bluuebunny/crossref_metadata_2025_split huggingface dataset:
Folding of the Pit1 homeodomain near the speed limit
Authors: Banachewicz Wiktor , Johnson Christopher M. , Fersht Alan R. 
Published: December 2010

Current questions in protein folding mechanisms include how fast can a protein fold and are there energy barriers for the folding and unfolding of ultrafast folding proteins? The small 3-helical engrailed homeodomain protein folds in 1.7 μs to form a well-characterized intermediate, which rearranges in 17 μs to native structure. We found that the homologous pituitary-specific transcription factor homeodomain (Pit1) folded in a similar manner, but in two better separated kinetic phases of 2.3 and 46 μs. The greater separation and better fluorescence changes facilitated a detailed kinetic analysis for the ultrafast phase for formation of the intermediate. Its folding rate constant changed little with denaturant concentration or mutation but unfolding was very sensitive to denaturant and energy changes on mutation. The folding rate constant of 3 × 10
5
s
-1
in water decreased with increasing viscosity, and was extrapolated to 4.4 × 10
5
s
-1
at zero viscosity. Thus, the formation of the intermediate was partly rate limited by chain diffusion and partly by an energy barrier to give a very diffuse transition state, which was followed by the formation of structure. Conversely, the unfolding reaction required the near complete disruption of the tertiary structure of the intermediate in a highly cooperative manner, being exquisitely sensitive to individual mutations. The folding is approaching, but has not reached, the downhill-folding scenario of energy landscape theory. Under folding conditions, there is a small energy barrier between the denatured and transition states but a larger barrier between native and transition states.
Cellular cohesiveness in benign and malignant thyroid follicular tumours varies significantly, but the difference is not useful in diagnosis of individual cases
Authors: Rozkos T. , Ryska A. , Cap J. , Sobande F. , Laco J. 
Published: December 2010

T. Rozkos, A. Ryska, J. Cap, F. Sobande and J. Laco  Cellular cohesiveness in benign and malignant thyroid follicular tumours varies significantly, but the difference is not useful in diagnosis of individual casesIntroduction:  The aim of our study was to search for new, readily available and statistically reliable cytological markers for differentiating benign and malignant follicular thyroid neoplasms pre‐operatively.Methods:  Cohesiveness of tumour cells in cytology slides from a series of 58 follicular tumours diagnosed between 1998 and 2004 inclusive was studied, including 48 follicular adenomas, and eight minimally invasive and two widely invasive follicular carcinomas. Photomicrographs of the cytology slides were taken and the digital images were analysed using computer image analysis software. We evaluated the relative proportions of cells arranged in groups of various sizes. The cohesiveness of the cells in cytological smears was then correlated with the immunohistochemical expression of E‐cadherin in corresponding histological slides.Results:  Cases from 15 men (26%) and 43 women (74%) with a mean age of 50 years (range, 19–79) were analysed. In follicular adenomas and carcinomas, respectively, isolated cells were seen in 16.8% and 24.7% (P = 0.028), groups of two to five cells in 9.7% and 11.5% (P = 0.145) and groups of more than five cells in 73.5% and 63.8% (P = 0.041). The mean cell count in groups with more than five cells was 46.5 and 27.0 in adenomas and carcinomas, respectively (P < 0.001). Cell cohesiveness, either as percentage of cells in groups of more than five (R2 = 0.026) or as mean cell count per group of more than five (R2 = 0.005), was not found to be dependent on the expression of E‐cadherin. Using a threshold of 13% isolated tumour cells in cytological smears, follicular adenomas and carcinomas could be distinguished with 90% sensitivity and 41% specificity.Conclusions:  Although we demonstrated a statistically significant difference in cell cohesion between follicular adenomas and carcinomas, these could not be distinguished in the clinical setting by evaluation of the percentage of isolated cells in cytological smears because the specificity was too low. The absence of correlation of cellular cohesiveness with E‐cadherin expression indicates that other factors are probably responsible for the loss of cohesiveness observed in follicular thyroid malignancy.
Discretely calibrated regulatory loops controlled by ppGpp partition gene induction across the ‘feast to famine’ gradient in Escherichia coli
Authors: Traxler Matthew F. , Zacharia Vineetha M. , Marquardt Stafford , Summers Sean M. , Nguyen Huyen‐Tran , Stark S. Elizabeth , Conway Tyrrell 
Published: December 2010

