bluuebunny/crossref_metadata_2025 huggingface dataset:
START: an automated tool for serial analysis of chromatin occupancy data
Authors: Marinescu Voichita D. , Kohane Isaac S. , Kim Tae-Kyung , Harmin David A. , Greenberg Michael E. , Riva Alberto
Published: February 2006

Summary: The serial analysis of chromatin occupancy technique (SACO) promises to become a widely used method for the unbiased genome-wide experimental identification of loci bound by a transcription factor of interest. We describe the first web-based automatic tool, termed sequence tag analysis and reporting tool (START), for processing SACO data generated by experiments performed for the yeast, fruit fly, mouse, rat or human genomes. The program uses as input sequences of inserts from a SACO library from which it extracts all SACO tags, maps them to genomic locations and annotates them. START returns detailed information about these tags including the genes, the genomic elements and the miRNA precursors found in their vicinity, and makes use of the MAPPER database to identify putative transcription factor binding sites located close to the tags.

Availability: The program is available at

Contact:  vdmarinescu@chip.org

Supplementary information: Supplementary information is available at
Refinement of optical map assemblies
Authors: Valouev Anton , Zhang Yu , Schwartz David C. , Waterman Michael S.
Published: February 2006

Motivation: Genomic mutations and variations provide insightful information about the functionality of sequence elements and their association with human diseases. Traditionally, variations are identified through analysis of short DNA sequences, usually shorter than 1000 bp per fragment. Optical maps provide both faster and more cost-efficient means for detecting such differences, because a single map can span over 1 million bp. Optical maps are assembled to cover the whole genome, and the accuracy of assembly is critical.

Results: We present a computationally efficient model-based method for improving quality of such assemblies. Our method provides very high accuracy even with moderate coverage (<20 ×). We utilize a hidden Markov model to represent the consensus map and use the expectation-Maximization algorithm to drive the refinement process. We also provide quality scores to assess the quality of the finished map.

Availability: Code is available from

Contact:  valouev@usc.edu
Incorporating biological knowledge into distance-based clustering analysis of microarray gene expression data
Authors: Huang Desheng , Pan Wei
Published: February 2006

Motivation: Because co-expressed genes are likely to share the same biological function, cluster analysis of gene expression profiles has been applied for gene function discovery. Most existing clustering methods ignore known gene functions in the process of clustering.

Results: To take advantage of accumulating gene functional annotations, we propose incorporating known gene functions into a new distance metric, which shrinks a gene expression-based distance towards 0 if and only if the two genes share a common gene function. A two-step procedure is used. First, the shrinkage distance metric is used in any distance-based clustering method, e.g. K-medoids or hierarchical clustering, to cluster the genes with known functions. Second, while keeping the clustering results from the first step for the genes with known functions, the expression-based distance metric is used to cluster the remaining genes of unknown function, assigning each of them to either one of the clusters obtained in the first step or some new clusters. A simulation study and an application to gene function prediction for the yeast demonstrate the advantage of our proposal over the standard method.

Contact:  weip@biostat.umn.edu
Avoiding model selection bias in small-sample genomic datasets
Authors: Berrar Daniel , Bradbury Ian , Dubitzky Werner
Published: February 2006

Motivation: Genomic datasets generated by high-throughput technologies are typically characterized by a moderate number of samples and a large number of measurements per sample. As a consequence, classification models are commonly compared based on resampling techniques. This investigation discusses the conceptual difficulties involved in comparative classification studies. Conclusions derived from such studies are often optimistically biased, because the apparent differences in performance are usually not controlled in a statistically stringent framework taking into account the adopted sampling strategy. We investigate this problem by means of a comparison of various classifiers in the context of multiclass microarray data.

Results: Commonly used accuracy-based performance values, with or without confidence intervals, are inadequate for comparing classifiers for small-sample data. We present a statistical methodology that avoids bias in cross-validated model selection in the context of small-sample scenarios. This methodology is valid for both k-fold cross-validation and repeated random sampling.

Contact:  dp.berrar@ulster.ac.uk
Automated classification of alternative splicing and transcriptional initiation and construction of visual database of classified patterns
Authors: Nagasaki Hideki , Arita Masanori , Nishizawa Tatsuya , Suwa Makiko , Gotoh Osamu
Published: February 2006

Motivation: Large-scale detection and classification of alternative splicing and transcriptional initiation (ASTI) is the first step towards detailed studies of the functional implication and mechanisms of these phenomena.

Results: We have developed an algorithm that classifies all observed units of ASTI into an extendable set of distinct types (e.g. cassette type) by converting a collection of alignments between a genomic DNA sequence and cDNA sequences into binary description. This description system can uniquely and compactly encode not only typical patterns but also any rare patterns that are usually collectively assigned to ‘others.’ More than 150 distinct ASTI types were found when this system was applied to genome-wide detection of ASTI units in human and five other eukaryotes.

Availability: The data detected by this system are available through ASTRA (), a database equipped with a Java-based browser that can interactively reorganize the order of displayed splicing patterns on demand.

Contact:  h-nagasaki@aist.go.jp
Mayday-a microarray data analysis workbench
Authors: Dietzsch Janko , Gehlenborg Nils , Nieselt Kay
Published: February 2006

Mayday is a workbench for visualization, analysis and storage of microarray data. It features a graphical user interface and supports the development and integration of existing and new analysis methods. Besides the infrastructural core functionality, Mayday offers a variety of plug-ins, such as various interactive viewers, a connection to the R statistical environment, a connection to SQL-based databases and different data mining methods, including WEKA-library based methods for classification and various clustering methods. In addition, so-called meta information objects are provided for annotation of the microarray data allowing integration of data from different sources, which is a feature that, for instance, is employed in the enhanced heatmap visualization.

