bluuebunny/crossref_metadata_2025_split huggingface dataset:
Aedes aegypti: An Emerging Model for Vector Mosquito Development: Figure 1.
Authors: Clemons Anthony , Haugen Morgan , Flannery Ellen , Tomchaney Michael , Kast Kristopher , Jacowski Caitlin , Le Christy , Mori Akio , Simanton Holland Wendy , Sarro Joseph , Severson David W. , Duman-Scheel Molly 
Published: October 2010

INTRODUCTIONBlood-feeding mosquitoes, including the dengue and yellow fever vectorAedes aegypti, transmit many of the world’s deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects. Targets of particular interest include genes that regulate development. However, although theAe. aegyptigenome project uncovered homologs of many known developmental regulatory genes, little is known of the genetic regulation of development inAe. aegyptior other vector mosquitoes. This article provides an overview of the background, husbandry, and potential uses ofAe. aegyptias a model species. Methods for culturing, collecting and fixing developing tissues, analyzing gene and protein expression, and knocking down genes are permitting detailed analyses of the functions of developmental regulatory genes and the selective inhibition of such genes duringAe. aegyptidevelopment. This methodology, much of which is applicable to other mosquito species, is useful to both the comparative development and vector research communities.
Stable Producer Cell Lines for Adeno-Associated Virus (AAV) Assembly
Authors: Chadeuf Gilliane , Salvetti Anna 
Published: October 2010

INTRODUCTIONStable producer cell lines containing both the rep and cap genes and recombinant adeno-associated virus (rAAV) vectors can be infected with a helper virus to provide reliable and efficient production of rAAV stocks. However, the development of these cell lines is time-consuming. The procedure described here is therefore recommended only for studies requiring the production of high amounts of rAAV, such as preclinical studies performed in large animals.
Imaging Collagen II Using Atomic Force Microscopy (AFM)
Authors: Plodinec Marija , Loparic Marko , Aebi Ueli 
Published: October 2010

INTRODUCTIONCollagen II is a fibrous protein that assembles from basic tropocollagen subunits to form extracellular supramolecular fiber networks within cartilage tissue. Tropocollagen subunits of ~300 nm in length self-assemble first into pentameric uniform microfibrils, which fuse into bigger collagen fibrils that can range from 10 nm to 500 nm in diameter. The collagen fibrils display a characteristic 67-nm repeat because of the staggering of individual collagen molecules with respect to each other. This protocol demonstrates how to prepare collagen protein samples for analysis by atomic force microscopy (AFM). It also describes the steps for generating AFM images of collagen samples during and after manipulation to analyze collagen self-assembly.
Efficient Genomic DNA Extraction from Low Target Concentration Bacterial Cultures Using SCODA DNA Extraction Technology
Authors: So Austin , Pel Joel , Rajan Sweta , Marziali Andre 
Published: October 2010

INTRODUCTIONMethods for the extraction of nucleic acids are straightforward in instances where there is ample nucleic acid mass in the sample and contamination is minimal. However, applications in areas such as metagenomics, life science research, clinical research, and forensics, that are limited by smaller amounts of starting materials or more dilute samples, require sample preparation methods that are more efficient at extracting nucleic acids. Synchronous coefficient of drag alteration (SCODA) is a novel electrophoretic nucleic acid purification technology that has been tested successfully with both highly contaminated and dilute samples and is a promising candidate for new sample preparation challenges. In this article, as an example of SCODA’s performance with limited sample material, we outline a genomic DNA (gDNA) extraction protocol from low abundance cultures of Escherichia coli DH10B. This method is equally well suited to high biomass samples.
Culturing and Egg Collection of Aedes aegypti
Authors: Clemons Anthony , Mori Akio , Haugen Morgan , Severson David W. , Duman-Scheel Molly 
Published: October 2010