SummaryBacteria comprehensively reorganize their global gene expression when faced with starvation. The alarmone ppGpp facilitates this massive response by co‐ordinating the downregulation of genes of the translation apparatus, and the induction of biosynthetic genes and the general stress response. Such a large reorientation requires the activities of multiple regulators, yet the regulatory network downstream of ppGpp remains poorly defined. Transcription profiling during isoleucine depletion, which leads to gradual starvation (over > 100 min), allowed us to identify genes that required ppGpp, Lrp and RpoS for their induction and to deduce the regulon response times. Although the Lrp and RpoS regulons required ppGpp for their activation, they were not induced simultaneously. The data suggest that metabolic genes, i.e. those of the Lrp regulon, require only a low level of ppGpp for their induction. In contrast, the RpoS regulon was induced only when high levels of ppGpp accumulated. We tested several predictions of a model that explains how bacteria allocate transcriptional resources between metabolism and stress response by discretely tuning two regulatory circuits to different levels of ppGpp. The emergent regulatory structure insures that stress survival circuits are only triggered if homeostatic metabolic networks fail to compensate for environmental deficiencies.
Spo0J regulates the oligomeric state of Soj to trigger its switch from an activator to an inhibitor of DNA replication initiation
Authors: Scholefield Graham , Whiting Rachel , Errington Jeff , Murray Heath 
Published: December 2010

SummaryControl of DNA replication initiation is essential for bacterial cells to co‐ordinate the faithful replication and segregation of their genetic material. The Bacillus subtilis ATPase Soj is a dynamic protein that regulates DNA replication initiation by either inhibiting or activating the DNA replication initiator protein DnaA. Here we report that the key event which switches Soj regulatory activity is a transition in its oligomeric state from a monomer to an ATP‐dependent homodimer capable of DNA binding. We show that the DNA binding activity of the Soj dimer is required both for activation of DNA replication initiation and for interaction with Spo0J. Finally, we demonstrate that Spo0J inhibits Soj dimerization by stimulating Soj ATPase activity. The data provide a molecular explanation for the dichotomous regulatory activities of Soj, as well as assigning unique Soj conformations to distinct cellular localization patterns. We discuss how the regulation of Soj ATPase activity by Spo0J could be utilized to control the initiation of DNA replication during the cell cycle.
Cooperative amino acid changes shift the response of the σ54‐dependent regulator XylR from natural m‐xylene towards xenobiotic 2,4‐dinitrotoluene
Authors: de las Heras Aitor , de Lorenzo Víctor 
Published: December 2010

SummaryXylR is a σ54‐dependent transcriptional factor of Pseudomonas putida that activates the Pu promoter of the TOL plasmid upon binding its natural effector, m‐xylene. The search for mutants of the signal‐sensing module of XylR that respond to the xenobiotic compound 2,4‐dinitrotoluene recurrently yields protein variants with a broad effector range. These mutants had amino acid changes not only in the effector recognition moiety (A module), but also in the inter‐domain B linker of the protein. A random mutagenesis and selection/counterselection setup was adopted to optimize the 2,4‐DNT reaction of XylRv17, one of the best 2,4‐DNT responders and thus recreate how this regulator can adjust its specificity to novel effectors by individual changes on the evolving protein. Site‐specific mutagenesis was then used to decipher the contribution of individual mutations in XylRv17 and in one of the mutants evolved from it (XylR28) to the 2,4‐DNT response. This approach allowed us to capture a new XylR version with novel mutations that fixed the protein in an intermediate stage of the progress from an effector‐promiscuous, pluri‐potent protein type to a more specific form where the natural response to m‐xylene was decreased and the non‐native acquired response to 2,4‐DNT was increased.
Two‐component regulatory systems in Pseudomonas aeruginosa: an intricate network mediating fimbrial and efflux pump gene expression
Authors: Sivaneson Melissa , Mikkelsen Helga , Ventre Isabelle , Bordi Christophe , Filloux Alain 
Published: December 2010