Contact:  nieselt@informatik.uni-tuebingen.de

Supplementary information: The software and more detailed information including screenshots and a user guide as well as test data can be found on the Mayday home page . The core is published under the GPL (GNU Public License) and the associated plug-ins under the LGPL (Lesser GNU Public License).
Promotion of harmful algal blooms by zooplankton predatory activity
Authors: Mitra Aditee , Flynn Kevin J
Published: March 2006

The relationship between algae and their zooplanktonic predators typically involves consumption of nutrients by algae, grazing of the algae by zooplankton which in turn enhances predator biomass, controls algal growth and regenerates nutrients. Eutrophication raises nutrient levels, but does not simply increase normal predator–prey activity; rather, harmful algal bloom (HAB) events develop often with serious ecological and aesthetic implications. Generally, HAB species are outwardly poor competitors for nutrients, while their development of grazing deterrents during nutrient stress ostensibly occurs too late, after the nutrients have largely been consumed already by fast-growing non-HAB species. A new mechanism is presented to explain HAB dynamics under these circumstances. Using a multi-nutrient predator–prey model, it is demonstrated that these blooms can develop through the self-propagating failure of normal predator–prey activity, resulting in the transfer of nutrients into HAB growth at the expense of competing algal species. Rate limitation of this transfer provides a continual level of nutrient stress that results in HAB species exhibiting grazing deterrents protecting them from top-down control. This process is self-stabilizing as long as nutrient demand exceeds supply, maintaining the unpalatable status of HABs; such events are most likely under eutrophic conditions with skewed nutrient ratios.
Transcriptional Profiling Implicates Novel Interactions between Abiotic Stress and Hormonal Responses in Thellungiella, a Close Relative of Arabidopsis
Authors: Wong Chui E. , Li Yong , Labbe Aurelie , Guevara David , Nuin Paulo , Whitty Brett , Diaz Claudia , Golding G. Brian , Gray Gordon R. , Weretilnyk Elizabeth A. , Griffith Marilyn , Moffatt Barbara A.
Published: February 2006

Thellungiella, an Arabidopsis (Arabidopsis thaliana)-related halophyte, is an emerging model species for studies designed to elucidate molecular mechanisms of abiotic stress tolerance. Using a cDNA microarray containing 3,628 unique sequences derived from previously described libraries of stress-induced cDNAs of the Yukon ecotype of Thellungiella salsuginea, we obtained transcript profiles of its response to cold, salinity, simulated drought, and rewatering after simulated drought. A total of 154 transcripts were differentially regulated under the conditions studied. Only six of these genes responded to all three stresses of drought, cold, and salinity, indicating a divergence among the end responses triggered by each of these stresses. Unlike in Arabidopsis, there were relatively few transcript changes in response to high salinity in this halophyte. Furthermore, the gene products represented among drought-responsive transcripts in Thellungiella associate a down-regulation of defense-related transcripts with exposure to water deficits. This antagonistic interaction between drought and biotic stress response may demonstrate Thellungiella's ability to respond precisely to environmental stresses, thereby conserving energy and resources and maximizing its survival potential. Intriguingly, changes of transcript abundance in response to cold implicate the involvement of jasmonic acid. While transcripts associated with photosynthetic processes were repressed by cold, physiological responses in plants developed at low temperature suggest a novel mechanism for photosynthetic acclimation. Taken together, our results provide useful starting points for more in-depth analyses of Thellungiella's extreme stress tolerance.
Calmodulin Interacts with and Regulates the RNA-Binding Activity of an Arabidopsis Polyadenylation Factor Subunit
Authors: Delaney Kimberly J. , Xu Ruqiang , Zhang Jingxian , Li Q. Quinn , Yun Kil-Young , Falcone Deane L. , Hunt Arthur G.
Published: February 2006

The Arabidopsis (Arabidopsis thaliana) gene that encodes the probable ortholog of the 30-kD subunit of the mammalian cleavage and polyadenylation specificity factor (CPSF) is a complex one, encoding small (approximately 28 kD) and large (approximately 68 kD) polypeptides. The small polypeptide (AtCPSF30) corresponds to CPSF30 and is the focus of this study. Recombinant AtCPSF30 was purified from Escherichia coli and found to possess RNA-binding activity. Mutational analysis indicated that an evolutionarily conserved central core of AtCPSF30 is involved in RNA binding, but that RNA binding also requires a short sequence adjacent to the N terminus of the central core. AtCPSF30 was found to bind calmodulin, and calmodulin inhibited the RNA-binding activity of the protein in a calcium-dependent manner. Mutational analysis showed that a small part of the protein, again adjacent to the N terminus of the conserved core, is responsible for calmodulin binding; point mutations in this region abolished both binding to and inhibition of RNA binding by calmodulin. Interestingly, AtCPSF30 was capable of self-interactions. This property also mapped to the central conserved core of the protein. However, calmodulin had no discernible effect on the self-association. These results show that the central portion of AtCPSF30 is involved in a number of important functions, and they raise interesting possibilities for both the interplay between splicing and polyadenylation and the regulation of these processes by stimuli that act through calmodulin.
HvVRN2Responds to Daylength, whereasHvVRN1Is Regulated by Vernalization and Developmental Status
Authors: Trevaskis Ben , Hemming Megan N. , Peacock W. James , Dennis Elizabeth S.
Published: February 2006

