bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein model refinement using an optimized physics-based all-atom force field
Authors: Jagielska Anna , Wroblewska Liliana , Skolnick Jeffrey
Published: June 2008

One of the greatest challenges in protein structure prediction is the refinement of low-resolution predicted models to high-resolution structures that are close to the native state. Although contemporary structure prediction methods can assemble the correct topology for a large fraction of protein domains, such approximate models are often not of the resolution required for many important applications, including studies of reaction mechanisms and virtual ligand screening. Thus, the development of a method that could bring those structures closer to the native state is of great importance. We recently optimized the relative weights of the components of the Amber ff03 potential on a large set of decoy structures to create a funnel-shaped energy landscape with the native structure at the global minimum. Such an energy function might be able to drive proteins toward their native structure. In this work, for a test set of 47 proteins, with 100 decoy structures per protein that have a range of structural similarities to the native state, we demonstrate that our optimized potential can drive protein models closer to their native structure. Comparing the lowest-energy structure from each trajectory with the starting decoy, structural improvement is seen for 70% of the models on average. The ability to do such systematic structural refinements by using a physics-based all-atom potential represents a promising approach to high-resolution structure prediction.
CTCF physically links cohesin to chromatin
Authors: Rubio Eric D. , Reiss David J. , Welcsh Piri L. , Disteche Christine M. , Filippova Galina N. , Baliga Nitin S. , Aebersold Ruedi , Ranish Jeffrey A. , Krumm Anton
Published: June 2008

Cohesin is required to prevent premature dissociation of sister chromatids after DNA replication. Although its role in chromatid cohesion is well established, the functional significance of cohesin's association with interphase chromatin is not clear. Using a quantitative proteomics approach, we show that the STAG1 (Scc3/SA1) subunit of cohesin interacts with the CCTC-binding factor CTCF bound to the c-myc insulator element. Both allele-specific binding of CTCF and Scc3/SA1 at the imprinted IGF2/H19 gene locus and our analyses of human DM1 alleles containing base substitutions at CTCF-binding motifs indicate that cohesin recruitment to chromosomal sites depends on the presence of CTCF. A large-scale genomic survey using ChIP-Chip demonstrates that Scc3/SA1 binding strongly correlates with the CTCF-binding site distribution in chromosomal arms. However, some chromosomal sites interact exclusively with CTCF, whereas others interact with Scc3/SA1 only. Furthermore, immunofluorescence microscopy and ChIP-Chip experiments demonstrate that CTCF associates with both centromeres and chromosomal arms during metaphase. These results link cohesin to gene regulatory functions and suggest an essential role for CTCF during sister chromatid cohesion. These results have implications for the functional role of cohesin subunits in the pathogenesis of Cornelia de Lange syndrome and Roberts syndromes.
Regulation of NKCC2 by a chloride-sensing mechanism involving the WNK3 and SPAK kinases
Authors: Ponce-Coria José , San-Cristobal Pedro , Kahle Kristopher T. , Vazquez Norma , Pacheco-Alvarez Diana , de los Heros Paola , Juárez Patricia , Muñoz Eva , Michel Gabriela , Bobadilla Norma A. , Gimenez Ignacio , Lifton Richard P. , Hebert Steven C. , Gamba Gerardo
Published: June 2008

The Na
+
:K
+
:2Cl
−
cotransporter (NKCC2) is the target of loop diuretics and is mutated in Bartter's syndrome, a heterogeneous autosomal recessive disease that impairs salt reabsorption in the kidney's thick ascending limb (TAL). Despite the importance of this cation/chloride cotransporter (CCC), the mechanisms that underlie its regulation are largely unknown. Here, we show that intracellular chloride depletion in
Xenopus laevis
oocytes, achieved by either coexpression of the K-Cl cotransporter KCC2 or low-chloride hypotonic stress, activates NKCC2 by promoting the phosphorylation of three highly conserved threonines (96, 101, and 111) in the amino terminus. Elimination of these residues renders NKCC2 unresponsive to reductions of [Cl
−
]
i
. The chloride-sensitive activation of NKCC2 requires the interaction of two serine-threonine kinases, WNK3 (related to WNK1 and WNK4, genes mutated in a Mendelian form of hypertension) and SPAK (a Ste20-type kinase known to interact with and phosphorylate other CCCs). WNK3 is positioned upstream of SPAK and appears to be the chloride-sensitive kinase. Elimination of WNK3's unique SPAK-binding motif prevents its activation of NKCC2, as does the mutation of threonines 96, 101, and 111. A catalytically inactive WNK3 mutant also completely prevents NKCC2 activation by intracellular chloride depletion. Together these data reveal a chloride-sensing mechanism that regulates NKCC2 and provide insight into how increases in the level of intracellular chloride in TAL cells, as seen in certain pathological states, could drastically impair renal salt reabsorption.
Genetic dissection of the biosynthetic route to gentamicin A2by heterologous expression of its minimal gene set
Authors: Park Je Won , Hong Jay Sung Joong , Parajuli Niranjan , Jung Won Seok , Park Sung Ryeol , Lim Si-Kyu , Sohng Jae Kyung , Yoon Yeo Joon
Published: June 2008