INTRODUCTIONBlood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world’s deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects, and genes that regulate development are of particular interest. This protocol describes methods for culturing Ae. aegypti and includes a procedure for egg collection that can be used in conjunction with fixation, immunohistochemistry, and in situ protocols.
Whole-Mount In Situ Hybridization for Analysis of Gene Expression during Aedes aegypti Development: Figure 1.
Authors: Haugen Morgan , Tomchaney Michael , Kast Kristopher , Flannery Ellen , Clemons Anthony , Jacowski Caitlin , Simanton Holland Wendy , Le Christy , Severson David , Duman-Scheel Molly 
Published: October 2010

INTRODUCTIONBlood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world’s deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects, and genes that regulate development are of particular interest. This protocol for whole-mount in situ hybridization can be used to analyze gene expression in Ae. aegypti embryos and larvae, a critical aspect of understanding developmental gene function in this vector mosquito.
Immunohistochemical Analysis of Protein Expression during Aedes aegypti Development: Figure 1.
Authors: Clemons Anthony , Flannery Ellen , Kast Kristopher , Severson David , Duman-Scheel Molly 
Published: October 2010

INTRODUCTIONBlood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world’s deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects, and gene products s that regulate development are of particular interest. This protocol for immunohistochemical analysis of protein expression can be used to analyze expression of developmental proteins of interest in Ae. aegypti embryos, larvae, and pupae, which will be critical for the development of markers for particular developing tissues.
Atomic Force Microscopy for Biological Imaging and Mechanical Testing across Length Scales
Authors: Plodinec Marija , Loparic Marko , Aebi Ueli 
Published: October 2010

INTRODUCTIONAtomic force microscopy (AFM) offers researchers a unique opportunity to visualize, manipulate, and quantitatively assess structural and mechanical aspects of native biological samples with nanometer resolution. An unparalleled advantage of AFM over other high-resolution microscopes is that biological specimens, ranging from tissues to cells to molecules, can be investigated in physiologically relevant aqueous environments. The AFM can be operated at 37°C, which makes it ideal for in situ cell or tissue studies. Combining an optical microscope with an AFM makes it possible to directly correlate structural/nanomechanical changes with optical/fluorescence images. This ability to simultaneously acquire structural and function information is unprecedented in biology. This article introduces the basics of AFM for imaging and investigating the properties of biological samples.
The Arabidopsis C3H2C3-Type RING E3 Ubiquitin Ligase AtAIRP1 Is a Positive Regulator of an Abscisic Acid-Dependent Response to Drought Stress
Authors: Ryu Moon Young , Cho Seok Keun , Kim Woo Taek 
Published: October 2010

AbstractUbiquitination is a eukaryotic posttranslational protein modification that is mediated by the cascade of E1, E2, and E3 ubiquitin (Ub) ligases and is involved in regulating numerous cellular functions. In this study, we obtained 100 different Arabidopsis (Arabidopsis thaliana) T-DNA insertion mutant plants in which RING E3 Ub ligase genes were suppressed and monitored their phenotypes in the presence of exogenous abscisic acid (ABA), a plant stress hormone. One of these loss-of-function mutants displayed ABA-insensitive phenotypes at the germination stage and was named atairp1 (for Arabidopsis ABA-insensitive RING protein 1). AtAIRP1 encodes a cytosolic protein containing a single C3H2C3-type RING motif with in vitro E3 Ub ligase activity. AtAIRP1 was significantly induced by ABA and drought stress. In contrast to atairp1 mutant plants, AtAIRP1-overexpressing transgenic plants (35S:AtAIRP1-sGFP) were hypersensitive to exogenous ABA in terms of radicle emergence, cotyledon development, root elongation, and stomatal closure. Ectopic expression of AtAIRP1-sGFP in atairp1 effectively rescued the loss-of-function ABA-insensitive phenotype. Both 35S:AtAIRP1-sGFP and atairp1/35S:AtAIRP1-sGFP plants accumulated higher amounts of hydrogen peroxide in response to exogenous ABA than did wild-type and atairp1 mutant plants. AtAIRP1 overexpressors were markedly tolerant to severe drought stress, as opposed to atairp1, which was highly susceptible. The levels of drought stress-related genes and basic leucine zipper transcription factor genes were up-regulated in the 35S:AtAIRP1-sGFP lines relative to wild-type and atairp1 mutant plants in response to ABA. Overall, these results suggest that AtAIRP1, a C3H2C3-type RING E3 Ub ligase, is a positive regulator in the Arabidopsis ABA-dependent drought response.
HIGS: Host-Induced Gene Silencing in the Obligate Biotrophic Fungal Pathogen Blumeria graminis
Authors: Nowara Daniela , Gay Alexandra , Lacomme Christophe , Shaw Jane , Ridout Christopher , Douchkov Dimitar , Hensel Götz , Kumlehn Jochen , Schweizer Patrick 
Published: October 2010

