bluuebunny/crossref_metadata_2025 huggingface dataset:
Proline inhibits aggregation during protein refolding
Authors: Samuel Dharmaraj , Ganesh Gopal , Yang Pey‐Wen , Chang Mei‐Ming , Wang Sue‐Lein , Hwang Kuo‐Chu , Yu Chin , Jayaraman Gurunathan , Kumar Thallampuranam Krishnaswamy S. , Trivedi Vishwa Deo , Chang Ding‐Kwo
Published: October 2010

The in vitro refolding of hen egg‐white lysozyme is studied in the presence of various osmolytes. Proline is found to prevent aggregation during protein refolding. However, other osmolytes used in this study fail to exhibit a similar property. Experimental evidence suggests that proline inhibits protein aggregation by binding to folding intermediate(s) and trapping the folding intermediate(s) into enzymatically inactive, “aggregation‐insensitive” state(s). However, elimination of proline from the refolded protein mixture results in significant recovery of the bacteriolytic activity. At higher concentrations (>1.5 M), proline is shown to form loose, higher‐order molecular aggregate(s). The supramolecular assembly of proline is found to possess an amphipathic character. Formation of higher‐order aggregates is believed to be crucial for proline to function as a protein folding aid. In addition to its role in osmoregulation under water stress conditions, the results of this study hint at the possibility of proline behaving as a protein folding chaperone.
Human RhoA/RhoGDI complex expressed in yeast: Gtp exchange is sufficient for translocation of RhoA to liposomes
Authors: Read Paul W. , Liu Xiaopu , Longenecker Kenton , Dipierro Charles G. , Walker Lori A. , Somlyo Avril V. , Somlyo Andrew P. , Nakamoto Robert K.
Published: October 2010

The human small GTPase, RhoA, expressed in Saccharomyces cerevisiae is post‐translationally processed and, when co‐expressed with its cytosolic inhibitory protein, RhoGDI, spontaneously forms a heterodimer in vivo. The RhoA/RhoGDI complex, purified to greater than 98% at high yield from the yeast cytosolic fraction, could be stoichiometrically ADP‐ribosylated by Clostridium botulinum C3 exoenzyme, contained stoichiometric GDP, and could be nucleotide exchanged fully with [3H]GDP or partially with GTP in the presence of submicromolar Mg2+. The GTP‐RhoA/RhoGDI complex hydrolyzed GTP with a rate constant of 4.5 × 10−5 s−1, considerably slower than free RhoA. Hydrolysis followed pseudo‐first‐order kinetics indicating that the RhoA hydrolyzing GTP was RhoGDI associated. The constitutively active G14V‐RhoA mutant expressed as a complex with RhoGDI and purified without added nucleotide also bound stoichiometric guanine nucleotide: 95% contained GDP and 5% GTP. Microinjection of the GTP‐bound G14V‐RhoA/RhoGDI complex (but not the GDP form) into serum‐starved Swiss 3T3 cells elicited formation of stress fibers and focal adhesions. In vitro, GTP‐bound‐RhoA spontaneously translocated from its complex with RhoGDI to liposomes, whereas GDP‐RhoA did not. These results show that GTP‐triggered translocation of RhoA from RhoGDI to a membrane, where it carries out its signaling function, is an intrinsic property of the RhoA/RhoGDI complex that does not require other protein factors or membrane receptors.
A new approach to the design of uniquely folded thermally stable proteins
Authors: Jiang Xin , Pistor Ernie , Farid Ramy S. , Farid Hany
Published: October 2010

A new computer program (CORE) is described that predicts core hydrophobic sequences of predetermined target protein structures. A novel scoring function is employed, which for the first time incorporates parameters directly correlated to free energies of unfolding (DLGu), melting temperatures (Tm), and cooperativity. Metropolis‐driven simulated annealing and low‐temperature Monte Carlo sampling are used to optimize this score, generating sequences predicted to yield uniquely folded, stable proteins with cooperative unfolding transitions. The hydrophobic core residues of four natural proteins were predicted using CORE with the backbone structure and solvent exposed residues as input. In the two smaller proteins tested (Gβl, 11 core amino acids; 434 cro, 10 core amino acids), the native sequence was regenerated as well as the sequence of known thermally stable variants that exhibit cooperative denaturation transitions. Previously designed sequences of variants with lower thermal stability and weaker cooperativity were not predicted. In the two larger proteins tested (myoglobin, 32 core amino acids; methionine aminopeptidase, 63 core amino acids), sequences with corresponding side‐chain conformations remarkably similar to that of native were predicted.
Direct measurement of islet amyloid polypeptide fibrillogenesis by mass spectrometry
Authors: Larson Jennifer L. , Ko Eric , Miranker Andrew D.
Published: October 2010

A novel method for monitoring fibrillogenesis is developed and applied to the amyloidogenic peptide, islet amyloid polypeptide (IAPP). The approach, based on electrospray ionization mass spectrometry, is complementary to existing assays of fibril formation as it monitors directly the population of precursor rather than product molecules. We are able to monitor fiber formation in two modes: a quenched mode in which fibril formation is halted by dilution into denaturant and a real time mode in which fibril formation is conducted within the capillary of the electrospray source. Central to the method is the observation that fibrillar IAPP does not compromise the ionization of monomeric IAPP. Furthermore, under mild ionization conditions, fibrillar IAPP does not dissociate and contribute to the monomeric signal. Critically, we introduce an internal standard, rat IAPP, for analysis on the mass spectrometer. This standard is sufficiently similar in sequence in that it ionizes identically to human IAPP. Furthermore, the sequence is sufficiently different in that it does not form fibrils and is distinguishable on the basis of mass. Applied to IAPP fibrillogenesis, our technique reveals that precursor consumption in seeded reactions obeys first‐order kinetics. Furthermore, a consistent level of monomer persists in both seeded and unseeded experiments after the fibril formation is complete. Given the inherent stability of fibrils, we expect this approach to be applicable to other amyloid systems.
A “structural” water molecule in the family of fatty acid binding proteins
Authors: Likić Vladimir A. , Prendergast Franklyn G. , Juranić Nenad , Macura Slobodan
Published: October 2010