Since the first use of streptomycin as an effective antibiotic drug in the treatment of tuberculosis, aminoglycoside antibiotics have been widely used against a variety of bacterial infections for over six decades. However, the pathways for aminoglycoside biosynthesis still remain unclear, mainly because of difficulty in genetic manipulation of actinomycetes producing this class of antibiotics. Gentamicin belongs to the group of 4,6-disubstituted aminoglycosides containing a characteristic core aminocyclitol moiety, 2-deoxystreptamine (2-DOS), and the recent discovery of its biosynthetic gene cluster inMicromonospora echinosporahas enabled us to decipher its biosynthetic pathway. To determine the minimal set of genes and their functions for the generation of gentamicin A2, the first pseudotrisaccharide intermediate in the biosynthetic pathway for the gentamicin complex, various sets of candidate genes fromM. echinosporaand other related aminoglycoside-producing strains were introduced into a nonaminoglycoside producing strain ofStreptomyces venezuelae. Heterologous expression of different combinations of putative 2-DOS biosynthetic genes revealed that a subset,gtmB-gtmA-gacH, is responsible for the biosynthesis of this core aminocyclitol moiety of gentamicin. Expression ofgtmGtogether withgtmB-gtmA-gacHled to production of 2′-N-acetylparomamine, demonstrating that GtmG acts as a glycosyltransferase that addsN-acetyl-d-glucosamine (GLcNA) to 2-DOS. Expression ofgtmMin a 2′-N-acetylparomamine-producing recombinantS. venezuelaestrain generated paromamine. Expression ofgtmEin an engineered paromamine-producing strain ofS. venezuelaesuccessfully generated gentamicin A2, indicating that GtmE is another glycosyltransferase that attachesd-xylose to paromamine. These results representin vivoevidence elucidating the complete biosynthetic pathway of the pseudotrisaccharide aminoglycoside.
Saccharomyces cerevisiae
septins: Supramolecular organization of heterooligomers and the mechanism of filament assembly
Authors: Bertin Aurelie , McMurray Michael A. , Grob Patricia , Park Sang-Shin , Garcia Galo , Patanwala Insiyyah , Ng Ho-leung , Alber Tom , Thorner Jeremy , Nogales Eva
Published: June 2008

Mitotic yeast cells express five septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1/Sep7). Only Shs1 is nonessential. The four essential septins form a complex containing two copies of each, but their arrangement was not known. Single-particle analysis by EM confirmed that the heterooligomer is octameric and revealed that the subunits are arrayed in a linear rod. Identity of each subunit was determined by examining complexes lacking a given septin, by antibody decoration, and by fusion to marker proteins (GFP or maltose binding protein). The rod has the order Cdc11–Cdc12–Cdc3–Cdc10–Cdc10–Cdc3–Cdc12–Cdc11 and, hence, lacks polarity. At low ionic strength, rods assemble end-to-end to form filaments but not when Cdc11 is absent or its N terminus is altered. Filaments invariably pair into long parallel “railroad tracks.” Lateral association seems to be mediated by heterotetrameric coiled coils between the paired C-terminal extensions of Cdc3 and Cdc12 projecting orthogonally from each filament. Shs1 may be able to replace Cdc11 at the end of the rod. Our findings provide insights into the molecular mechanisms underlying the function and regulation of cellular septin structures.
Drug-sensitiveFGFR2mutations in endometrial carcinoma
Authors: Dutt Amit , Salvesen Helga B. , Chen Tzu-Hsiu , Ramos Alex H. , Onofrio Robert C. , Hatton Charlie , Nicoletti Richard , Winckler Wendy , Grewal Rupinder , Hanna Megan , Wyhs Nicolas , Ziaugra Liuda , Richter Daniel J. , Trovik Jone , Engelsen Ingeborg B. , Stefansson Ingunn M. , Fennell Tim , Cibulskis Kristian , Zody Michael C. , Akslen Lars A. , Gabriel Stacey , Wong Kwok-Kin , Sellers William R. , Meyerson Matthew , Greulich Heidi
Published: June 2008

Oncogenic activation of tyrosine kinases is a common mechanism of carcinogenesis and, given the druggable nature of these enzymes, an attractive target for anticancer therapy. Here, we show that somatic mutations of the fibroblast growth factor receptor 2 (FGFR2) tyrosine kinase gene,FGFR2, are present in 12% of endometrial carcinomas, with additional instances found in lung squamous cell carcinoma and cervical carcinoma. TheseFGFR2mutations, many of which are identical to mutations associated with congenital craniofacial developmental disorders, are constitutively activated and oncogenic when ectopically expressed in NIH 3T3 cells. Inhibition of FGFR2 kinase activity in endometrial carcinoma cell lines bearing suchFGFR2mutations inhibits transformation and survival, implicatingFGFR2as a novel therapeutic target in endometrial carcinoma.
Orphan nuclear receptor NR4A2 expressed in T cells from multiple sclerosis mediates production of inflammatory cytokines
Authors: Doi Yoshimitsu , Oki Shinji , Ozawa Tomoko , Hohjoh Hirohiko , Miyake Sachiko , Yamamura Takashi
Published: June 2008

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) mediated by Th17 and Th1 cells. DNA microarray analysis previously showed that NR4A2, an orphan nuclear receptor, is strongly up-regulated in the peripheral blood T cells of MS. Here, we report that NR4A2 plays a pivotal role for mediating cytokine production from pathogenic T cells. In experimental autoimmune encephalomyelitis (EAE), an animal model of MS, NR4A2, was selectively up-regulated in the T cells isolated from the CNS. Strikingly, a forced expression of NR4A2 augmented promoter activities of IL-17 and IFN-γ genes, leading to an excessive production of these cytokines. Conversely, treatment with siRNA for NR4A2, resulted in a significant reduction in the production of IL-17 and IFN-γ. Furthermore, treatment with NR4A2 siRNA reduced the ability of encephalitogenic T cells to transfer EAE in recipient mice. Thus, NR4A2 is an essential transcription factor for triggering the inflammatory cascade of MS/EAE and may serve as a therapeutic target.
In situ
microbial metabolism as a cause of gas anomalies in ice
Authors: Rohde Robert A. , Price P. Buford , Bay Ryan C. , Bramall Nathan E.
Published: June 2008