Two genetic loci control the vernalization response in winter cereals; VRN1, which encodes an AP1-like MADS-box transcription factor, and VRN2, which has been mapped to a chromosome region containing ZCCT zinc finger transcription factor genes. We examined whether daylength regulates expression of HvVRN1 and HvVRN2. In a vernalization-responsive winter barley (Hordeum vulgare), expression of HvVRN1 is regulated by vernalization and by development, but not by daylength. Daylength affected HvVRN1 expression in only one of six vernalization-insensitive spring barleys examined and so cannot be a general feature of regulation of this gene. In contrast, daylength is the major determinant of expression levels of two ZCCT genes found at the barley VRN2 locus, HvZCCTa and HvZCCTb. In winter barley, high levels of HvZCCTa and HvZCCTb expression were detected only when plants were grown in long days. During vernalization in long-day conditions, HvVRN1 is induced and expression of HvZCCTb is repressed. During vernalization under short days, induction of HvVRN1 occurs without changes in HvZCCTa and HvZCCTb expression. Analysis of HvZCCTa and HvZCCTb expression levels in a doubled haploid population segregating for different vernalization and daylength requirements showed that HvVRN1 genotype determines HvZCCTa and HvZCCTb expression levels. We conclude that the vernalization response is mediated through HvVRN1, whereas HvZCCTa and HvZCCTb respond to daylength cues to repress flowering under long days in nonvernalized plants.
Characterization of 43 Non-Protein-Coding mRNA Genes in Arabidopsis, Including the MIR162a-Derived Transcripts
Authors: Hirsch Judith , Lefort Vincent , Vankersschaver Marion , Boualem Adnane , Lucas Antoine , Thermes Claude , d'Aubenton-Carafa Yves , Crespi Martin
Published: February 2006

Messenger RNAs that do not contain a long open reading frame (ORF) or non-protein-coding RNAs (npcRNAs) are an emerging novel class of transcripts. Their functions may involve the RNA molecule itself and/or short ORF-encoded peptides. npcRNA genes are difficult to identify using standard gene prediction programs that rely on the presence of relatively long ORFs. Here, we used detailed bioinformatic analyses of expressed sequence tag/cDNA databases to detect a restricted set of npcRNAs in the Arabidopsis (Arabidopsis thaliana) genome and further characterized these transcripts using a combination of bioinformatic and molecular approaches. Compositional analyses revealed strong nucleotide strand asymmetries in the npcRNAs, as well as a biased GC content, suggesting the existence of functional constraints on these RNAs. Thirteen of these transcripts display tissue-specific expression patterns, and three are regulated in conditions affecting root architecture. The npcRNA 78 gene contains the miR162 sequence in an alternative intron and corresponds to the MIR162a locus. Although DICER-LIKE 1 (DCL1) mRNA is known to be regulated by miR162-guided cleavage, its level does not change in a mir162a mutant. Alternative splicing of npcRNA 78 leads to several transcript isoforms, which all accumulate in a dcl1 mutant. This suggests that npcRNA 78 is a genuine substrate of DCL1 and that splicing of this microRNA primary transcript and miR162 processing are competitive nuclear events. Our results provide new insights into Arabidopsis npcRNA biology and the potential roles of these genes.
Adducts of Oxylipin Electrophiles to Glutathione Reflect a 13 Specificity of the Downstream Lipoxygenase Pathway in the Tobacco Hypersensitive Response
Authors: Davoine Céline , Falletti Olivier , Douki Thierry , Iacazio Gilles , Ennar Najla , Montillet Jean-Luc , Triantaphylidès Christian
Published: February 2006

The response to reactive electrophile species (RES) is now considered as part of the plant response to pathogen and insect attacks. Thanks to a previously established high-performance liquid chromatography tandem mass spectrometry methodology, we have investigated the production of oxylipin RES adducts to glutathione (GSH) during the hypersensitive response (HR) of plants. We have observed that RES conjugation to GSH in tobacco (Nicotiana tabacum) leaves is facile and nonspecific. In cryptogein-elicited tobacco leaves, we show that the oxylipin RES adducts to GSH are produced in correlation with GSH consumption, increase in glutathione S-transferase activity, and the appearance of the cell death symptoms. In this model, the adducts arise mainly from the downstream 13 lipoxygenase (LOX) metabolism, although the induced 9 LOX pathway leads massively to the accumulation of upstream metabolites. The main adducts were obtained from 2-hexenal and 12-oxo-phytodienoic acid. They accumulate transiently as 1-hexanol-3-GSH, a reduced adduct, and 12-oxo-phytodienoic acid-GSH, respectively. RES conjugation does not initiate cell death but explains part of the GSH depletion that accompanies HR cell death. The nature of these GSH conjugates shows the key role played by the 13 LOX pathway in RES signaling in the tobacco HR.
Arabidopsis Hsa32, a Novel Heat Shock Protein, Is Essential for Acquired Thermotolerance during Long Recovery after Acclimation
Authors: Charng Yee-yung , Liu Hsiang-chin , Liu Nai-yu , Hsu Fu-chiun , Ko Swee-suak
Published: February 2006