Summary Pseudomonas aeruginosa is responsible for chronic and acute infections in humans. Chronic infections are associated with production of fimbriae and the formation of a biofilm. The two‐component system Roc1 is named after its role in the regulation of cup genes, which encode components of a machinery allowing assembly of fimbriae. A non‐characterized gene cluster, roc2, encodes components homologous to the Roc1 system. We show that cross‐regulation occurs between the Roc1 and Roc2 signalling pathways. We demonstrate that the sensors RocS2 and RocS1 converge on the response regulator RocA1 to control cupC gene expression. This control is independent of the response regulator RocA2. Instead, we show that these sensors act via the RocA2 response regulator to repress the mexAB‐oprM genes. These genes encode a multidrug efflux pump and are upregulated in the rocA2 mutant, which is less susceptible to antibiotics. It has been reported that in cystic fibrosis lungs, in which P. aeruginosa adopts the biofilm lifestyle, most isolates have an inactive MexAB‐OprM pump. The concomitant RocS2‐dependent upregulation of cupC genes (biofilm formation) and downregulation of mexAB‐oprM genes (antibiotic resistance) is in agreement with this observation. It suggests that the Roc systems may sense the environment in the cystic fibrosis lung.
Gene expression profiling of yeasts overexpressing wild type or misfolded Pma1 variants reveals activation of the Hog1 MAPK pathway
Authors: Eraso Pilar , Mazón María J. , Posas Francesc , Portillo Francisco 
Published: December 2010

SummaryDominant negative PMA1 mutants render misfolded proteins that are retained in the endoplasmic reticulum (ER) and slowly degraded by ER‐associated degradation. Accumulation of misfolded proteins in the ER activates an ER‐to‐nucleus signalling pathway termed the unfolded protein response (UPR). We have used a PMA1‐D378T dominant negative mutant to analyse its impact on UPR induction. Our results show that overexpression of the misfolded mutant Pma1 does not lead to activation of the UPR. In addition, in mutants with a constitutively activated UPR the turnover rate of the mutant ATPase is not altered. To determine if the expression of the misfolded mutant protein induces some other kind of response we performed global gene expression analysis experiments in yeasts overexpressing either wild type or dominant lethal PMA1 alleles. The results suggest that the high osmolarity glycerol (Hog1) mitogen‐activated protein kinase (MAPK) pathway is activated by both wild type and mutant ATPases. We show that expression of the PMA1 alleles induces phosphorylation of Hog1 and activation of the Hog1 MAPK cascade. This activation is mediated by the Sln1 branch of the stress‐dependent Hog1 MAPK network. Finally, we show that at least two other plasma membrane proteins are also able to activate the Hog1 MAPK system.
Amelioration of intestinal colitis by macrophage migration inhibitory factor isolated from intestinal parasites through Toll‐like receptor 2
Authors: CHO M. K. , LEE C. H. , YU H. S. 
Published: December 2010

SummaryIn a previous study, we cloned type II MIFs (As‐MIF) from Anisakis simplex 3rd stage larva and expressed a recombinant protein that suppressed allergic airway inflammation via regulatory T (CD4+CD25+Foxp3+ T; Treg)‐cell recruitment. In this study, in an effort to evaluate the function of rAs‐MIF on another immune disease, we induced intestinal inflammation in mice using dextran sodium sulphate (DSS) with or without the application of rAs‐MIF treatment to the mice. As a consequence, weight losses were recovered, and the value of disease activity index (DAI) was reduced by rAs‐MIF treatment during the experimental period. The levels of TGF‐β and IL‐10 in the spleens and mesenteric lymph nodes (MLN) from the rAs‐MIF‐treated mice were higher, but the levels of IFN‐γ, IL‐6 and IL‐13 were lower than those of the mice treated with DSS but not with rAs‐MIF. Additionally, the Treg cells observed were greatly increased in the MLNs of the rAs‐MIF‐treated mice than those of mice not treated with rAs‐MIF. The results of our in vitro experiments showed that the elevated IL‐10 production induced by rAs‐MIF was generated via toll‐like receptor 2. In conclusion, rAs‐MIF appears to ameliorate DSS‐induced colitis and may prove useful as a therapeutic agent for the treatment of intestinal inflammatory disease.
The DUF579 domain containing proteins IRX15 and IRX15‐L affect xylan synthesis in Arabidopsis
Authors: Jensen Jacob K. , Kim Hoon , Cocuron Jean‐Christophe , Orler Robert , Ralph John , Wilkerson Curtis G. 
Published: December 2010