Abstract
Powdery mildew fungi are obligate biotrophic pathogens that only grow on living hosts and cause damage in thousands of plant species. Despite their agronomical importance, little direct functional evidence for genes of pathogenicity and virulence is currently available because mutagenesis and transformation protocols are lacking. Here, we show that the accumulation in barley (Hordeum vulgare) and wheat (Triticum aestivum) of double-stranded or antisense RNA targeting fungal transcripts affects the development of the powdery mildew fungus Blumeria graminis. Proof of concept for host-induced gene silencing was obtained by silencing the effector gene Avra10, which resulted in reduced fungal development in the absence, but not in the presence, of the matching resistance gene Mla10. The fungus could be rescued from the silencing of Avra10 by the transient expression of a synthetic gene that was resistant to RNA interference (RNAi) due to silent point mutations. The results suggest traffic of RNA molecules from host plants into B. graminis and may lead to an RNAi-based crop protection strategy against fungal pathogens.
Identification of tissue transglutaminase‐reactive lysine residues in glyceraldehyde‐3‐phosphate dehydrogenase
Authors: Orru Stefania , Ruoppolo Margherita , Francese Simona , Vitagliano Luigi , Marino Gennaro , Esposito Carla 
Published: October 2010

AbstractPolyglutamine domains are excellent substrates for tissue transglutaminase resulting in the formation of cross‐links with polypeptides containing lysyl residues. This finding suggests that tissue transglutaminase may play a role in the pathology of neurodegenerative diseases associated with polyglutamine expansion. The glycolytic enzyme GAPDH previously was shown to tightly bind several proteins involved in such diseases. The present study confirms that GAPDH is an in vitro lysyl donor substrate of tissue transglutaminase. A dansylated glutamine‐containing peptide was used as probe for labeling the amino‐donor sites. SDS gel electrophoresis of a time‐course reaction mixture revealed the presence of both fluorescent GAPDH monomers and high molecular weight polymers. Western blot analysis performed using antitransglutaminase antibodies reveals that tissue transglutaminase takes part in the formation of heteropolymers. The reactive amino‐donor sites were identified using mass spectrometry. Here, we report that of the 26 lysines present in GAPDH, K191, K268, and K331 were the only amino‐donor residues modified by tissue transglutaminase.
Unique oligomeric intermediates of bovine liver catalase
Authors: Prakash Koodathingal , Prajapati Shashi , Ahmad Atta , Jain S.K. , Bhakuni Vinod 
Published: October 2010

AbstractCatalases, although synthesized from single genes and built up from only one type of subunit, exist in heterogeneous form with respect to their conformations and association states in biological systems. This heterogeneity is not of genetic origin, but rather reflects the instability of this oligomeric heme enzyme. To understand better the factors that stabilize the various association states of catalase, we performed studies on the multimeric intermediates that are stabilized during guanidine‐hydrochloride‐ and urea‐induced unfolding of bovine liver catalase (BLC). For the first time, we have observed an enzymatically active, folded dimer of native BLC. This dimer has slightly higher enzymatic activity and altered structural properties compared to the native tetramer. Comparative studies of the effect of NaCl, GdmCl, and urea on BLC show that cation binding to negatively charged groups present in amino acid side chains of the enzyme leads to stabilization of an enzymatically active, folded dimer of BLC. Besides the folded dimer, an enzymatically active expanded tetramer and a partially unfolded, enzymatically inactive dimer of BLC were also observed. A complete recovery of native enzyme was observed on refolding of expanded tetramers and folded dimers; however, a very low recovery (maximum of ∼5%) of native enzyme was observed on refolding of partially unfolded dimers and fully unfolded monomers.
Early formation of a beta hairpin during folding of staphylococcal nuclease H124L as detected by pulsed hydrogen exchange
Authors: Walkenhorst William F. , Edwards Jason A. , Markley John L. , Roder Heinrich 
Published: October 2010