Plants and animals share similar mechanisms in the heat shock (HS) response, such as synthesis of the conserved HS proteins (Hsps). However, because plants are confined to a growing environment, in general they require unique features to cope with heat stress. Here, we report on the analysis of the function of a novel Hsp, heat-stress-associated 32-kD protein (Hsa32), which is highly conserved in land plants but absent in most other organisms. The gene responds to HS at the transcriptional level in moss (Physcomitrella patens), Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa). Like other Hsps, Hsa32 protein accumulates greatly in Arabidopsis seedlings after HS treatment. Disruption of Hsa32 by T-DNA insertion does not affect growth and development under normal conditions. However, the acquired thermotolerance in the knockout line was compromised following a long recovery period (>24 h) after acclimation HS treatment, when a severe HS challenge killed the mutant but not the wild-type plants, but no significant difference was observed if they were challenged within a short recovery period. Quantitative hypocotyl elongation assay also revealed that thermotolerance decayed faster in the absence of Hsa32 after a long recovery. Similar results were obtained in Arabidopsis transgenic plants with Hsa32 expression suppressed by RNA interference. Microarray analysis of the knockout mutant indicates that only the expression of Hsa32 was significantly altered in HS response. Taken together, our results suggest that Hsa32 is required not for induction but rather maintenance of acquired thermotolerance, a feature that could be important to plants.
The reb1-1 Mutation of Arabidopsis. Effect on the Structure and Localization of Galactose-Containing Cell Wall Polysaccharides
Authors: Nguema-Ona Eric , Andème-Onzighi Christine , Aboughe-Angone Sophie , Bardor Muriel , Ishii Tadashi , Lerouge Patrice , Driouich Azeddine
Published: February 2006

The Arabidopsis (Arabidopsis thaliana) root epidermal bulger1-1 (reb1-1) mutant (allelic to root hair defective1 [rhd1]) is characterized by a reduced root elongation rate and by bulging of trichoblast cells. The REB1/RHD1 gene belongs to a family of UDP-d-Glucose 4-epimerases involved in the synthesis of d-Galactose (Gal). Our previous study showed that certain arabinogalactan protein epitopes were not expressed in bulging trichoblasts of the mutant. In this study, using a combination of microscopical and biochemical methods, we have investigated the occurrence and the structure of three major Gal-containing polysaccharides, namely, xyloglucan (XyG), rhamnogalacturonan (RG)-I, and RG-II in the mutant root cell walls. Our immunocytochemical data show that swollen trichoblasts were not stained with the monoclonal antibody CCRC-M1 specific for α-l-Fucp-(1→2)-β-d-Galp side chains of XyG, whereas they were stained with anti-XyG antibodies specific for XyG backbone. In addition, analysis of a hemicellulosic fraction from roots demonstrates the presence of two structurally different XyGs in reb1-1. One is structurally similar to wild-type XyG and the other is devoid of fuco-galactosylated side chains and has the characteristic of being insoluble. Similar to anti-XyG antibodies, anti-bupleuran 2IIC, a polyclonal antibody specific for galactosyl epitopes associated with pectins, stained all root epidermal cells of both wild type and reb1-1. Similarly, anti-RG-II antibodies also stained swollen trichoblasts in the mutant. In addition, structural analysis of pectic polymers revealed no change in the galactosylation of RG-I and RG-II isolated from reb1-1 root cells. These findings demonstrate that the reb1-1 mutation affects XyG structure, but not that of pectic polysaccharides, thus lending support to the hypothesis that biosynthesis of Gal as well as galactosylation of complex polysaccharides is regulated at the polymer level.
An Insight into the Molecular Basis of Salt Tolerance of l-myo-Inositol 1-P Synthase (PcINO1) from Porteresia coarctata (Roxb.) Tateoka, a Halophytic Wild Rice
Authors: Ghosh Dastidar Krishnarup , Maitra Susmita , Goswami Lily , Roy Debjani , Das Kali Pada , Majumder Arun Lahiri
Published: February 2006

The molecular basis of salt tolerance of l-myo-inositol 1-P synthase (MIPS; EC 5.5.1.4) from Porteresia coarctata (Roxb.) Tateoka (PcINO1, AF412340) earlier reported from this laboratory, has been analyzed by in vitro mutant and hybrid generation and subsequent biochemical and biophysical studies of the recombinant proteins. A 37-amino acid stretch between Trp-174 and Ser-210 has been confirmed as the salt-tolerance determinant domain in PcINO1 both by loss or gain of salt tolerance by either deletion or by addition to salt-sensitive MIPS(s) of Oryza (OsINO1) and Brassica juncea (BjINO1). This was further verified by growth analysis under salt environment of Schizosaccharomyces pombe transformed with the various gene constructs and studies on the differential behavior of mutant and wild proteins by Trp fluorescence, aggregation, and circular dichroism spectra in the presence of salt. 4,4′-Dianilino-1,1′-binaphthyl-5,5-disulfonic acid binding experiments revealed a lower hydrophobic surface on PcINO1 than OsINO1, contributed by this 37-amino acid stretch explaining the differential behavior of OsINO1 and PcINO1 both with respect to their enzymatic functions and thermodynamic stability in high salt environment. Detailed amino acid sequence comparison and modeling studies revealed the interposition of polar and charged residues and a well-connected hydrogen-bonding network formed by Ser and Thr in this stretch of PcINO1. On the contrary, hydrophobic residues clustered in two continuous stretches in the corresponding region of OsINO1 form a strong hydrophobic patch on the surface. It is conceivable that salt-tolerant MIPS proteins may be designed out of the salt-sensitive plant MIPS proteins by replacement of the corresponding amino acid stretch by the designated 37-amino acid stretch of PcINO1.
ArabidopsisSHORT HYPOCOTYL UNDER BLUE1 Contains SPX and EXS Domains and Acts in Cryptochrome Signaling
Authors: Kang Xiaojun , Ni Min
Published: February 2006