A single water molecule (w135), buried within the structure of rat intestinal fatty acid binding protein (I‐FABP), is investigated by NMR, molecular dynamics simulations, and analysis of known crystal structures. An ordered water molecule was found in structurally analogous position in 24 crystal structures of nine different members of the family of fatty acid binding proteins. There is a remarkable conservation of the local structure near the w135 binding site among different proteins from this family. NMR cross‐relaxation measurements imply that w135 is present in the I‐FABP:ANS (1‐sulfonato‐8‐(1′)anilinonaphthalene) complex in solution with the residence time of >300 ps. Mean‐square positional fluctuations of w135 oxygen observed in MD simulations (0.18 and 0.13 Å2) are comparable in magnitude to fluctuations exhibited by the backbone atoms and result from highly constrained binding pocket as revealed by Voronoi volumes (averages of 27.0 ± 1.8 Å3 and 24.7 ± 2.2 Å3 for the two simulations). Escape of w135 from its binding pocket was observed only in one MD simulation. The escape process was initiated by interactions with external water molecules and was accompanied by large deformations in β‐strands D and E. Immediately before the release, w135 assumed three distinct states that differ in hydrogen bonding topology and persisted for about 15 ps each. Computer simulations suggest that escape of w135 from the I‐FABP matrix is primarily determined by conformational fluctuations of the protein backbone and interactions with external water molecules.
The effect of nucleotide bias upon the composition and prediction of transmembrane helices
Authors: Stevens Timothy J. , Arkin Isaiah T.
Published: October 2010

Transmembrane helices are the most readily predictable secondary structure components of proteins. They can be predicted to a high degree of accuracy in a variety of ways. Many of these methods compare new sequence data with the sequence characteristics of known transmembrane domains. However, the known transmembrane sequences are not necessarily representative of a particular organism. We attempt to demonstrate that parameters optimized for the known transmembrane domains are far from optimal when predicting transmembrane regions in a given genome. In particular, we have tested the effect of nucleotide bias upon the composition and hence the prediction characteristics of transmembrane helices. Our analysis shows that nucleotide bias of a genome has a strong and predictable influence upon the occurrences of several of the most important hydrophobic amino acids found within transmembrane helices. Thus, we show that nucleotide bias should be taken into account when determining putative transmembrane domains from sequence data.
Cloning, expression, purification, and preliminary characterization of a putative hemoglobin from the cyanobacterium synechocystis sp. PCC 6803
Authors: Scott Nancy L. , Lecomte JULIETTE T. J.
Published: October 2010

The genome of the unicellular cyanobacterium Synechocystis sp. PCC 6803 contains a gene (slr2097, glbN) encoding a 123 amino‐acid product with sequence similarity to globins. Related proteins from cyanobacteria, ciliates, and green algae bind oxygen and have a pronounced tendency to coordinate the heme iron with two protein ligands. To study the structural and functional properties of Synechocystis sp. PCC 6803 hemoglobin, slr2097 was cloned and overexpressed in Escherichia coli. Purification of the hemoglobin was performed after addition of hemin to the clarified cell lysate. Recombinant, heme‐reconstituted ferric Synechocystis sp. PCC 6803 hemoglobin was found to be a stable helical protein, soluble to concentrations higher than 500 μM. At neutral pH, it yielded an electronic absorption spectrum typical of a low‐spin ferric species, with maxima at 410 and 546 nm. The proton NMR spectrum revealed sharp lines spread over a chemical shift window narrower than 40 ppm, in support of low‐spin hexacoordination of the heme iron. Nuclear Overhauser effects demonstrated that the heme is inserted in the protein matrix to produce one major equilibrium form. Addition of dithionite resulted in an absorption spectrum with maxima at 426, 528, and 560 nm. This reduced form appeared capable of carbon monoxide binding. Optical data also suggested that cyanide ions could bind to the heme in the ferric state. The spectral properties of the putative Synechocystis sp. PCC 6803 hemoglobin confirmed that it can be used for further studies of an ancient hemoprotein structure.
Mutational analysis of the major coat protein of M13 identifies residues that control protein display
Authors: Weiss Gregory A. , Wells James A. , Sidhu Sachdev S.
Published: October 2010