SummaryXylan is the principal hemicellulose in the secondary cell walls of eudicots and in the primary and secondary cell walls of grasses and cereals. The biosynthesis of this important cell wall component has yet to be fully determined although a number of proteins have been shown to be required for xylan synthesis. To discover new genes involved in xylan biosynthesis we explored the psyllium (Plantago ovata Forsk) seed mucilaginous layer through EST profiling. This tissue synthesizes large amounts of a complex heteroxylan over a short period of time. By comparing abundant transcripts in this tissue with abundant transcripts specifically present during secondary cell wall formation in Arabidopsis thaliana, where glucuronoxylan biosynthesis is pronounced, we identified two Arabidopsis genes likely involved in xylan biosynthesis. These genes encode proteins containing a Domain of Unknown Function (DUF) 579 and were designated IRREGULAR XYLEM (IRX) 15 and IRX15‐LIKE (IRX15‐L). We obtained Arabidopsis T‐DNA knockout lines for the two genes and analyzed their lower stems for changes in neutral monosaccharide composition. No changes were observed in each of these mutants, although the irx15 irx15‐L double mutant displayed a moderate reduction in stem xylose. Further characterization of the irx15 irx15‐L mutant revealed irregular secondary cell wall margins in fiber cells and a lower xylan degree of polymerization. Through these studies we conclude that IRX15 and IRX15‐L function in a redundant manner and are involved in xylan biosynthesis.
Automated analysis of hypocotyl growth dynamics during shade avoidance in Arabidopsis
Authors: Cole Benjamin , Kay Steve A. , Chory Joanne 
Published: December 2010

SummaryPlants that are adapted to environments where light is abundant are especially sensitive to competition for light from neighboring vegetation. As a result, these plants initiate a series of changes known as the shade avoidance syndrome, during which plants elongate their stems and petioles at the expense of leaf development. Although the developmental outcomes of exposure to prolonged shade are known, the signaling dynamics during the initial exposure of seedlings to shade is less well studied. Here, we report the development of a new software‐based tool, called HyDE (Hypocotyl Determining Engine) to measure hypocotyl lengths of time‐resolved image stacks of Arabidopsis wild‐type and mutant seedlings. We show that Arabidopsis grows rapidly in response to the shade stimulus, with measurable growth after just 45 min shade exposure. Similar to other mustard species, this growth response occurs in multiple distinct phases, including two phases of rapid growth and one phase of slower growth. Using mutants affected in shade avoidance phenotypes, we demonstrate that most of this early growth requires new auxin biosynthesis via the indole‐3‐pyruvate pathway. When activity of this pathway is reduced, the first phase of elongation growth is absent, and this is correlated with reduced activity of auxin‐regulated genes. Finally, we show that varying shade intensity and duration can affect the shape and magnitude of the growth response, indicating a broad range of the elongation response to shade.
OsbHLH148, a basic helix‐loop‐helix protein, interacts with OsJAZ proteins in a jasmonate signaling pathway leading to drought tolerance in rice
Authors: Seo Ju‐Seok , Joo Joungsu , Kim Min‐Jeong , Kim Yeon‐Ki , Nahm Baek Hie , Song Sang Ik , Cheong Jong‐Joo , Lee Jong Seob , Kim Ju‐Kon , Choi Yang Do 
Published: December 2010

SummaryJasmonates play important roles in development, stress responses and defense in plants. Here, we report the results of a study using a functional genomics approach that identified a rice basic helix‐loop‐helix domain gene, OsbHLH148, that conferred drought tolerance as a component of the jasmonate signaling module in rice. OsbHLH148 transcript levels were rapidly increased by treatment with methyl jasmonate (MeJA) or abscisic acid, and abiotic stresses including dehydration, high salinity, low temperature and wounding. Transgenic over‐expression of OsbHLH148 in rice confers plant tolerance to drought stress. Expression profiling followed by DNA microarray and RNA gel‐blot analyses of transgenic versus wild‐type rice identified genes that are up‐regulated by OsbHLH148 over‐expression. These include OsDREB and OsJAZ genes that are involved in stress responses and the jasmonate signaling pathway, respectively. OsJAZ1, a rice ZIM domain protein, interacted with OsbHLH148 in yeast two‐hybrid and pull‐down assays, but it interacted with the putative OsCOI1 only in the presence of coronatine. Furthermore, the OsJAZ1 protein was degraded by rice and Arabidopsis extracts in the presence of coronatine, and its degradation was inhibited by MG132, a 26S proteasome inhibitor, suggesting 26S proteasome‐mediated degradation of OsJAZ1 via the SCFOsCOI1 complex. The transcription level of OsJAZ1 increased upon exposure of rice to MeJA. These results show that OsJAZ1 could act as a transcriptional regulator of the OsbHLH148‐related jasmonate signaling pathway leading to drought tolerance. Thus, our study suggests that OsbHLH148 acts on an initial response of jasmonate‐regulated gene expression toward drought tolerance, constituting the OsbHLH148–OsJAZ–OsCOI1 signaling module in rice.
Cyanobacterial flavodoxin complements ferredoxin deficiency in knocked‐down transgenic tobacco plants
Authors: Blanco Nicolás E. , Ceccoli Romina D. , Segretin María E. , Poli Hugo O. , Voss Ingo , Melzer Michael , Bravo‐Almonacid Fernando F. , Scheibe Renate , Hajirezaei Mohammad‐Reza , Carrillo Néstor 
Published: December 2010