Photomorphogenesis is regulated by red/far-red light–absorbing phytochromes and blue/UV-A light–absorbing cryptochromes. We isolated an Arabidopsis thaliana blue light mutant, short hypocotyl under blue1 (shb1), a knockout allele. However, shb1-D, a dominant allele, exhibited a long-hypocotyl phenotype under red, far-red, and blue light. The phenotype conferred by shb1-D was caused by overaccumulation of SHB1 transcript and recapitulated by overexpression of SHB1 in Arabidopsis. Therefore, SHB1 acts in cryptochrome signaling but overexpression may expand its signaling activity to red and far-red light. Consistent with this, overexpression of SHB1 enhanced the expression of PHYTOCHROME-INTERACTING FACTOR4 (PIF4) under red light. PIF4 appears to specifically mediate SHB1 regulation of hypocotyl elongation and CHLOROPHYLL a/b BINDING PROTEIN3 or CHALCONE SYNTHASE expression under red light. Overexpression of SHB1 also promoted proteasome-mediated degradation of phytochrome A and hypocotyl elongation under far-red light. Under blue light, shb1 suppressed LONG HYPOCOTYL IN FAR-RED LIGHT1 (HFR1) expression and showed several deetiolation phenotypes similar to hfr1-201. However, the hypocotyl and cotyledon-opening phenotypes of shb1 were opposite to those of hfr1-201, and HFR1 acts downstream of SHB1. SHB1 encodes a nuclear and cytosolic protein that has motifs homologous with SYG1 protein family members. Therefore, our studies reveal a signaling step in regulating cryptochrome- and possibly phytochrome-mediated light responses.
“Zero‐length” cross‐linking in solid state as an approach for analysis of protein–protein interactions
Authors: El‐Shafey Ahmed , Tolic Nikola , Young Malin M. , Sale Kenneth , Smith Richard D. , Kery Vladimir
Published: February 2006

We have developed a new approach for the analysis of interacting interfaces in protein complexes and protein quaternary structure based on cross‐linking in the solid state. Protein complexes are freeze‐dried under vacuum, and cross‐links are introduced in the solid phase by dehydrating the protein in a nonaqueous solvent creating peptide bonds between amino and carboxyl groups of the interacting peptides. Cross‐linked proteins are digested into peptides with trypsin in both H216O and H218O and then readily distinguished in mass spectra by characteristic 8 atomic mass unit (amu) shifts reflecting incorporation of two 18O atoms into each C terminus of proteolytic peptides. Computer analysis of mass spectrometry (MS) and MS/MS data is used to identify the cross‐linked peptides. We demonstrated specificity and reproducibility of our method by cross‐linking homo‐oligomeric protein complexes of glutathione‐S‐transferase (GST) from Schistosoma japonicum alone or in a mixture of many other proteins. Identified cross‐links were predominantly of amide origin, but six esters and thioesters were also found. The cross‐linked peptides were validated against the GST monomer and dimer X‐ray structures and by experimental (MS/MS) analyses. Some of the identified cross‐links matched interacting peptides in the native 3D structure of GST, indicating that the structure of GST and its oligomeric complex remained primarily intact after freeze‐drying. The pattern of oligomeric GST obtained in solid state was the same as that obtained in solution by Ru (II) Bpy32+ catalyzed, oxidative “zero‐length” cross‐linking, confirming that it is feasible to use our strategy for analyzing the molecular interfaces of interacting proteins or peptides.
Physical characterization of MxiH and PrgI, the needle component of the type III secretion apparatus from Shigella and Salmonella
Authors: Darboe Numukunda , Kenjale Roma , Picking Wendy L. , Picking William D. , Middaugh C. Russell
Published: February 2006

Shigella and Salmonella use similar type III secretion systems for delivering effector proteins into host cells. This secretion system consists of a base anchored in both bacterial membranes and an extracellular “needle” that forms a rod‐like structure exposed on the pathogen surface. The needle is composed of multiple subunits of a single protein and makes direct contact with host cells to facilitate protein delivery. The proteins that make up the needle of Shigella and Salmonella are MxiH and PrgI, respectively. These proteins are attractive vaccine candidates because of their essential role in virulence and surface exposure. We therefore isolated, purified, and characterized the monomeric forms of MxiH and PrgI. Their far‐UV circular dichroism spectra show structural similarities with hints of subtle differences in their secondary structure. Both proteins are highly helical and thermally unstable, with PrgI having a midpoint of thermal unfolding (Tm) near 37°C and MxiH having a value near 42°C. The two proteins also have comparable intrinsic stabilities as measured by chemically induced (urea) unfolding. MxiH, however, with a free energy of unfolding (ΔG°0,un) of 1.6 kcal/mol, is slightly more stable than PrgI (1.2 kcal/mol). The relatively low m‐values obtained for the urea‐induced unfolding of the proteins suggest that they undergo only a small change in solvent‐accessible surface area. This argues that when MxiH and PrgI are incorporated into the needle complex, they obtain a more stable structural state through the introduction of protein–protein interactions.
Role of unfolded state heterogeneity and en‐route ruggedness in protein folding kinetics
Authors: Ellison Paul A. , Cavagnero Silvia
Published: February 2006