Isolated spikes of anomalously high concentrations of N
2
O have been reported at depths in Greenland and Antarctic ice cores corresponding to narrow time intervals over the past ≈10
5
years. Now, using a calibrated spectrofluorimeter to map protein-bound Trp, a proxy for microbes, versus depth in the 3,053-m GISP2 ice core, we find six depths at which localized spikes of high cell concentrations coincide with N
2
O spikes. We show that the excess gases are consistent with accumulation of
in situ
metabolic wastes during residence times of the excess microbes in the ice. Because of sparseness of N
2
O measurements and our spectrofluorimetry versus depth, the total number of microbially produced N
2
O spikes in GISP2 is probably much larger than six. Spikes of excess methanogens coincident with CH
4
spikes are found at three depths in the bottom 3% of GISP2, most likely because of methanogenic metabolism in the underlying silty ice, followed by turbulent flow of the lowest ≈90 m of ice. The apparent rates of
in situ
production of N
2
O and CH
4
spikes by metabolism are observed to be consistent with a single activation energy,
U
, and maintain proportionality to exp(−
U
/
RT
) over the entire temperature range down to −40°C. Fluorescence of nonmicrobial aerosols in GISP2 ice is distinguishable from microbial fluorescence by its different emission spectra. Our spectrofluorimetric scans throughout the GISP2 ice core lead us to conclude that both microbes and nonmicrobial aerosols are deposited in discontinuous bursts, which may provide a tool for studying wind storms in the distant past.
Fractional diffusion models of non-local perturbative transport: numerical results and application to JET experiments*
Authors: del-Castillo-Negrete D. , Mantica P. , Naulin V. , Rasmussen J.J.
Published: June 2008

Perturbative experiments in magnetically confined fusion plasmas have shown that edge cold pulses travel to the centre of the device on a time scale much faster than expected on the basis of diffusive transport. An open issue is whether the observed fast pulse propagation is due to non-local transport mechanisms or if it could be explained on the basis of local transport models. To elucidate this distinction, perturbative experiments involving ICRH power modulation in addition to cold pulses have been conducted in JET for the same plasma. Local transport models have found problematic the reconciliation of the fast propagation of cold pulses with the comparatively slower propagation of heat waves generated by power modulation. In this paper, a non-local model based on the use of fractional diffusion operators is used to describe these experiments. A numerical study of the parameter dependence of the pulse speed and the amplitude and phase of the heat wave is also presented.
Dependence of spontaneous growth and suppression of the magnetic island on beta and collisionality in the LHD
Authors: Narushima Y. , Watanabe K.Y. , Sakakibara S. , Narihara K. , Yamada I. , Suzuki Y. , Ohdachi S. , Ohyabu N. , Yamada H. , Nakamura Y.
Published: June 2008

The dynamics of the magnetic island structure in the plasma are investigated in plasmas with a wide range of beta and collisionality. The perturbed magnetic field is diagnosed by a toroidal array of flux loops installed in the vacuum vessel on the Large Helical Device (LHD). It is found that the magnetic island grows with beta at relatively low beta values. In contrast, when the beta exceeds a critical value, the sign of the perturbed magnetic field suddenly reverses and its strength saturates to the magnetic field perturbation required to cancel the external perturbation. This suggests spontaneous healing of the magnetic island.
Vacuole Membrane Protein 1 Is an Endoplasmic Reticulum Protein Required for Organelle Biogenesis, Protein Secretion, and Development
Authors: Calvo-Garrido Javier , Carilla-Latorre Sergio , Lázaro-Diéguez Francisco , Egea Gustavo , Escalante Ricardo
Published: June 2008

Vacuole membrane protein 1 (Vmp1) is membrane protein of unknown molecular function that has been associated with pancreatitis and cancer. The social amoeba Dictyostelium discoideum has a vmp1-related gene that we identified previously in a functional genomic study. Loss-of-function of this gene leads to a severe phenotype that compromises Dictyostelium growth and development. The expression of mammalian Vmp1 in a vmp1−Dictyostelium mutant complemented the phenotype, suggesting a functional conservation of the protein among evolutionarily distant species and highlights Dictyostelium as a valid experimental system to address the function of this gene. Dictyostelium Vmp1 is an endoplasmic reticulum protein necessary for the integrity of this organelle. Cells deficient in Vmp1 display pleiotropic defects in the secretory pathway and organelle biogenesis. The contractile vacuole, which is necessary to survive under hypoosmotic conditions, is not functional in the mutant. The structure of the Golgi apparatus, the function of the endocytic pathway and conventional protein secretion are also affected in these cells. Transmission electron microscopy of vmp1−cells showed the accumulation of autophagic features that suggests a role of Vmp1 in macroautophagy. In addition to these defects observed at the vegetative stage, the onset of multicellular development and early developmental gene expression are also compromised.
Live Observation of Forespore Membrane Formation in Fission Yeast
Authors: Nakamura Taro , Asakawa Haruhiko , Nakase Yukiko , Kashiwazaki Jun , Hiraoka Yasushi , Shimoda Chikashi
Published: June 2008