AbstractPulsed hydrogen exchange methods were used to follow the formation of structure during the refolding of acid‐denatured staphylococcal nuclease containing a stabilizing Leu substitution at position 124 (H124L SNase). The protection of more than 60 backbone amide protons in uniformly15N‐labeled H124L SNase was monitored as a function of refolding time by heteronuclear two‐dimensional NMR spectroscopy. As found in previous studies of staphylococcal nuclease, partial protection was observed for a subset of amide protons even at the earliest folding time point (10 msec). Protection indicative of marginally stable hydrogen‐bonded structure in an early folding intermediate was observed at over 30 amide positions located primarily in the β‐barrel and to a lesser degree in the α‐helical domain of H124L SNase. To further characterize the folding intermediate, protection factors for individual amide sites were measured by varying the pH of the labeling pulse at a fixed refolding time of 16 msec. Protection factors >5.0 were observed only for amide positions in a β‐hairpin formed by strands 2 and 3 of the β‐barrel domain and a single site near the C‐terminus. The results indicate that formation of stable hydrogen‐bonded structure in a core region of the β‐sheet is among the earliest structural events in the folding of SNase and may serve as a nucleation site for further structure formation.
Composites of local structure propensities: Evidence for local encoding of long‐range structure
Authors: Shortle David 
Published: October 2010

AbstractTo estimate how extensively the ensemble of denatured‐state conformations is constrained by local side‐chain–backbone interactions, propensities of each of the 20 amino acids to occur in mono‐ and dipeptides mapped to discrete regions of the Ramachandran map are computed from proteins of known structure. In addition, propensities are computed for the trans, gauche−, and gauche+ rotamers, with or without consideration of the values of phi and psi. These propensities are used in scoring functions for fragment threading, which estimates the energetic favorability of fragments of protein sequence to adopt the native conformation as opposed to hundreds of thousands of incorrect conformations. As finer subdivisions of the Ramachandran plot, neighboring residue phi/psi angles, and rotamers are incorporated, scoring functions become better at ranking the native conformation as the most favorable. With the best composite propensity function, the native structure can be distinguished from 300,000 incorrect structures for 71% of the 2130 arbitrary protein segments of length 40, 48% of 2247 segments of length 30, and 20% of 2368 segments of length 20. A majority of fragments of length 30–40 are estimated to be folded into the native conformation a substantial fraction of the time. These data suggest that the variations observed in amino acid frequencies in different phi/psi/chi1 environments in folded proteins reflect energetically important local side‐chain–backbone interactions, interactions that may severely restrict the ensemble of conformations populated in the denatured state to a relatively small subset with nativelike structure.
Structure determinants of substrate specificity of hydroxynitrile lyase from Manihot esculenta
Authors: Lauble Hanspeter , Miehlich Burkhard , Förster Siegfried , Kobler Christoph , Wajant Harald , Effenberger Franz 
Published: October 2010

AbstractTryptophan 128 of hydroxynitrile lyase of Manihot esculenta (MeHNL) covers a significant part of a hydrophobic channel that gives access to the active site of the enzyme. This residue was therefore substituted in the mutant MeHNL‐W128A by alanine to study its importance for the substrate specificity of the enzyme. Wild‐type MeHNL and MeHNL‐W128A showed comparable activity on the natural substrate acetone cyanohydrin (53 and 40 U/mg, respectively). However, the specific activities of MeHNL‐W128A for the unnatural substrates mandelonitrile and 4‐hydroxymandelonitrile are increased 9‐fold and ∼450‐fold, respectively, compared with the wild‐type MeHNL. The crystal structure of the MeHNL‐W128A substrate‐free form at 2.1 Å resolution indicates that the W128A substitution has significantly enlarged the active‐site channel entrance, and thereby explains the observed changes in substrate specificity for bulky substrates. Surprisingly, the MeHNL‐W128A–4‐hydroxybenzaldehyde complex structure at 2.1 Å resolution shows the presence of two hydroxybenzaldehyde molecules in a sandwich type arrangement in the active site with an additional hydrogen bridge to the reacting center.
On the non‐respect of the thermodynamic cycle by DsbA variants
Authors: Moutiez Mireille , Quéméneur Eric , Burova Tatiana V. , Haertlé Thomas 
Published: October 2010