We have reported variants of the M13 bacteriophage major coat protein (P8) that enable high copy display of monomeric and oligomeric proteins, such as human growth hormone and steptavidin, on the surface of phage particles (Sidhu SS, Weiss GA, Wells JA. 2000. High copy display of large proteins on phage for functional selections. J Mol Biol 296:487–495). Here, we explore how an optimized P8 variant (opti‐P8) could evolve the ability to efficiently display a protein fused to its N‐terminus. Reversion of individual opti‐P8 residues back to the wild‐type P8 residue identifies a limited set of hydrophobic residues responsible for the high copy protein display. These hydrophobic amino acids bracket a conserved hydrophobic face on the P8 alpha helix thought to be in contact with the phage coat. Mutations additively combine to promote high copy protein display, which was further enhanced by optimization of the linker between the phage coat and the fusion protein. These data are consistent with a model in which protein displayenhancing mutations allow for better packing of the fusion protein into the phage coat. The high tolerance for phage coat protein mutations observed here suggests that filamentous phage coat proteins could readily evolve new capabilities.
A method to predict residues conferring functional differences between related proteins: Application to MAP kinase pathways
Authors: Caffrey Daniel R. , Shields Denis C. , O'Neill LUKE A. J.
Published: October 2010

Physicochemical properties are potentially useful in predicting functional differences between aligned protein subfamilies. We present a method that considers physicochemical properties from ancestral sequences predicted to have given rise to the subfamilies of interest by gene duplication. Comparison between two map kinases subfamilies, p38 and ERK, revealed a region that had an excess of change in properties after gene duplication followed by conservation within the two subfamilies. This region corresponded to that experimentally defined as important for substrate and pathway specificity. The derived scores for the region of interest were found to differ significantly in their distribution compared to the rest of the protein when the Kolmogorov‐Smirnov test was applied (p = 0.005). Thus, the incorporation of ancestral physicochemical properties is useful in predicting functional differences between protein subfamilies. In addition, the method was applied to the MKK and MAPK components of the p38 and JNK pathways. These proteins showed a similar pattern in their evolution and regions predicted to confer functional differences are discussed.
Backbone dynamics measurements on leukemia inhibitory factor, a rigid four‐helical bundle cytokine
Authors: Yao Shenggen , Smith David K. , Hinds Mark G. , Zhang Jian‐Guo , Nicola Nicos A. , Norton Raymond S.
Published: October 2010

The backbone dynamics of the four‐helical bundle cytokine leukemia inhibitory factor (LIF) have been investigated using 15N NMR relaxation and amide proton exchange measurements on a murine—human chimera, MH35‐LIF. For rapid backbone motions (on a time scale of 10 ps to 100 ns), as probed by 15N relaxation measurements, the dynamics parameters were calculated using the model‐free formalism incorporating the model selection approach. The principal components of the inertia tensor of MH35‐LIF, as calculated from its NMR structure, were 1:0.98:0.38. The global rotational motion of the molecule was, therefore, assumed to be axially symmetric in the analysis of its relaxation data. This yielded a diffusion anisotropy D⟂D⟂ of 1.31 and an effective correlation time (4D⟂ + 2D⟂)−1 of 8.9 ns. The average values of the order parameters (S2) for the four helices, the long interhelical loops, and the N‐terminus were 0.91, 0.84, and 0.65, respectively, indicating that LIF is fairly rigid in solution, except at the N‐terminus. The S2 values for the long interhelical loops of MH35‐LIF were higher than those of their counterparts in short‐chain members of the four‐helical bundle cytokine family. Residues involved in LIF receptor binding showed no consistent pattern of backbone mobilities, with S2 values ranging from 0.71 to 0.95, but residues contributing to receptor binding site III had relatively lower S2 values, implying higher amplitude motions than for the backbone of sites I and II. In the relatively slow motion regime, backbone amide exchange measurements showed that a number of amides from the helical bundle exchanged extremely slowly, persisting for several months in 2H2O at 37 °C. Evidence for local unfolding was considered, and correlations among various structure‐related parameters and the backbone amide exchange rates were examined. Both sets of data concur in showing that LIF is one of the most rigid four‐helical bundle cytokines.
15N and 1H NMR study of histidine containing protein (hpr) from staphylococcus carnosus at high pressure
Authors: Kalbitzer Hans Robert , Görler Adrian , Li Hua , Dubovskii Peter V. , Hengstenberg Wolfgang , Kowolik Claudia , Yamada Hiroaki , Akasaka Kazuyuki
Published: October 2010

The pressure‐induced changes in 15N enriched HPr from Staphylococcus carnosus were investigated by two‐dimensional (2D) heteronuclear NMR spectroscopy at pressures ranging from atmospheric pressure up to 200 MPa. The NMR experiments allowed the simultaneous observation of the backbone and side‐chain amide protons and nitrogens. Most of the resonances shift downfield with increasing pressure indicating generalized pressure‐induced conformational changes. The average pressure‐induced shifts for amide protons and nitrogens are 0.285 ppm GPa−1 at 278 K and 2.20 ppm GPa−1, respectively. At 298 K the corresponding values are 0.275 and 2.41 ppm GPa−1. Proton and nitrogen pressure coefficients show a significant but rather small correlation (0.31) if determined for all amide resonances. When restricting the analysis to amide groups in the β‐pleated sheet, the correlation between these coefficients is with 0.59 significantly higher. As already described for other proteins, the amide proton pressure coefficients are strongly correlated to the corresponding hydrogen bond distances, and thus are indicators for the pressure‐induced changes of the hydrogen bond lengths. The nitrogen shift changes appear to sense other physical phenomena such as changes of the local backbone conformation as well. Interpretation of the pressure‐induced shifts in terms of structural changes in the HPr protein suggests the following picture: the four‐stranded β‐pleated sheet of HPr protein is the least compressible part of the structure showing only small pressure effects. The two long helices a and c show intermediary effects that could be explained by a higher compressibility and a concomitant bending of the helices. The largest pressure coefficients are found in the active center region around His15 and in the regulatory helix b which includes the phosphorylation site Ser46 for the HPr kinase. This suggests that this part of the structure occurs in a number of different structural states whose equilibrium populations are shifted by pressure. In contrast to the surrounding residues of the active center loop that show large pressure effects, Ile14 has a very small proton and nitrogen pressure coefficient. It could represent some kind of anchoring point of the active center loop that holds it in the right place in space, whereas other parts of the loop adapt themselves to changing external conditions.
Crystal structure of adenosine kinase from Toxoplasma gondii at 1.8 Å resolution
Authors: Cook William J. , Delucas Lawrence J. , Chattopadhyay Debasish
Published: October 2010