Sporulation in the fission yeast Schizosaccharomyces pombe is a unique biological process in that the plasma membrane of daughter cells is assembled de novo within the mother cell cytoplasm. A double unit membrane called the forespore membrane (FSM) is constructed dynamically during meiosis. To obtain a dynamic view of FSM formation, we visualized FSM in living cells by using green fluorescent protein fused with Psy1, an FSM-resident protein, together with the nucleus or microtubules. The assembly of FSM initiates in prophase II, and four FSMs in a cell expand in a synchronous manner at the same rate throughout meiosis II. After the meiosis II completes, FSMs continue to expand until closure to form the prespore, a spore precursor. Prespores are initially ellipsoidal, and eventually become spheres. FSM formation was also observed in the sporulation-deficient mutants spo3, spo14, and spo15. In the spo15 mutant, the initiation of FSM formation was completely blocked. In the spo3 mutant, the FSM expanded normally during early meiosis II, but it was severely inhibited during late and postmeiosis, whereas in the spo14 mutant, membrane expansion was more severely inhibited throughout meiosis II. These observations suggest that FSM expansion is composed of two steps, early meiotic FSM expansion and late and post meiotic FSM expansion. Possible regulatory mechanisms of FSM formation in fission yeast are discussed.
Cdc2 and Mos Regulate Emi2 Stability to Promote the Meiosis I–Meiosis II Transition
Authors: Tang Wanli , Wu Judy Qiju , Guo Yanxiang , Hansen David V. , Perry Jennifer A. , Freel Christopher D. , Nutt Leta , Jackson Peter K. , Kornbluth Sally
Published: June 2008

The transition of oocytes from meiosis I (MI) to meiosis II (MII) requires partial cyclin B degradation to allow MI exit without S phase entry. Rapid reaccumulation of cyclin B allows direct progression into MII, producing a cytostatic factor (CSF)-arrested egg. It has been reported that dampened translation of the anaphase-promoting complex (APC) inhibitor Emi2 at MI allows partial APC activation and MI exit. We have detected active Emi2 translation at MI and show that Emi2 levels in MI are mainly controlled by regulated degradation. Emi2 degradation in MI depends not on Ca2+/calmodulin-dependent protein kinase II (CaMKII), but on Cdc2-mediated phosphorylation of multiple sites within Emi2. As in MII, this phosphorylation is antagonized by Mos-mediated recruitment of PP2A to Emi2. Higher Cdc2 kinase activity in MI than MII allows sufficient Emi2 phosphorylation to destabilize Emi2 in MI. At MI anaphase, APC-mediated degradation of cyclin B decreases Cdc2 activity, enabling Cdc2-mediated Emi2 phosphorylation to be successfully antagonized by Mos-mediated PP2A recruitment. These data suggest a model of APC autoinhibition mediated by stabilization of Emi2; Emi2 proteins accumulate at MI exit and inhibit APC activity sufficiently to prevent complete degradation of cyclin B, allowing MI exit while preventing interphase before MII entry.
MPIDB: the microbial protein interaction database
Authors: Goll Johannes , Rajagopala Seesandra V. , Shiau Shen C. , Wu Hank , Lamb Brian T. , Uetz Peter
Published: June 2008

Summary: The microbial protein interaction database (MPIDB) aims to collect and provide all known physical microbial interactions. Currently, 22 530 experimentally determined interactions among proteins of 191 bacterial species/strains can be browsed and downloaded. These microbial interactions have been manually curated from the literature or imported from other databases (IntAct, DIP, BIND, MINT) and are linked to 24 060 experimental evidences (PubMed ID, PSI-MI methods). In contrast to these databases, interactions in MPIDB are further supported by 8150 additional evidences based on interaction conservation, co-purification and 3D domain contacts (iPfam, 3did).

Availability:  http://www.jcvi.org/mpidb/

Contact:  jgoll@jcvi.org
Wax-ester mobilization by female Calanus finmarchicus (Gunnerus) during spring ascendance and advection to the Faroe Shelf
Authors: Madsen Matias L. , Gaard Eilif , Hansen Benni W.
Published: June 2008

Madsen, M. L., Gaard, E., and Hansen, B. W. 2008. Wax-ester mobilization by female Calanus finmarchicus (Gunnerus) during spring ascendance and advection to the Faroe Shelf. – ICES Journal of Marine Science, 65: 1112–1121. Time of ascendance and initiation of reproduction in Calanus finmarchicus is closely correlated with the phytoplankton spring bloom. However, significant egg production can take place before the bloom, fuelled by mobilizing stored wax-ester lipids. Calanus finmarchicus from stations on the Faroe Shelf were compared with specimens collected off the shelf. Biological parameters such as gut contents, egg production, developmental stage, and lipid content were determined and correlated with phytoplankton concentration and spatial distribution along two transects in late April 2003 across the shelf northeast and southwest of the Faroe Islands. Grazing by C. finmarchicus on phytoplankton was significantly lower northeast of the Faroe plateau. However, the egg production was generally high for a pre-bloom situation, with significantly higher rates of egg production on the shelf than off it, along both transects. Wax-ester content of female C. finmarchicus was significantly higher and more variable at off-shelf stations than on the shelf (<2 µg female−1). From this, we suggest that off-shelf C. finmarchicus had recently emerged from overwintering depths, in contrast to individuals from stations on the shelf, which had been in the upper water masses for some time. Females (from off-shelf stations) most likely supported the initial egg production from their wax-ester reserves.
Vulimiri Ramalingaswami. 8 August 1921 — 28 May 2001
Authors: Menon M. G. K. , Tandon P. N.
Published: June 2008

Dr Vulimiri Ramalingaswami was born on 8 August 1921 in Srikakulam in Andhra Pradesh (India). He was known to his family and friends as ‘Rama’ and that is how he will be referred to in this memoir. Rama was undoubtedly the most outstanding, multifaceted medical personality of independent India, as can be seen from the following brief introduction.
Engineered telomere degradation models dyskeratosis congenita
Authors: Hockemeyer Dirk , Palm Wilhelm , Wang Richard C. , Couto Suzana S. , de Lange Titia
Published: June 2008

Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome characterized by cutaneous symptoms, including hyperpigmentation and nail dystrophy. Some forms of DC are caused by mutations in telomerase, the enzyme that counteracts telomere shortening, suggesting a telomere-based disease mechanism. However, mice with extensively shortened telomeres due to telomerase deficiency do not develop the characteristics of DC, raising questions about the etiology of DC and/or mouse models for human telomere dysfunction. Here we describe mice engineered to undergo telomere degradation due to the absence of the shelterin component POT1b. When combined with reduced telomerase activity, POT1b deficiency elicits several characteristics of DC, including hyperpigmentation and fatal bone marrow failure at 4–5 mo of age. These results provide experimental support for the notion that DC is caused by telomere dysfunction, and demonstrate that key aspects of a human telomere-based disease can be modeled in the mouse.
On the origin of new genes in Drosophila
Authors: Zhou Qi , Zhang Guojie , Zhang Yue , Xu Shiyu , Zhao Ruoping , Zhan Zubing , Li Xin , Ding Yun , Yang Shuang , Wang Wen
Published: June 2008

Several mechanisms have been proposed to account for the origination of new genes. Despite extensive case studies, the general principles governing this fundamental process are still unclear at the whole-genome level. Here, we unveil genome-wide patterns for the mutational mechanisms leading to new genes and their subsequent lineage-specific evolution at different time nodes in the Drosophila melanogaster species subgroup. We find that (1) tandem gene duplication has generated ∼80% of the nascent duplicates that are limited to single species (D. melanogaster or Drosophila yakuba); (2) the most abundant new genes shared by multiple species (44.1%) are dispersed duplicates, and are more likely to be retained and be functional; (3) de novo gene origination from noncoding sequences plays an unexpectedly important role during the origin of new genes, and is responsible for 11.9% of the new genes; (4) retroposition is also an important mechanism, and had generated ∼10% of the new genes; (5) ∼30% of the new genes in the D. melanogaster species complex recruited various genomic sequences and formed chimeric gene structures, suggesting structure innovation as an important way to help fixation of new genes; and (6) the rate of the origin of new functional genes is estimated to be five to 11 genes per million years in the D. melanogaster subgroup. Finally, we survey gene frequencies among 19 globally derived strains for D. melanogaster-specific new genes and reveal that 44.4% of them show copy number polymorphisms within a population. In conclusion, we provide a panoramic picture for the origin of new genes in Drosophila species.
RNA-seq: An assessment of technical reproducibility and comparison with gene expression arrays
Authors: Marioni John C. , Mason Christopher E. , Mane Shrikant M. , Stephens Matthew , Gilad Yoav
Published: June 2008

Ultra-high-throughput sequencing is emerging as an attractive alternative to microarrays for genotyping, analysis of methylation patterns, and identification of transcription factor binding sites. Here, we describe an application of the Illumina sequencing (formerly Solexa sequencing) platform to study mRNA expression levels. Our goals were to estimate technical variance associated with Illumina sequencing in this context and to compare its ability to identify differentially expressed genes with existing array technologies. To do so, we estimated gene expression differences between liver and kidney RNA samples using multiple sequencing replicates, and compared the sequencing data to results obtained from Affymetrix arrays using the same RNA samples. We find that the Illumina sequencing data are highly replicable, with relatively little technical variation, and thus, for many purposes, it may suffice to sequence each mRNA sample only once (i.e., using one lane). The information in a single lane of Illumina sequencing data appears comparable to that in a single array in enabling identification of differentially expressed genes, while allowing for additional analyses such as detection of low-expressed genes, alternative splice variants, and novel transcripts. Based on our observations, we propose an empirical protocol and a statistical framework for the analysis of gene expression using ultra-high-throughput sequencing technology.
Drought Induction of Arabidopsis 9-cis-Epoxycarotenoid Dioxygenase Occurs in Vascular Parenchyma Cells
Authors: Endo Akira , Sawada Yoshiaki , Takahashi Hirokazu , Okamoto Masanori , Ikegami Keiichi , Koiwai Hanae , Seo Mitsunori , Toyomasu Tomonobu , Mitsuhashi Wataru , Shinozaki Kazuo , Nakazono Mikio , Kamiya Yuji , Koshiba Tomokazu , Nambara Eiji
Published: June 2008

The regulation of abscisic acid (ABA) biosynthesis is essential for plant responses to drought stress. In this study, we examined the tissue-specific localization of ABA biosynthetic enzymes in turgid and dehydrated Arabidopsis (Arabidopsis thaliana) plants using specific antibodies against 9-cis-epoxycarotenoid dioxygenase 3 (AtNCED3), AtABA2, and Arabidopsis aldehyde oxidase 3 (AAO3). Immunohistochemical analysis revealed that in turgid plants, AtABA2 and AAO3 proteins were localized in vascular parenchyma cells most abundantly at the boundary between xylem and phloem bundles, but the AtNCED3 protein was undetectable in these tissues. In water-stressed plants, AtNCED3 was detected exclusively in the vascular parenchyma cells together with AtABA2 and AAO3. In situ hybridization using the antisense probe for AtNCED3 showed that the drought-induced expression of AtNCED3 was also restricted to the vascular tissues. Expression analysis of laser-microdissected cells revealed that, among nine drought-inducible genes examined, the early induction of most genes was spatially restricted to vascular cells at 1 h and then some spread to mesophyll cells at 3 h. The spatial constraint of AtNCED3 expression in vascular tissues provides a novel insight into plant systemic response to drought stresses.
Tracking Monolignols during Wood Development in Lodgepole Pine
Authors: Kaneda Minako , Rensing Kim H. , Wong John C.T. , Banno Brian , Mansfield Shawn D. , Samuels A. Lacey
Published: June 2008

