evaluation method to roughly estimate machine tool performances during its early design stage. In this paper, the above-mentioned design tool is applied to find suitable miniaturizing strategies. By applying the design tool to the miniaturization of machine tools, it is possible to determine which of the design candidates have the best theoretical performance and which of the local error factors would significantly affect machine performance. From the results of calculation, the tool can clarify the difference of effect of error sources on performances between normal machine tools and miniature machine tools. This leads to some suggestions regarding structures, sizes and suitable machine components. Design guidelines for miniature machine tools can be obtained from the information.