Human infection with Toxoplasma gondii is an important cause of morbidity and mortality. Protozoan parasites such as T. gondii are incapable of de novo purine biosynthesis and must acquire purines from their host, so the purine salvage pathway offers a number of potential targets for antiparasitic chemotherapy. In T. gondii tachyzoites, adenosine is the predominantly salvaged purine nucleoside, and thus adenosine kinase is a key enzyme in the purine salvage pathway of this parasite. The structure of T. gondii adenosine kinase was solved using molecular replacement and refined by simulated annealing at 1.8 Å resolution to an R‐factor of 0.214. The overall structure and the active site geometry are similar to human adenosine kinase, although there are significant differences. The T. gondii adenosine kinase has several unique features compared to the human sequence, including a five‐residue deletion in one of the four linking segments between the two domains, which is probably responsible for a major change in the orientation of the two domains with respect to each other. These structural differences suggest the possibility of developing specific inhibitors of the parasitic enzyme.
Analysis of knowledge‐based protein‐ligand potentials using a self‐consistent method
Authors: Shimada J. , Ishchenko A.V. , Shakhnovich E.I.
Published: October 2010

We propose a self‐consistent approach to analyze knowledge‐based atom‐atom potentials used to calculate protein‐ligand binding energies. Ligands complexed to actual protein structures were first built using the SMoG growth procedure (DeWitte & Shakhnovich, 1996) with a chosen input potential. These model protein‐ligand complexes were used to construct databases from which knowledge‐based protein‐ligand potentials were derived. We then tested several different modifications to such potentials and evaluated their performance on their ability to reconstruct the input potential using the statistical information available from a database composed of model complexes. Our data indicate that the most significant improvement resulted from properly accounting for the following key issues when estimating the reference state: (1) the presence of significant nonenergetic effects that influence the contact frequencies and (2) the presence of correlations in contact patterns due to chemical structure. The most successful procedure was applied to derive an atom‐atom potential for real protein‐ligand complexes. Despite the simplicity of the model (pairwise contact potential with a single interaction distance), the derived binding free energies showed a statistically significant correlation (∼0.65) with experimental binding scores for a diverse set of complexes.
Structure of an analog of fusion peptide from hemagglutinin
Authors: Dubovskii Peter V. , Li Hua , Takahashi Sho , Arseniev Alexander S. , Akasaka Kazuyuki
Published: October 2010

A 20‐residue peptide E5 containing five glutamates, an analog of the fusion peptide of influenza virus hemagglutinin (HA) exhibiting fusion activity at acidic pH lower than 6.0–6.5 was studied by circular dichroism (CD), Fourier transform infrared, and 1H‐NMR spectroscopy in water, water/trifluoroethanol (TFE) mixtures, dodecylphosphocholine (DPC) micelles, and phospholipid vesicles. E5 became structurally ordered at pH ≤6 and the helical content in the peptide increased in the row: water < water/tfe < dpc ∼ phospholipid vesicle while the amount of β‐structure was approximately reverse. 1H‐NMR data and line‐broadening effect of 5‐, 16‐doxylstearates on proton resonances of DPC bound peptide showed E5 forms amphiphilic α‐helix in residues 2–18, which is flexible in 11–18 part. The analysis of the proton chemical shifts of DPC bound and CD intensity at 220 nm of phospholipid bound E5 showed that the pH dependence of helical content is characterized by the same pKa ≈︁5.6. Only Glu11 and Glu15 in DPC bound peptide showed such elevated pKas, presumably due to transient hydrogen bond(s) Glu11 (Glu15) δCOO−(H+) … HN Glu15 that dispose(s) the side chain of Glu11 (Glu15) residue(s) close to the micelle/water interface. These glutamates are present in the HA‐fusion peptide and the experimental half‐maximal pH of fusion for HA and E5 peptides is ∼5.6. Therefore, a specific anchorage of these peptides onto membrane necessary for fusion is likely driven by the protonation of the carboxylate group of Glu11 (Glu 15) residue (s) participating in transient hydrogen bond(s).
Folding of a dimeric β‐barrel: Residual structure in the urea denatured state of the human papillomavirus E2 DNA binding domain
Authors: Alonso Leonardo G. , Prat‐Gay Gonzalo De , Bycroft Mark , Mok Yu‐Keung , Lima L. Mauricio T.R.
Published: October 2010