Secondary xylem (wood) formation in gymnosperms requires that the tracheid protoplasts first build an elaborate secondary cell wall from an array of polysaccharides and then reinforce it with lignin, an amorphous, three-dimensional product of the random radical coupling of monolignols. The objective of this study was to track the spatial distribution of monolignols during development as they move from symplasm to apoplasm. This was done by feeding [3H]phenylalanine ([3H]Phe) to dissected cambium/developing wood from lodgepole pine (Pinus contorta var latifolia) seedlings, allowing uptake and metabolism, then rapidly freezing the cells and performing autoradiography to detect the locations of the monolignols responsible for lignification. Parallel experiments showed that radioactivity was incorporated into polymeric lignin and a methanol-soluble pool that was characterized by high-performance liquid chromatography. [3H]Phe was incorporated into expected lignin precursors, such as coniferyl alcohol and p-coumaryl alcohol, as well as pinoresinol. Coniferin, the glucoside of coniferyl alcohol, was detected by high-performance liquid chromatography but was not radioactively labeled. With light microscopy, radiolabeled phenylpropanoids were detected in the rays as well as the tracheids, with the two cell types showing differential sensitivity to inhibitors of protein translation and phenylpropanoid metabolism. Secondary cell walls of developing tracheids were heavily labeled when incubated with [3H]Phe. Inside the cell, cytoplasm was most strongly labeled followed by Golgi and low-vacuole label. Inhibitor studies suggest that the Golgi signal could be attributed to protein, rather than phenylpropanoid, origins. These data, produced with the best microscopy tools that are available today, support a model in which unknown membrane transporters, rather than Golgi vesicles, export monolignols.
The Relationship between Auxin Transport and Maize Branching
Authors: Gallavotti Andrea , Yang Yan , Schmidt Robert J. , Jackson David
Published: June 2008

Maize (Zea mays) plants make different types of vegetative or reproductive branches during development. Branches develop from axillary meristems produced on the flanks of the vegetative or inflorescence shoot apical meristem. Among these branches are the spikelets, short grass-specific structures, produced by determinate axillary spikelet-pair and spikelet meristems. We investigated the mechanism of branching in maize by making transgenic plants expressing a native expressed endogenous auxin efflux transporter (ZmPIN1a) fused to yellow fluorescent protein and a synthetic auxin-responsive promoter (DR5rev) driving red fluorescent protein. By imaging these plants, we found that all maize branching events during vegetative and reproductive development appear to be regulated by the creation of auxin response maxima through the activity of polar auxin transporters. We also found that the auxin transporter ZmPIN1a is functional, as it can rescue the polar auxin transport defects of the Arabidopsis (Arabidopsis thaliana) pin1-3 mutant. Based on this and on the groundbreaking analysis in Arabidopsis and other species, we conclude that branching mechanisms are conserved and can, in addition, explain the formation of axillary meristems (spikelet-pair and spikelet meristems) that are unique to grasses. We also found that BARREN STALK1 is required for the creation of auxin response maxima at the flanks of the inflorescence meristem, suggesting a role in the initiation of polar auxin transport for axillary meristem formation. Based on our results, we propose a general model for branching during maize inflorescence development.
ArabidopsisDREB2A-Interacting Proteins Function as RING E3 Ligases and Negatively Regulate Plant Drought Stress–Responsive Gene Expression
Authors: Qin Feng , Sakuma Yoh , Tran Lam-Son Phan , Maruyama Kyonoshin , Kidokoro Satoshi , Fujita Yasunari , Fujita Miki , Umezawa Taishi , Sawano Yoriko , Miyazono Ken-ichi , Tanokura Masaru , Shinozaki Kazuo , Yamaguchi-Shinozaki Kazuko
Published: June 2008

The DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN2A (DREB2A) transcription factor controls water deficit–inducible gene expression and requires posttranslational modification for its activation. The activation mechanism is not well understood; however, the stability of this protein in the nucleus was recently found to be important for its activation. Here, we report the isolation of Arabidopsis thaliana DREB2A-INTERACTING PROTEIN1 (DRIP1) and DRIP2, C3HC4 RING domain–containing proteins that interact with the DREB2A protein in the nucleus. An in vitro ubiquitination assay showed that they function as E3 ubiquitin ligases and are capable of mediating DREB2A ubiquitination. Overexpression of DRIP1 in Arabidopsis delayed the expression of DREB2A-regulated drought-responsive genes. Drought-inducible gene expression was slightly enhanced in the single T-DNA mutants of drip1-1 and drip2-1. By contrast, significantly enhanced gene expression was revealed in the drip1 drip2 double mutant under dehydration stress. Collectively, these data imply that DRIP1 and DRIP2 function negatively in the response of plants to drought stress. Moreover, overexpression of full-length DREB2A protein was more stable in drip1-1 than in the wild-type background. These results suggest that DRIP1 and DRIP2 act as novel negative regulators in drought-responsive gene expression by targeting DREB2A to 26S proteasome proteolysis.
ArabidopsisDDB1-CUL4 ASSOCIATED FACTOR1 Forms a Nuclear E3 Ubiquitin Ligase with DDB1 and CUL4 That Is Involved in Multiple Plant Developmental Processes
Authors: Zhang Yu , Feng Suhua , Chen Fangfang , Chen Haodong , Wang Jia , McCall Chad , Xiong Yue , Deng Xing Wang
Published: June 2008