In order to improve our understanding of the physical bases of protein folding, there is a compelling need for better connections between experimental and computational approaches. This work addresses the role of unfolded state conformational heterogeneity and en‐route intermediates, as an aid for planning and interpreting protein folding experiments. The expected kinetics were modeled for different types of energy landscapes, including multiple parallel folding routes, preferential paths dominated by one primary folding route, and distributed paths with a wide spectrum of microscopic folding rate constants. In the presence of one or more preferential routes, conformational exchange among unfolded state populations slows down the observed rates for native protein formation. We find this to be a general phenomenon, taking place even when unfolded conformations interconvert much faster than the “escape” rate constants to folding. Dramatic kinetic deceleration is expected in the presence of an increasing number of folding‐incompetent unfolded conformations. This argues for the existence of parallel folding paths involving several folding‐competent unfolded conformations, during the early stages of protein folding. Deviations from single‐exponential behavior are observed for unfolded conformations exchanging at comparable rates or more slowly than folding events. Analysis of the effect of en‐route (on‐path) intermediate formation and landscape ruggedness on folding kinetics leads to the following unexpected conclusions: (1) intermediates, which often retard native state formation, may in some cases accelerate folding, and (2) rugged landscapes, usually associated with stretched exponentials, display single‐exponential behavior in the presence of late high‐friction paths.
Amyloid β‐protein monomer structure: A computational and experimental study
Authors: Baumketner Andrij , Bernstein Summer L. , Wyttenbach Thomas , Bitan Gal , Teplow David B. , Bowers Michael T. , Shea Joan‐Emma
Published: February 2006

The structural properties of the Aβ42 peptide, a main constituent of the amyloid plaques formed in Alzheimer's disease, were investigated through a combination of ion‐mobility mass spectrometry and theoretical modeling. Replica exchange molecular dynamics simulations using a fully atomic description of the peptide and implicit water solvent were performed on the −3 charge state of the peptide, its preferred state under experimental conditions. Equilibrated structures at 300 K were clustered into three distinct families with similar structural features within a family and with significant root mean square deviations between families. An analysis of secondary structure indicates the Aβ42 peptide conformations are dominated by loops and turns but show some helical structure in the C‐terminal hydrophobic tail. A second calculation on Aβ42 in a solvent‐free environment yields compact structures turned “inside out” from the solution structures (hydrophobic parts on the outside, polar parts on the inside). Ion mobility experiments on the Aβ42 −3 charge state electrosprayed from solution yield a bimodal arrival time distribution. This distribution can be quantitatively fit using cross‐sections from dehydrated forms of the three families of calculated solution structures and the calculated solvent‐free family of structures. Implications of the calculations on the early stages of aggregation of Aβ42 are discussed.
Automated extraction of backbone deuteration levels from amide H/2H mass spectrometry experiments
Authors: Hotchko Matthew , Anand Ganesh S. , Komives Elizabeth A. , Ten Eyck Lynn F.
Published: February 2006

A Fourier deconvolution method has been developed to explicitly determine the amount of backbone amide deuterium incorporated into protein regions or segments by hydrogen/deuterium (H/D) exchange with high‐resolution mass spectrometry. Determination and analysis of the level and number of backbone amide exchanging in solution provide more information about the solvent accessibility of the protein than do previous centroid methods, which only calculate the average deuterons exchanged. After exchange, a protein is digested into peptides as a way of determining the exchange within a local area of the protein. The mass of a peptide upon deuteration is a sum of the natural isotope abundance, fast exchanging side‐chain hydrogens (present in MALDI‐TOF H/2H data) and backbone amide exchange. Removal of the components of the isotopic distribution due to the natural isotope abundances and the fast exchanging side‐chains allows for a precise quantification of the levels of backbone amide exchange, as is shown by an example from protein kinase A. The deconvoluted results are affected by overlapping peptides or inconsistent mass envelopes, and evaluation procedures for these cases are discussed. Finally, a method for determining the back exchange corrected populations is presented, and its effect on the data is discussed under various circumstances.
On the precision of experimentally determined protein folding rates and ϕ‐values
Authors: De Los Rios Miguel A. , Muralidhara B.K. , Wildes David , Sosnick Tobin R. , Marqusee Susan , Wittung‐Stafshede Pernilla , Plaxco Kevin W. , Ruczinski Ingo
Published: February 2006