The dimeric β‐barrel is a characteristic topology initially found in the transcriptional regulatory domain of the E2 DNA binding domain from papillomaviruses. We have previously described the kinetic folding mechanism of the human HPV‐16 domain, and, as part of these studies, we present a structural characterization of the urea‐denatured state of the protein. We have obtained a set of chemical shift assignments for the C‐terminal domain in urea using heteronuclear NMR methods and found regions with persistent residual structure. Based on chemical shift deviations from random coil values, 3Jnhnα coupling constants, heteronuclear single quantum coherence peak intensities, and nuclear Overhauser effect data, we have determined clusters of residual structure in regions corresponding to the DNA binding helix and the second β‐strand in the folded conformation. Most of the structures found are of nonnative nature, including turn‐like conformations. Urea denaturation at equilibrium displayed a loss in protein concentration dependence, in absolute parallel to a similar deviation observed in the folding rate constant from kinetic experiments. These results strongly suggest an alternative folding pathway in which a dimeric intermediate is formed and the rate‐limiting step becomes first order at high protein concentrations. The structural elements found in the denatured state would collide to yield productive interactions, establishing an intermolecular folding nucleus at high protein concentrations. We discuss our results in terms of the folding mechanism of this particular topology in an attempt to contribute to a better understanding of the folding of dimers in general and intertwined dimeric proteins such as transcription factors in particular.
The crystal structures of glutathione S‐transferases isozymes 1–3 and 1–4 from Anopheles dirus species B
Authors: Oakley Aaron J. , Harnnoi Thasaneeya , Udomsinprasert Rungrutai , Jirajaroenrat Kanya , Ketterman Albert J. , Wilce Matthew C.J.
Published: October 2010

Glutathione S‐transferases (GSTs) are dimeric proteins that play an important role in cellular detoxification. Four GSTs from the mosquito Anopheles dirus species B (Ad), an important malaria vector in South East Asia, are produced by alternate splicing of a single transcription product and were previously shown to have detoxifying activity towards pesticides such as DDT. We have determined the crystal structures for two of these alternatively spliced proteins, AdGST1–3 (complexed with glutathione) and AdGST1–4 (apo form), at 1.75 and 2.45 Å resolution, respectively. These GST isozymes show differences from the related GST from the Australian sheep blowfly Lucilia cuprina; in particular, the presence of a C‐terminal helix forming part of the active site. This helix causes the active site of the Anopheles GSTs to be enclosed. The glutathione‐binding helix α2 and flanking residues are disordered in the AdGST1–4 (apo) structure, yet ordered in the AdGST1–3 (GSH‐bound) structure, suggesting that insect GSTs operate with an induced fit mechanism similar to that found in the plant phi‐ and human pi‐class GSTs. Despite the high overall sequence identities, the active site residues of AdGST1–4 and AdGST1–3 have different conformations.
Preferred proline puckerings in cis and trans peptide groups: Implications for collagen stability
Authors: Vitagliano Luigi , Berisio Rita , Mastrangelo Antonio , Mazzarella Lelio , Zagari Adriana
Published: October 2010

The interplay between side‐chain and main‐chain conformations is a distinctive characteristic of proline residues. Here we report the results of a statistical analysis of proline conformations using a large protein database. In particular, we found that proline residues with the preceding peptide bond in the cis state preferentially adopt a down puckering. Indeed, out of 178 cis proline residues, as many as 145 (81%) are down. By analyzing the 1–4 and 1–5 nonbonding distances between backbone atoms, we provide a structural explanation for the observed trend. The observed correlation between proline puckering and peptide bond conformation suggests a new mechanism to explain the reported shift of the cis‐trans equilibrium in proline derivatives. The implications of these results for the current models of collagen stability are also discussed.
The rsmA‐like gene rsmAXoo of Xanthomonas oryzae pv. oryzae regulates bacterial virulence and production of diffusible signal factor
Authors: ZHU PEI‐LIANG , ZHAO SHUAI , TANG JI‐LIANG , FENG JIA‐XUN
Published: October 2010

The plant‐pathogenic prokaryote Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight, one of the most destructive diseases of rice. A nonpolar mutant of the rsmA‐like gene rsmAXoo of the Xoo Chinese strain 13751 was constructed by homologous integration with a suicide plasmid. Virulence tests on a host plant, namely the hybrid rice cultivar Teyou 63, showed that the mutant had lost its virulence almost completely, whereas tests on a nonhost, namely castor‐oil plant (Ricinus communis), showed that the mutant had also lost the ability to induce a hypersensitive response in the nonhost. In addition, the rsmAXoo mutant produced significantly smaller amounts of the diffusible signal factor, extracellular endoglucanase, amylase and extracellular polysaccharide, but showed significantly higher glycogen accumulation, bacterial aggregation and cell adhesion. The expression of most hrp genes, genes encoding AvrBs3/PthA family members, rpfB, xrvA, glgA, eglXoB and XOO0175 (encoding an α‐amylase) was down‐regulated in the rsmAXoo mutant. All phenotypes and expression levels of the tested genes in the rsmAXoo mutant were restored to their levels in the wild‐type by the presence of rsmAXoo in trans. These results indicate that rsmAXoo is essential for the virulence of Xoo.
Two nucleotide positions in the Citrus exocortis viroid RNA associated with symptom expression in Etrog citron but not in experimental herbaceous hosts
Authors: MURCIA NUBIA , BERNAD LUCÍA , DURAN‐VILA NÚRIA , SERRA PEDRO
Published: October 2010