The human DDB1-CUL4 ASSOCIATED FACTOR (DCAF) proteins have been reported to interact directly with UV-DAMAGED DNA BINDING PROTEIN1 (DDB1) through the WDxR motif in their WD40 domain and function as substrate-recognition receptors for CULLIN4-based E3 ubiquitin ligases. Here, we identified and characterized a homolog of human DCAF1/VprBP in Arabidopsis thaliana. Yeast two-hybrid analysis demonstrated the physical interaction between DCAF1 and DDB1 from Arabidopsis, which is likely mediated via the WD40 domain of DCAF1 that contains two WDxR motifs. Moreover, coimmunoprecipitation assays showed that DCAF1 associates with DDB1, RELATED TO UBIQUITIN–modified CUL4, and the COP9 signalosome in vivo but not with CULLIN-ASSOCIATED and NEDDYLATION-DISSOCIATED1, CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1), or the COP10-DET1-DDB1 complex, supporting the existence of a distinct Arabidopsis CUL4 E3 ubiquitin ligase, the CUL4-DDB1-DCAF1 complex. Transient expression of fluorescently tagged DCAF1, DDB1, and CUL4 in onion epidermal cells showed their colocalization in the nucleus, consistent with the notion that the CUL4-DDB1-DCAF1 complex functions as a nuclear E3 ubiquitin ligase. Genetic and phenotypic analysis of two T-DNA insertion mutants of DCAF1 showed that embryonic development of the dcaf1 homozygote is arrested at the globular stage, indicating that DCAF1 is essential for plant embryogenesis. Reducing the levels of DCAF1 leads to diverse developmental defects, implying that DCAF1 might be involved in multiple developmental pathways.
ArabidopsisNuclear-Encoded Plastid Transit Peptides Contain Multiple Sequence Subgroups with Distinctive Chloroplast-Targeting Sequence Motifs
Authors: Lee Dong Wook , Kim Jong Kyoung , Lee Sumin , Choi Seungjin , Kim Sanguk , Hwang Inhwan
Published: June 2008

The N-terminal transit peptides of nuclear-encoded plastid proteins are necessary and sufficient for their import into plastids, but the information encoded by these transit peptides remains elusive, as they have a high sequence diversity and lack consensus sequences or common sequence motifs. Here, we investigated the sequence information contained in transit peptides. Hierarchical clustering on transit peptides of 208 plastid proteins showed that the transit peptide sequences are grouped to multiple sequence subgroups. We selected representative proteins from seven of these multiple subgroups and confirmed that their transit peptide sequences are highly dissimilar. Protein import experiments revealed that each protein contained transit peptide–specific sequence motifs critical for protein import into chloroplasts. Bioinformatics analysis identified sequence motifs that were conserved among members of the identified subgroups. The sequence motifs identified by the two independent approaches were nearly identical or significantly overlapped. Furthermore, the accuracy of predicting a chloroplast protein was greatly increased by grouping the transit peptides into multiple sequence subgroups. Based on these data, we propose that the transit peptides are composed of multiple sequence subgroups that contain distinctive sequence motifs for chloroplast targeting.
Synchrotron X-ray powder diffraction and extended X-ray absorption fine structure spectroscopy studies on nanocrystalline ZrO2–CaO solid solutions
Authors: Fábregas Ismael Oscar , Lamas Diego Germán , Walsöe de Reca Noemí Elizabeth , Fantini Márcia C.A. , Craievich Aldo F. , Prado Rogério Junqueira
Published: June 2008

The crystal structure and the local atomic order of a series of nanocrystalline ZrO2–CaO solid solutions with varying CaO content were studied by synchrotron radiation X-ray powder diffraction and extended X-ray absorption fine structure (EXAFS) spectroscopy. These samples were synthesized by a pH-controlled nitrate–glycine gel-combustion process. For CaO contents up to 8 mol%, the t′ form of the tetragonal phase (c/a> 1) was identified, whereas for 10 and 12 mol% CaO, the t′′ form (c/a= 1; oxygen anions displaced from their ideal positions in the cubic phase) was detected. Finally, the cubic phase was observed for solid solutions with CaO content of 14 mol% CaO or higher. The t′/t′′ and t′′/cubic compositional boundaries were determined to be at 9 (1) and 13 (1) mol% CaO, respectively. The EXAFS study demonstrated that this transition is related to a tetragonal-to-cubic symmetry change of the first oxygen coordination shell around the Zr atoms.
Fast and easy Bragg reflex search for Berg–Barrett topography of misoriented crystals
Authors: Andreas Birk , Kuetgens Ulrich , Becker Peter
Published: June 2008

A fast search method for X-ray Bragg reflexes of misoriented crystals is presented. The method is also applicable to determine the accuracy of the crystal orientation. This technique is demonstrated by means of a simple setup for Berg–Barrett X-ray topography, consisting of a rotating sample holder on a two-circle diffractometer. The imaged area of the topograms is about 1 cm2, and the quality is comparable to the topograms taken by white-beam synchrotron X-ray topography.
Point-focusing monochromator crystal realized by hot plastic deformation of a Ge wafer
Authors: Okuda Hiroshi , Nakajima Kazuo , Fujiwara Kozo , Morishita Kohei , Ochiai Shojiro
Published: June 2008