SummaryFerredoxins are the main electron shuttles in chloroplasts, accepting electrons from photosystem I and delivering them to essential oxido‐reductive pathways in the stroma. Ferredoxin levels decrease under adverse environmental conditions in both plants and photosynthetic micro‐organisms. In cyanobacteria and some algae, this decrease is compensated for by induction of flavodoxin, an isofunctional flavoprotein that can replace ferredoxin in many reactions. Flavodoxin is not present in plants, but tobacco lines expressing a plastid‐targeted cyanobacterial flavodoxin developed increased tolerance to environmental stress. Chloroplast‐located flavodoxin interacts productively with endogenous ferredoxin‐dependent pathways, suggesting that its protective role results from replacement of stress‐labile ferredoxin. We tested this hypothesis by using RNA antisense and interference techniques to decrease ferredoxin levels in transgenic tobacco. Ferredoxin‐deficient lines showed growth arrest, leaf chlorosis and decreased CO2 assimilation. Chlorophyll fluorescence measurements indicated impaired photochemistry, over‐reduction of the photosynthetic electron transport chain and enhanced non‐photochemical quenching. Expression of flavodoxin from the nuclear or plastid genome restored growth, pigment contents and photosynthetic capacity, and relieved the electron pressure on the electron transport chain. Tolerance to oxidative stress also recovered. In the absence of flavodoxin, ferredoxin could not be decreased below 45% of physiological content without fatally compromising plant survival, but in its presence, lines with only 12% remaining ferredoxin could grow autotrophically, with almost wild‐type phenotypes. The results indicate that the stress tolerance conferred by flavodoxin expression in plants stems largely from functional complementation of endogenous ferredoxin by the cyanobacterial flavoprotein.
Trisomy 7 mosaicism prenatally misdiagnosed and maternal uniparental disomy in a child with pigmentary mosaicism and Russell– Silver syndrome
Authors: Petit F , Holder‐Espinasse M , Duban‐Bedu B , Bouquillon S , Boute‐Benejean O , Bazin A , Rouland V , Manouvrier‐Hanu S , Delobel B 
Published: December 2010

Petit F, Holder‐Espinasse M, Duban‐Bedu B, Bouquillon S, Boute‐Benejean O, Bazin A, Rouland V, Manouvrier‐Hanu S, Delobel B. Trisomy 7 mosaicism prenatally misdiagnosed and maternal uniparental disomy in a child with pigmentary mosaicism and Russell–Silver syndrome.Prenatal diagnosis of true mosaic trisomy 7 is rare in amniotic fluid and can be misinterpreted as pseudomosaic. The phenotype is highly variable and may be modified by a maternal uniparental disomy of chromosome 7 leading to mild Russell–Silver syndrome (RSS). We report here the third postnatal case of mosaic trisomy 7 with maternal uniparental disomy of chromosome 7 in a boy presenting a mild RSS. Fetal karyotype performed in amniocentesis for intrauterine growth retardation was considered normal. Mosaic trisomy 7 was diagnosed after birth, on fibroblasts karyotype performed for blaschkolinear pigmentary skin anomalies and failure to thrive. Maternal uniparental disomy of chromosome 7 was observed in blood sample. Retrospectively, trisomic 7 cells were identified in one prenatal long‐term flask culture revealing a prenatal diagnosis failure. This report emphasizes the difficulty of assessing fetal mosaicism and distinguishing it from pseudomosaicism in cultured amniocytes. It is important to search for uniparental disomy as an indirect clue of trisomy 7 mosaicism and a major prognosis element. Although there are only few prenatal informative cases, detection of trisomy 7 in amniocentesis appears to be associated with a relatively good outcome when maternal uniparental disomy has been ruled out.