Citrus exocortis viroid (CEVd) is the causal agent of exocortis disease of citrus. CEVd has a wide host range that includes woody and herbaceous species. A new CEVd strain (CEVdCOL), phylogenetically clustering with CEVd variants of Class A inducing severe symptoms in tomato, was identified in Colombia and shown to induce only extremely mild symptoms in Etrog citron indicator plants. Using site‐directed mutagenesis, two nucleotide substitutions (314A → G and 315U → A) in the lower strand of the P domain of the predicted CEVdCOL secondary structure resulted in a severe artificial CEVdMCOL variant. Conversely, two nucleotide exchanges (314G → A and 315A → U) in the same region of the severe variant CEVdE‐117 resulted in a symptomless artificial CEVdME‐117 variant. Infectivity assays conducted with the natural and mutated variants showed that all induced severe symptoms in Gynura aurantiaca, tomato and chrysanthemum. This is the first report of the identification of pathogenic determinants of CEVd in citrus, and shows that these pathogenicity determinants are host dependent.
Elsinoë fawcettii and Elsinoë australis: the fungal pathogens causing citrus scab
Authors: CHUNG KUANG‐REN
Published: October 2010

Elsinoë fawcettii and E. australis are important pathogens of citrus. Both species are known to produce red or orange pigments, called elsinochrome. Elsinochrome is a nonhost‐selective phytotoxin and is required for full fungal virulence and lesion formation. This article discusses the taxonomy, epidemiology, genetics and pathology of the pathogens. It also provides a perspective on the cellular toxicity, biosynthetic regulation and pathological role of elsinochrome phytotoxin.

Taxonomy: Elsinoë fawcettii (anamorph: Sphaceloma fawcettii) and E. australis (anamorph: S. australis) are classified in the Phylum Ascomycota, Class Dothideomycetes, Order Myriangiales and Family Elsinoaceae.

Host range: Elsinoë fawcettii causes citrus scab (formerly sour orange scab and common scab) on various species and hybrids in the Rutaceae family worldwide, whereas E. australis causes sweet orange scab, primarily on sweet orange and some mandarins, and has a limited geographical distribution.

Disease symptoms: Citrus tissues infested with Elsinoë often display erumpent scab pustules with a warty appearance.

Toxin production: Elsinochrome and many perylenequinone‐containing phytotoxins of fungal origin are grouped as photosensitizing compounds that are able to absorb light energy, react with oxygen molecules and produce reactive oxygen species, such as superoxide and singlet oxygen. Elsinochrome has been documented to cause peroxidation of cell membranes and to induce rapid electrolyte leakage from citrus tissues. Elsinochrome biosynthesis and conidiation are coordinately regulated in E. fawcettii, and the environmental and physiological inducers commonly involved in both processes have begun to be elucidated.
Tobacco leaf spot and root rot caused by Rhizoctonia solani Kühn
Authors: GONZALEZ MARLENY , PUJOL MERARDO , METRAUX JEAN‐PIERRE , GONZALEZ‐GARCIA VICENTE , BOLTON MELVIN D. , BORRÁS‐HIDALGO ORLANDO
Published: October 2010

Rhizoctonia solani Kühn is a soil‐borne fungal pathogen that causes disease in a wide range of plants worldwide. Strains of the fungus are traditionally grouped into genetically isolated anastomosis groups (AGs) based on hyphal anastomosis reactions. This article summarizes aspects related to the infection process, colonization of the host and molecular mechanisms employed by tobacco plants in resistance against R. solani diseases.

Taxonomy: Teleomorph: Thanatephorus cucumeris (Frank) Donk; anamorph: Rhizoctonia solani Kühn; Kingdom Fungi; Phylum Basidiomycota; Class Agaricomycetes; Order Cantharellales; Family Ceratobasidiaceae; genus Thanatephorus.

Identification: Somatic hyphae in culture and hyphae colonizing a substrate or host are first hyaline, then buff to dark brown in colour when aging. Hyphae tend to form at right angles at branching points that are usually constricted. Cells lack clamp connections, but possess a complex dolipore septum with continuous parenthesomes and are multinucleate. Hyphae are variable in size, ranging from 3 to 17 µm in diameter. Although the fungus does not produce any conidial structure, ellipsoid to globose, barrel‐shaped cells, named monilioid cells, 10–20 µm wide, can be produced in chains and can give rise to sclerotia. Sclerotia are irregularly shaped, up to 8–10 mm in diameter and light to dark brown in colour.

Disease symptoms: Symptoms in tobacco depend on AG as well as on the tissue being colonized. Rhizoctonia solani AG‐2‐2 and AG‐3 infect tobacco seedlings and cause damping off and stem rot. Rhizoctonia solani AG‐3 causes ‘sore shin’ and ‘target spot’ in mature tobacco plants. In general, water‐soaked lesions start on leaves and extend up the stem. Stem lesions vary in colour from brown to black. During late stages, diseased leaves are easily separated from the plant because of severe wilting. In seed beds, disease areas are typically in the form of circular to irregular patches of poorly growing, yellowish and/or stunted seedlings.

Resistance: Knowledge is scarce regarding the mechanisms associated with resistance to R. solani in tobacco. However, recent evidence suggests a complex response that involves several constitutive factors, as well as induced barriers controlled by multiple defence pathways.

