bluuebunny/crossref_metadata_2025 huggingface dataset:
Reduced positive affect (anhedonia) predicts major clinical events following implantation of coronary‐artery stents
Authors: Denollet J. , Pedersen S. S. , Daemen J. , De Jaegere P. , Serruys P. W. , Van Domburg R. T.
Published: August 2010

Objective.  Emotional distress has been related to clinical events in patients with coronary artery disease, but the influence of positive affect (i.e. mood states such as activity, joy and cheerfulness) has received little attention. Therefore, we wanted to investigate the role of positive affect on clinical outcome after percutaneous coronary intervention (PCI) with stent implantation in these patients.

Design.  Prospective follow‐up study. At baseline, patients from the Rapamycin‐Eluting Stent Evaluated At Rotterdam Cardiology Hospital (RESEARCH) registry completed measures of positive affect, depression and anxiety post‐PCI. Patients with reduced positive affect scored 1 SD below the mean score.

Setting.  University Hospital; Thoraxcenter of the Department of Cardiology.

Subjects.  874 patients (72% men; 62.2 ± 10.9 years) from the RESEARCH registry.

Main outcome measure.  Death or myocardial infarction (MI) 2 years post‐PCI.

Results.  At follow‐up, there were 52 clinical events (deaths n = 27, MIs n = 25). Reduced positive affect and depression/anxiety were associated with poor prognosis, but reduced positive affect was the only independent predictor of events. The incidence of death/MI in adequate versus reduced positive affect patients was 4% (29/663) vs. 11% (23/211); HR = 2.55 (95% CI 1.46–4.34, P = 0.001), adjusting for clinical variables. Reduced positive affect and diabetes were independent prognostic factors, and patients with one (HR = 2.84, 95% CI 1.58–5.10) or both (HR = 5.61, 95% CI 2.25–13.99) of these factors had a higher risk when compared with nondiabetic patients with adequate positive affect, P ≤ 0.003.

Conclusions.  Reduced positive affect independently predicted death/MI following stent implantation, and improved risk stratification above and beyond diabetes.
Two SNPs in the promoter region of the CTLA‐4 gene affect binding of transcription factors and are associated with human myasthenia gravis
Authors: Wang XB. , Pirskanen R. , Giscombe R. , Lefvert A. K.
Published: August 2010

Objectives.  The molecular mechanisms underlying the regulation of the CD152 (CTLA‐4) gene are largely unknown. Two single nucleotide polymorphisms (SNPs) located in the promoter region are suspected to contribute to the pathogenesis of myasthenia gravis (MG) through regulation of gene expression.

Setting, Subjects and Design.  One hundred and sixty‐five unrelated Swedish‐Caucasian patients with MG (103 females and 62 males, age 17 to 92 years) and 148 ethnically matched healthy individuals were studied. Gene typing of two SNPs (T/C−1772 and A/G−1661) and quantification of soluble CD152 were performed in the patients. Besides the association studies, the function of these two SNPs is characterized.

Results.  We present new genetic associations of two SNPs in the CD152 gene with human MG. These SNPs located in the promoter region are involved in transcriptional binding activity for Nuclear Factor I (NF‐1) and c/EBPbeta, as demonstrated using chromatin immunoprecipitation and electromobility shift assay. MG patients with the T/C−1772 polymorphism have elevated levels of sCD152 in sera.

Conclusions.  The two SNPs in the promoter region are associated with MG and might cause abnormal alternative splicing and affect the expression of CD152, thereby contributing to the pathogenesis of MG.
Antitissue transglutaminase antibodies in acute coronary syndrome: an alert signal of myocardial tissue lesion?
Authors: Di Tola M. , Barillà F. , Trappolini M. , Palumbo H. F. , Gaudio C. , Picarelli A.
Published: August 2010

Background and aim.  Antitransglutaminase, previously considered identical to antiendomysial in coeliac sprue (CS), have been reported in end‐stage heart failure. To clarify the above‐mentioned data, we evaluated these antibodies in a cohort of cardiological patients with respect to troponin I, creatine kinase (CK), MB fraction creatine kinase (CK‐MB mass) and myoglobin.

Methods.  Forty‐one patients with acute coronary syndrome (ACS), 39 with dilated cardiomyopathy (DCM), 45 with CS and 58 blood donors (BDs) were evaluated. Antitransglutaminase and antiendomysial antibodies were tested in serum of the patients being studied.

Results.  High‐positive antitransglutaminase values were found in CS, whilst low‐positive values were also found in ACS and DCM. In patients at the second ACS, antibody levels were higher than in those at the first cardiac event. In patients with infarct Q, antitransglutaminase were higher than those in infarct non‐Q, in which antibody levels were higher than those in unstable angina. A correlation between antitransglutaminase and troponin I, CK, CK‐MB mass and myoglobin was found. Finally, antibody levels rose to reach a peak at 30 days from the cardiac event, whereas after further 150 days, approached the values of BDs. Antiendomysial were detectable only in CS.