ϕ‐Values, a relatively direct probe of transition‐state structure, are an important benchmark in both experimental and theoretical studies of protein folding. Recently, however, significant controversy has emerged regarding the reliability with which ϕ‐values can be determined experimentally: Because ϕ is a ratio of differences between experimental observables it is extremely sensitive to errors in those observations when the differences are small. Here we address this issue directly by performing blind, replicate measurements in three laboratories. By monitoring within‐ and between‐laboratory variability, we have determined the precision with which folding rates and ϕ‐values are measured using generally accepted laboratory practices and under conditions typical of our laboratories. We find that, unless the change in free energy associated with the probing mutation is quite large, the precision of ϕ‐values is relatively poor when determined using rates extrapolated to the absence of denaturant. In contrast, when we employ rates estimated at nonzero denaturant concentrations or assume that the slopes of the chevron arms (mf and mu) are invariant upon mutation, the precision of our estimates of ϕ is significantly improved. Nevertheless, the reproducibility we thus obtain still compares poorly with the confidence intervals typically reported in the literature. This discrepancy appears to arise due to differences in how precision is calculated, the dependence of precision on the number of data points employed in defining a chevron, and interlaboratory sources of variability that may have been largely ignored in the prior literature.
Solution structure and backbone dynamics of an N‐terminal ubiquitin‐like domain in the GLUT4‐regulating protein, TUG
Authors: Tettamanzi M. Cristina , Yu Chenfei , Bogan Jonathan S. , Hodsdon Michael E.
Published: February 2006

The GLUT4‐regulating protein, TUG, functions to retain GLUT4‐containing membrane vesicles intracellularly and, in response to insulin stimulation, releases these vesicles to the cellular exocytic machinery for translocation to the plasma membrane. As part of our on going effort to describe the molecular basis for TUG function, we have determined the tertiary structure and characterized the backbone dynamics for an N‐terminal ubiquitin‐like domain (TUG–UBL1) using NMR spectroscopy. A well‐ordered conformation is observed for residues 10–83 of full‐length TUG, and confirms a β‐grasp or ubiquitin‐like topology. Although not required for in vitro association with GLUT4, the functional role of the TUG–UBL1 domain has not yet been described. We undertook a limited literature review of similar N‐terminal UBL domains and noted that a majority participate in protein–protein interactions, generally functioning as adaptor modules to physically associate the over all activity of the protein with a specific cellular process, such as the ubiquitin–proteasome pathway. In consistent fashion, TUG–UBL1 is not expected to participate in a covalent protein modification reaction as it lacks the characteristic C‐terminal diglycine (“GG”) motif required for conjugation to an acceptor lysine, and also lacks the three most common acceptor lysine residues involved in polyubiquitination. Additionally, analysis of the TUG–UBL1 molecular surface reveals a lack of conservation of the “Ile‐44 hydrophobic face” typically involved in ubiquitin recognition. Instead, we speculate on the possible significance of a concentrated area of negative electrostatic potential with increased backbone mobility, both of which are features suggestive of a potential protein–protein interaction site.
Substitutions of prolines examine their role in kinetic trap formation of the caspase recruitment domain (CARD) of RICK
Authors: Chen Yun‐Ru , Clark A. Clay
Published: February 2006

Caspase recruitment domains (CARDs) are small helical protein domains that adopt the Greek key fold. For the two CARDs studied to date, RICK‐CARD and caspase‐1‐CARD (CP1‐CARD), the proteins unfold by an apparent two‐state process at equilibrium. However, the folding kinetics are complex for both proteins and may contain kinetically trapped species on the folding pathway. In the case of RICK‐CARD, the time constants of the slow refolding phases are consistent with proline isomerism. RICK‐CARD contains three prolines, P47 in turn 3, and P85 and P87. The latter two prolines constitute a nonconserved PxP motif in helix 6. To examine the role of the prolines in the complex folding kinetics of RICK‐CARD, we generated seven proline‐to‐alanine mutants, including three single mutants, three double mutants, and one triple mutant. We examined the spectroscopic properties, equilibrium folding, binding to CP1‐CARD, and folding kinetics. The results show that P85 is critical for maintaining the function of the protein and that all mutations decrease the stability. Results from single mixing and sequential mixing stopped‐flow studies strongly suggest the presence of parallel folding pathways consisting of at least two unfolded populations. The mutations affect the distribution of the two unfolded species, thereby affecting the population that folds through each channel. The two conformations also are present in the triple mutant, demonstrating that interconversion between them is not due to prolyl isomerism. Overall, the data show that the complex folding pathway, especially formation of kinetically trapped species, is not due to prolyl isomerism.
The early history of the ubiquitin field
Authors: Varshavsky Alexander
Published: February 2006

This is a personal account of the early history of ubiquitin research, by one of its protagonists. The field of ubiquitin and regulated protein degradation was created in the 1980s, largely through the complementary discoveries by the laboratory of A. Hershko (Technion, Haifa, Israel) and by my laboratory, then at MIT (Cambridge, MA). I describe the elegant insights by Hershko and his colleagues that yielded the initial understanding of ubiquitin conjugation and ubiquitin‐mediated proteolysis in cell extracts, including the identification of E1, E2, and E3 enzymes. These advances were followed by a set of interconnected discoveries in my laboratory that revealed the biology of the ubiquitin system, i.e., its necessity for the protein degradation in vivo, its specific physiological functions (in the cell cycle, DNA repair, protein synthesis, transcriptional regulation, and stress responses), the source of its selectivity (specific degradation signals in short‐lived proteins), and its key mechanistic attributes, such as the polyubiquitin chain and the subunit selectivity of protein degradation. The above biological (function‐based) insights produced the main discovery of the physiological regulation by intracellular protein degradation. These advances caused the enormous expansion of the ubiquitin field in the 1990s. Together with the initial discovery of ubiquitin‐mediated proteolysis by Hershko and coworkers, our biological discoveries in the 1980s led to a radically changed understanding of the logic of intracellular circuits, as it became clear that the control through regulated protein degradation rivals, and often surpasses in significance, the classical regulation through transcription and translation.
Alarm calls of wintering great tits Parus major: warning of mate, reciprocal altruism or a message to the predator?
Authors: Krams Indrikis , Krama Tatjana , Igaune Kristine
Published: March 2006