Management: This fungus can survive for many years in soil as mycelium, and also by producing sclerotia, which makes the management of the disease using conventional means very difficult. Integrated pest management has been most successful; it includes timely fungicide applications, crop rotation and attention to soil moisture levels. Recent developments in biocontrol may provide other tools to control R. solani in tobacco.
RNA‐mediated gene silencing in the cereal fungal pathogen Cochliobolus sativus
Authors: LENG YUEQIANG , WU CHENGXIANG , LIU ZHAOHUI , FRIESEN TIMOTHY L. , RASMUSSEN JACK B. , ZHONG SHAOBIN
Published: October 2010

A high‐throughput RNA‐mediated gene silencing system was developed for Cochliobolus sativus (anamorph: Bipolaris sorokiniana), the causal agent of spot blotch, common root rot and black point in barley and wheat. The green fluorescent protein gene (GFP) and the proteinaceous host‐selective toxin gene (ToxA) were first introduced into C. sativus via the polyethylene glycol (PEG)‐mediated transformation method. Transformants with a high level of expression of GFP or ToxA were generated. A silencing vector (pSGate1) based on the Gateway cloning system was developed and used to construct RNA interference (RNAi) vectors. Silencing of GFP and ToxA in the transformants was demonstrated by transformation with the RNAi construct expressing hairpin RNA (hpRNA) of the target gene. The polyketide synthase gene (CsPKS1), involved in melanin biosynthesis pathways in C. sativus, was also targeted by transformation with the RNAi vector (pSGate1‐CsPKS1) encoding hpRNA of the CsPKS1 gene. The transformants with pSGate1‐CsPKS1 exhibited an albino phenotype or reduced melanization, suggesting effective silencing of the endogenous CsPKS1 in C. sativus. Sectors exhibiting the wild‐type phenotype of the fungus appeared in some of the CsPKS1‐silenced transformants after subcultures as a result of inactivation or deletions of the RNAi transgene. The gene silencing system established provides a useful tool for functional genomics studies in C. sativus and other filamentous fungi.
Hormone receptor‐like in 96 and Broad‐Complex modulate phenobarbital induced transcription of cytochrome P450CYP6D1inDrosophilaS2 cells
Authors: Lin G. G.‐H. , Kozaki T. , Scott J. G.
Published: October 2010

Phenobarbital (PB) is a prototypical inducer for studies of xenobiotic responses in animals. In mammals, the nuclear receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR) have been identified as key transcription factors regulating PB induced transcription of xenobiotic responsive genes. In insects, much less is known about the transcription factors involved in regulating PB induced transcription, although CAR and PXR have a single orthologue hormone receptor‐like in 96 (HR96) inDrosophila melanogaster. Using dual luciferase reporter assays inDrosophilaSchneider (S2) cells, constructs containing variable lengths of the promoter of the PB inducible cytochrome P450CYP6D1were evaluated in the presence and absence of PB. The promoter region between −330 and −280 (relative to the position of transcription start site, +1) was found to be critical for PB induction. Putative binding sites forDrosophilaBroad‐Complex (BR‐C) and deformed (Dfd) were identified within this promoter region using TFsearch. RNA interference (RNAi) treatment of S2 cells in conjunction withCYP6D1promoter assays showed that suppression ofDrosophilaHR96 and BR‐C transcription in S2 cells resulted in a significant decrease and increase, respectively, of PB induction. Effects of HR96 and BR‐C in mediating PB induction were PB specific and PB dependent. This represents new functional evidence thatDrosophilaHR96 and BR‐C can act as an activator and repressor, respectively, in regulating PB induced transcription in insects.
Polymorphisms, haplotypes and mutations in the protamine 1 and 2 genes
Authors: Jodar M. , Oriola J. , Mestre G. , Castillo J. , Giwercman A. , Vidal‐Taboada J. M. , Ballescà J. L. , Oliva R.
Published: October 2010

Protamines are the most abundant nuclear proteins and alterations in their expression have been described in infertile patients. Also, protamine haplo‐insufficient mice have been described as infertile. Therefore, the protamine 1 and 2 genes have been considered important candidates in different mutational studies. In this article, we review all published articles related to protamine gene mutations and report new data on mutations from patients and controls drawn from the Spanish and Swedish populations. Sequencing of the protamine 1 and 2 genes in a total of 209 infertile patients and 152 fertility‐proven controls from the Spanish and Swedish populations identified two novel and rare non‐pathogenic missense mutations (R17C and R38M) in the protamine 1 gene and several additional polymorphisms. Furthermore, we have identified and we report for the first time five novel rare haplotypes encompassing the protamine 1 and 2 genes. A review of all available protamine gene mutational studies indicates that none of the reported missense mutations can be considered of proven pathogenicity. However, it is interesting to note that rare protamine 1 promoter variants have been reported only in infertile patients, but not in fertile control groups. Pathogenic high penetrance protamine gene missense mutations, if any, must be extremely rare. However, the detected presence of rare variants and haplotypes in infertile patients deserves further investigation.
Breed‐associated phenotypes in canine atopic dermatitis
Authors: Wilhem Sylvia , Kovalik Marcel , Favrot Claude
Published: October 2010

Canine atopic dermatitis is a multifaceted disease, whose clinical presentation may be affected by numerous factors, including the genetic background of the animal, the environment, the offending allergens and flare factors. In particular, breed‐associated differences have often been mentioned but never defined precisely. Using a large data set of atopic dogs, we document in this study the clinical presentation of nine often‐affected breeds and demonstrate the existence of substantial differences between the clinical phenotype of each breed and the whole population. Some of the differences may be due to genetic differences while others are most likely to be associated with variations in environmental factors.
Reptiles with dermatological lesions: a retrospective study of 301 cases at two university veterinary teaching hospitals (1992–2008)
Authors: White Stephen D. , Bourdeau Patrick , Bruet Vincent , Kass Philip H. , Tell Lisa , Hawkins Michelle G.
Published: October 2010