AbstractThe mechanism of the disulfide‐bond forming enzyme DsbA depends on the very low pKa of a cysteine residue in its active‐site and on the relative instability of the oxidized enzyme compared to the reduced one. A thermodynamic cycle has been used to correlate its redox properties to the difference in the free energies of folding (ΔΔGred/ox) of the oxidized and reduced forms. However, the relation was proved unsatisfied for a number of DsbA variants. In this study, we investigate the thermodynamic and redox properties of a highly destabilized variant DsbAP151A (substitution of cis‐Pro151 by an alanine) by the means of intrinsic tryptophan fluorescence and by high‐sensitivity differential scanning calorimetry (HS‐DSC). When the value of ΔΔGred/ox obtained fluorimetrically for DsbAP151A does not correlate with the value expected from its redox potential, the value of ΔΔGred/ox provided by HS‐DSC are in perfect agreement with the predicted thermodynamic cycle for both wild‐type and variant. HS‐DSC data indicate that oxidized wild‐type enzyme and the reduced forms of both wild‐type and variant unfold according to a two‐state mechanism. Oxidized DsbAP151A shows a deviation from two‐state behavior that implies the loss of interdomain cooperativity in DsbA caused by Pro151 substitution. The presence of chaotrope in fluorimetric measurements could facilitate domain uncoupling so that the fluorescence probe (Trp76) does not reflect the whole unfolding process of DsbAP151A anymore. Thus, theoretical thermodynamic cycle is respected when an appropriate method is applied to DsbA unfolding under conditions in which protein domains still conserve their cooperativity.
Structural comparisons of TIM barrel proteins suggest functional and evolutionary relationships between β‐galactosidase and other glycohydrolases
Authors: Juers Douglas H. , Huber Reuben E. , Matthews Brian W. 
Published: October 2010

Abstractβ‐Galactosidase (lacZ) from Escherichia coli is a 464 kDa homotetramer. Each subunit consists of five domains, the third being an α/β barrel that contains most of the active site residues. A comparison is made between each of the domains and a large set of proteins representative of all structures from the protein data bank. Many structures include an α/β barrel. Those that are most similar to the α/β barrel of E. coli β‐galactosidase have similar catalytic residues and belong to the so‐called “4/7 superfamily” of glycosyl hydrolases. The structure comparison suggests that β‐amylase should also be included in this family. Of three structure comparison methods tested, the “ProSup” procedure of Zu‐Kang and Sippl and the “Superimpose” procedure of Diederichs were slightly superior in discriminating the members of this superfamily, although all procedures were very powerful in identifying related protein structures. Domains 1, 2, and 4 of E. coli β‐galactosidase have topologies related to “jelly‐roll barrels” and “immunoglobulin constant” domains. This fold also occurs in the cellulose binding domains (CBDs) of a number of glycosyl hydrolases. The fold of domain 1 of E. coli β‐galactosidase is closely related to some CBDs, and the domain contributes to substrate binding, but in a manner unrelated to cellulose binding by the CBDs. This is typical of domains 1, 2, 4, and 5, which appear to have been recruited to play roles in β‐galactosidase that are unrelated to the functions that such domains provide in other contexts. It is proposed that β‐galactosidase arose from a prototypical single domain α/β barrel with an extended active site cleft. The subsequent incorporation of elements from other domains could then have reduced the size of the active site from a cleft to a pocket to better hydrolyze the disaccharide lactose and, at the same time, to facilitate the production of inducer, allolactose.