When a predator is not an immediate threat, a prey may produce relatively loud alarm calls because the risk is low. Since such calls could nevertheless attract acoustically oriented predators, the cost of predator attraction must be outweighed by factors beneficial to the caller. In this field study we elicited low‐risk alarm calls by temporarily catching wintering adult male great tits Parus major at feeders both within and outside their territories. We tested whether the alarm calls of dominant males can be explained in terms of mate warning, reciprocal altruism or notifying the predator of detection. If alarms are intended to warn mates, males accompanied by their mates should give alarm calls both within and outside home range, even if other permanent flock members are absent. If alarms are to be explained by reciprocal altruism, male great tits should give low‐risk alarm calls when accompanied by permanent flock members other than mate within and not outside of the home‐range. If alarm calling is a message to a predator, males should call when foraging alone. We found that male great tits gave low‐risk alarm calls when accompanied by their mates, independent of feeder location. They also gave low‐risk alarm calls within home ranges in the presence of other permanent flock members when mates were absent. In contrast, only a few males gave calls when foraging alone within their home ranges, or when in the company of unfamiliar great tits outside their usual home‐range. The results suggest that the utterance of alarm calls may be explained as mate protection and reciprocal altruism among familiar individuals.
Adult‐onset Still's disease with prurigo pigmentosa‐like skin eruption
Authors: TOMARU Kahori , NAGAI Yayoi , OHYAMA Naoko , HASEGAWA Michiko , ENDO Yukie , TAMURA Atsushi , ISHIKAWA Osamu
Published: March 2006

A 34‐year‐old woman with adult‐onset Still's disease (AOSD) developed prurigo pigmentosa‐like lesions on her chest and upper back in addition to the typical rash of AOSD. A biopsy specimen taken from the upper back showed characteristic features of prurigo pigmentosa. The eruption and fever subsided immediately after the administration of 40 mg/day prednisolone, but arthralgia persisted even after intravenous pulse methylprednisolone therapy in combination with immunosuppressive drugs. Various atypical skin rashes, including prurigo pigmentosa‐like lesions, have been reported in association with AOSD. Therefore, one should carefully follow the clinical course of a patient in order not to overlook these atypical cutaneous manifestations of AOSD.
Fusion of ETV6 with an intronic sequence of the BAZ2A gene in a paediatric pre‐B acute lymphoblastic leukaemia with a cryptic chromosome 12 rearrangement
Authors: Panagopoulos Ioannis , Strömbeck Bodil , Isaksson Margareth , Heldrup Jesper , Olofsson Tor , Johansson Bertil
Published: March 2006

ETV6 at 12p13 is rearranged in a variety of haematological malignancies and solid tumours, with more than 20 different partners having been reported. These fusions result in either chimeric proteins or activation of the partner gene. However, there are a few examples of abnormalities resulting in truncated and, most likely, unproductive ETV6 proteins, suggesting that haploinsufficiency of ETV6 and/or the partner is leukaemogenic. We present a novel ETV6 rearrangement, identified in a paediatric pre‐B acute lymphoblastic leukaemia. Fluorescence in situ hybridisation (FISH) and molecular genetic analyses revealed a fusion of ETV6 and BAZ2A (at 12q13), generated through a cryptic rearrangement between 12p13 and 12q13, consisting of exons 1 and 2 of ETV6 and a sequence from intron 1 of BAZ2A. This transcript is not expected to produce any chimeric protein, but may encode a truncated form of ETV6, containing the first 54 amino acids (aa), followed by 16 aa from the 3′ fusion sequence, reminiscent of ETV6 fusions with MDS2, LOC115548, PER1, and STL. The rearrangement might also modify the regulation of BAZ2A by either activating a cryptic promoter or by coming under the control of the ETV6 promoter. The present case emphasises that ‘unproductive’ETV6 rearrangements may play an important pathogenetic role in leukaemia.
A porous‐matrix sensor to measure the matric potential of soil water in the field
Authors: Whalley W. R. , Clark L. J. , Take W. A. , Bird N. R. A. , Leech P. K. , Cope R. E. , Watts C. W.
Published: March 2006

The matric potential of soil water is probably the most useful assessment of soil water status. However, the water‐filled tensiometer (the benchmark instrument for measuring matric potential) typically only operates in the range 0 to −85 kPa. In this paper, we report the development of a porous‐matrix sensor to measure matric potential in the approximate range −50 to −300 kPa. The sensor uses a dielectric probe to measure the water content of a ceramic material with known water retention characteristics. The calculation of matric potential takes into account hysteresis through the application of an appropriate model to measured wetting and drying loops. It is important that this model uses closed, rather than open, scanning loops. The calibrated sensors were tested in the field and the output compared with data from water‐filled tensiometers and dielectric measurements of soil water content. These comparisons indicated that conventional tensiometers gave stable but false readings of matric potential when soil dried to matric potentials more negative than −80 kPa. The porous‐matrix sensors appeared to give reliable readings of matric potential in soil down to −300 kPa and also responded appropriately to repeated wetting and drying. This porous‐matrix sensor has considerable potential to help understand plant responses to drying soil.