Pre-polished Ge(111) single-crystal wafers were deformed just below the melting temperature to prepare point-focusing Johansson monochromator crystals. The (111) lattice plane had curvature 2Rin the focusing plane andRperpendicular to it, with a hemispherical inner surface with a radius ofR= 600 mm. By using CuKα radiation, the diverging X-ray beam was focused onto a small spot.
Branching and twinning in α-cristobalite dendrites: an electron backscatter diffraction study
Authors: Zhao Shanrong , Yang Mingling , Xu Haijun , Zhang Suxin
Published: June 2008

In this paper, the electron backscatter diffraction (EBSD) technique is used to determine the orientations of α-cristobalite dendrites. The two branches of the cross-shaped α-cristobalite dendrite extend along the crystallographic directions [100] and [010], but no branches develop along 〈001〉. A new kind of twinning relationship is found between two α-cristobalite dendrites. The twin axes are [110], [111] and [\bar 1\bar 11], and the twin planes are (111) and (\bar 1\bar 11). It is shown that crystallographic symmetry controls the dendrite morphology and dendrite aggregation.
X-ray diffuse scattering from HMTA: analysis via a Monte Carlo model
Authors: Goossens D. J. , Heerdegen A. P. , Welberry T. R.
Published: June 2008

Hexamethylenetetramine (HMT, C6H12N4, also referred to as urotropin) and azelaic acid [A, HOOC—(CH2)7—COOH] form a co-crystal or adduct (HMTA, also referred to as urotropin azelate) which exhibits several structural phases as a function of temperature. At room temperature, the structure is orthorhombic, but shows substantial disorder. Here, this disorder is explored by analyzing the diffuse scattering from single crystals of HMTA via Monte Carlo simulation. The disorder is in part occupational, with two orientations of azelaic acid occurring, and in part thermally induced, which is to say dynamic. The occupational disorder can be thought of as a combination of limited-range in-plane (bc plane) negative correlations combined with effectively zero correlation between planes (along a), rather like stacking faults. Size effect, the cross-correlation between molecular orientation and displacement from average position, is required to reproduce the observed diffuse scattering.
Pressure-freezing with conformational conversion of 3-aminopropan-1-ol molecules
Authors: Gajda Roman , Katrusiak Andrzej
Published: June 2008

3-Aminopropan-1-ol, NH2(CH2)3OH, was pressure-frozen and its structure determined at 0.2, 0.9 and 1.31 GPa by single-crystal X-ray diffraction. The freezing pressure of 0.13 GPa at 296 K was measured by ruby fluorescence in the diamond–anvil cell and from compressibility measurement in the piston-and-cylinder reaction press. The molecules assume an extended conformation in the crystalline state, different from the pseudo-ring conformers, with the terminal groups linked by an intramolecular hydrogen bond, present in the gaseous and liquid states. The polar arrangement in the 3-aminopropan-1-ol crystals is explained in terms of the pattern of intermolecular hydrogen bonds.
Selection and censorship: librarians and their collections
Authors: McMenemy David
Published: June 2008

Purpose

The purpose of this paper is to discuss the issues raised in the report, Hate on the State, published in 2007.

Design/methodology/approach

The paper is a reflective viewpoint that debates the points made in the report and their wider implications for the profession.

Findings

The report raised key issues around censorship, and the role of the librarian in selecting a balanced collection for their public. These concerns need to be debated at length, since they represent fundamental professional concerns.

Practical implications

The paper debates key professional concerns and how changes in stock procurement may lead to future occurrences of such controversies.

Originality/value

This is the only editorial to date related to this report in library literature.
Identification and management of high‐potential professional services
Authors: Smedlund Anssi
Published: June 2008

Purpose

In professional services, innovation occurs in the delivery. The purpose of this paper is to provide a framework for classifying innovation in professional services by taking into account the strength of the relationship with the client in addition to the incremental‐radical nature of innovation.

Design/methodology/approach

Based on the framework provided, there are operational, experimental, tactical, and high‐potential professional services, the characteristics of which are illustrated both conceptually and with a case example.

Findings

Professional service goes through different stages during its lifespan. A high‐potential service is a stage where the nature of the innovation is radical and the relationship with the client is strong. It is suggested that high‐potential services create a future competitive advantage not only for the service provider, but also for the client.

Research limitations/implications

A qualitative network approach with a focal actor perspective, in which the networks are illustrated from the supplier's point of view, is used. The characteristics of a high‐potential service are connected to the development stage of the service.

Practical implications

Classification of professional services is provided, and the importance of high‐potential services is highlighted. Advice for managers is given for designing suitable management initiatives in professional services in all their stages: idea, development, and commercialization.

Originality/value

The approach introduced in this paper addresses three gaps in the existing research: it helps to characterize and theorize the concept of innovation in professional services; it draws attention to the important role of the client in professional services, and it expands the management of innovation in professional services to include network relationships.
Workforce modernization in the police service: prospects for reform?
Authors: Loveday Barry , Williams Steve , Scott Peter
Published: June 2008

Purpose

The aim of this paper is to examine the significance and the implications of efforts to institute workforce modernization within the police service in England and Wales.

Design/methodology/approach

The approach taken uses an analysis of the modernization proposals advanced by Her Majesty's Inspectorate of Constabulary as its starting point.

Findings

The development of workforce modernization in the police service would appear to have eroded the hitherto “reform‐resistant” nature of policing, however political factors continue to impede reform.

Research limitations/implications

Although more evidence concerning the scale and the outcomes of the reform process would be desirable, the main implication of this paper is that workforce modernization in the police is viable, but constrained by political factors.

Originality/value

Empirically, the paper focuses on developments in a sector – the police service – that has been neglected by the existing literature on workforce modernization; theoretically, it demonstrates the important influence often exercised by political contingencies over public sector workforce reform.