This retrospective study reviews the medical records of 301 reptiles with dermatological lesions that were examined at the Veterinary Medical Teaching Hospital, University of California at Davis (VMTH‐UCD) and the Unité de Dermatologie‐Parasitologie‐Mycologie, Ecole Nationale Vétérinaire de Nantes (UDPM‐ENVN) from 1 January 1992 to 1 July 2008. The most common reptile groups differed between the two hospitals, with lizards being the most common at the VMTH‐UCD and chelonians at the UDPM‐ENVN. At the VMTH‐UCD, boa constrictors (Boa constrictor), ball pythons (Python regius) and other Python species were over‐represented, and box turtles (Terrapene carolina) were under‐represented in the dermatological lesion caseload. When institutional data were combined, 47% of all reptiles at both institutions with confirmed or suspected cases of sepsis had petechiae, with the highest association seen in chelonians at 82%. Dependent on institution and reptile group, from 29% to 64% of the cases had underlying husbandry issues. Sixty‐two per cent of all cases were alive at final status. Veterinarians treating reptiles with skin disease should be aware of the following: (i) that boa constrictors and Python species may be predisposed to dermatological lesions; (ii) that client education is important for proper husbandry; and (iii) that there is a possible association between petechiae and sepsis, especially in chelonians. The conjectural association between certain skin lesions and sepsis remains to be confirmed by systematically derived data that demonstrate a causal relationship between the two.
Sutureless thyroidectomy: surgical technique
Authors: O'Neill Christine J. , Chang Lin‐Yun , Suliburk James W. , Sidhu Stan B. , Delbridge Leigh W. , Sywak Mark S.
Published: October 2010

The technique of thyroid surgery has continued to evolve over the last 200 years. Sutureless thyroidectomy is one of the recent developments in surgical technique and is made possible by the use of vessel sealing devices. The most frequently used devices include a bipolar coagulation device (Ligasure Precise) and ultrasonic dissecting shears (Harmonic Scalpel FOCUS). The use of this technology is safe with minimal morbidity and has been shown in numerous studies to significantly decrease operative time. Over 3000 thyroid and parathyroid procedures, both open and minimally invasive, have been performed with vessel sealing devices within the University of Sydney Endocrine Surgery Unit since the technology was introduced in 2005. This manuscript discusses the technique of sutureless thyroidectomy focusing on points of difference in comparison with conventional thyroidectomy using ligatures or clips.
Does surgical decompression in Ludwig's angina decrease hospital length of stay?
Authors: Rowe David Phillip , Ollapallil Jacob
Published: October 2010

Introduction:  Ludwig's angina (LA) is an uncommon and potentially life‐threatening condition of the upper aero‐digestive tract that often requires the coordinated efforts of the surgical, anesthetic and intensive care teams to optimize management. The purpose of the present study was to investigate the documented clinical features and the surgical and airway management of LA at Alice Springs Hospital for the purpose of assessing surgical outcomes with particular reference to length of stay (LOS).

Methods:  Retrospective chart review from January 1998 to January 2008 examined patients admitted with LA at Alice Springs Hospital. Documented clinical features, interventions, and operative findings including floor of mouth swelling, Mallampati score, and airway compromise were collected. Outcomes, with particular respect to LOS, for those who received intravenous (IV) or inhalational induction and those that received awake fibre‐optic intubations were compared.

Results:  Of 30 patients with LA, 28 (93%) were managed with operative drainage with a LOS in the intensive care unit (ICU) of 2 days and a hospital LOS of 5 days. Seven received awake fibre‐optic intubation and 21 had IV or inhalational anesthesia with none requiring tracheotomy. There was no statistical difference in LOS between those patients whose microbiological culture results showed no growth and those whose cultures had positive growth.

Discussion:  Management was generally operative decompression with IV antibiotics. LOS is not affected by the presence or absence of culture positive infection. It is proposed that operative intervention is safe, effective, and is associated with shorter patient stays in the intensive care unit and the hospital overall.
Role of metabolically generated reactive oxygen species for lipotoxicity in pancreatic β‐cells
Authors: Gehrmann W. , Elsner M. , Lenzen S.
Published: October 2010

Chronically elevated concentrations of non‐esterified fatty acids (NEFAs) in type 2 diabetes may be involved in β‐cell dysfunction and apoptosis. It has been shown that long‐chain saturated NEFAs exhibit a strong cytotoxic effect upon insulin‐producing cells, while short‐chain as well as unsaturated NEFAs are well tolerated. Moreover, long‐chain unsaturated NEFAs counteract the toxicity of palmitic acid. Reactive oxygen species (ROS) formation and gene expression analyses together with viability assays in different β‐cell lines showed that the G‐protein‐coupled receptors 40 and 120 do not mediate lipotoxicity. This is independent from the role, which these receptors, specifically GPR40, play in the potentiation of glucose‐induced insulin secretion by saturated and unsaturated long‐chain NEFAs. Long‐chain NEFAs are not only metabolized in the mitochondria but also in peroxisomes. In contrast to mitochondrial β‐oxidation, the acyl‐coenzyme A (CoA) oxidases in the peroxisomes form hydrogen peroxide and not reducing equivalents. As β‐cells almost completely lack catalase, they are exceptionally vulnerable to hydrogen peroxide generated in peroxisomes. ROS generation in the respiratory chain is less important because overexpression of catalase and superoxide dismutase in the mitochondria do not provide protection. Thus, peroxisomally generated hydrogen peroxide is the likely ROS that causes pancreatic β‐cell dysfunction and ultimately β‐cell death.