Conclusions.  Data highlight that antitransglutaminase can occur in cardiological patients, and that these antibodies are related to the severity/extent of the myocardial tissue lesion. This feature suggests a loss of specificity for antitransglutaminase in CS. Furthermore, the possibility of employing these antibodies in the long‐term follow‐up of ACS, could become an object of interesting discussion.
Understanding ageing from an evolutionary perspective
Authors: Kirkwood T. B. L.
Published: August 2010

There is clear heritability of human longevity. However, the genetics of ageing is likely to be complex. Evolution theory tells us not to expect genes that have been selected to promote ageing. Ageing is not programmed but results from accumulation of somatic damage, owing to limited investments in maintenance and repair. Genes controlling the levels of activities, such as DNA repair and antioxidant defence, thus regulate longevity. In addition, there may be contributions either from late‐acting deleterious genes that escape the force of natural selection or that trade benefit at an early age against harm at older ages. In some species, there is evidence that genes have evolved to detect and respond to changes in the environment, e.g. food supply. Evolutionary understanding can also help to understand important features of the human life history such as menopause.
Genomic studies in ageing research: the need to integrate genetic and gene expression approaches
Authors: Passtoors W. M. , Beekman M. , Gunn D. , Boer J. M. , Heijmans B. T. , Westendorp R. G. J. , Zwaan B. J. , Slagboom P. E.
Published: August 2010

Genome‐wide and hypothesis‐based approaches to the study of ageing and longevity have been dominated by genetic investigations. To identify essential mechanisms of a complex trait such as ageing in higher species, a holistic understanding of interacting pathways is required. More information on such interactions is expected to be obtained from global gene expression analysis if combined with genetic studies. Genetic sequence variation often provides a functional gene marker for the trait, whereas a gene expression profile may provide a quantitative biomarker representing complex cellular pathway interactions contributing to the trait. Thus far, gene expression studies have associated multiple pathways to ageing including mitochondrial electron transport and the oxidative stress response. However, most of the studies are underpowered to detect small age‐changes. A systematic survey of gene expression changes as a function of age in human individuals and animal models is lacking. Well designed gene expression studies, especially at the level of biological processes, will provide hypotheses on gene–environmental interactions determining biological ageing rate. Cross‐sectional studies monitoring the profile as a chronological marker of ageing must be integrated with prospective studies indicating which profiles represent biomarkers preceding and predicting physiological decline and mortality. New study designs such as the Leiden Longevity Study, including two generations of subjects from longevity families, aim to achieve these combined approaches.
The lactose operon‐controlling elements: a complex paradigm
Authors: Reznikoff William S.
Published: August 2010

The lactose‐controlling elements have been considered to be the simple paradigm of a cis‐acting genetic regulatory system, containing a promoter whose activity is modulated by an operator and a catabolite gene activator protein (CAP)‐binding site. The reality is considerably more complex. We now know that transcription is negatively regulated as a result of the repressor binding to three binding sites: the operator, a secondary repressor‐binding site within the lacZ gene and a tertiary repressor‐binding site upstream near lacl. In addition to the promoter, the lac‐controlling elements contain five promoter‐like elements. The physiological role, if any, of these promoter‐like elements is not clear, although three of them can be activated by single base pair changes to give high levels of in vivo expression. Finally, the positive activator protein CAP has been found to bind to a secondary site which is coincident with the operator. No role has been identified for this secondary CAP‐DNA complex.
Genetics of resistance to third‐generation cephalosporins in clinical isolates of Streptococcus pneumoniae
Authors: Muñoz Rosario , Dowson Christopher G. , Daniels Maggie , Coffey Tracey J. , Martin Christiane , Hakenbeck Regine , Spratt Brian G.
Published: August 2010

Resistance to third‐generation cephalosporins in a clinical isolate of Streptococcus pneumoniae was shown to be due to the production of altered forms of penicillin‐binding proteins (PBPs) 2X and 1A. The cloned PBP2X gene from the resistant strain was able to transform a susceptible strain to an intermediate level of resistance. The resulting transformant could be transformed to the full level of resistance of the clinical isolate using the cloned PBP1A gene from the latter strain. Chromosomal DNA from the resistant strain (and from other resistant strains) could readily transform a susceptible strain to the full level of resistance to third‐generation cephalosporins (>250‐fold for cefotaxime; >100‐fold for ceftriaxone) in a single step (transformation frequency of about 10‐5). The resistant transformants obtained with chromosomal DNA were shown by gene fingerprinting to have gained both the PBP1A and PBP2X genes from the DNA donor.
Fur regulation in Yersinia species
Authors: Staggs Teanna M. , Perry Robert D.
Published: August 2010

The effects of iron have been linked with several phenomena including regulation of membrane proteins; however, the mechanism of iron regulation is not well characterized in Yersinia pestis. It is well known that in Escherichia coli, the fur gene product mediates negative transcriptional regulation of several genes in response to iron. We have cloned a Y. pestis fur gene which is highly homologous to the E coli fur regulatory gene. The sequence of the Y. pestis fur gene exhibits 75% homology to the E. coli gene at the nucleotide level, and 84% homology at the predicted amino acid level. The Y. pestis fur gene is transcribed as a single gene message of approximately 0.5 kb which encodes an approximately 16 kDa protein when expressed in E coli minicells. A Yersinia enterocolitica fur mutant exhibits hypersensitivity to the Y. pestis bacteriocin, pesticin; the cloned Y. pestis fur gene restores wild‐type levels of pesticin sensitivity. Furthermore, iron regulation of at least five surface proteins in this Y. enterocolitica fur mutant is restored by transcomplementation with the Y. pestis fur gene. These data indicate that Y. pestis and Y. enterocolitica possess homologous Fur systems which regulate expression of proteins in response to iron availability.
RecA protein of Escherichia coli and chromosome partitioning
Authors: Zyskind Judith W. , Svitil Amy L. , Stine W. Blaine , Biery Matthew C. , Smith Douglas W.
Published: August 2010

Escherichia coli cells deficient in RecA protein frequently contain an abnormal number of chromosomes after completion of ongoing rounds of DNA replication. This suggests that RecA protein may be required for correct timing of initiation of DNA replication; however, we show here that initiation of DNA replication is properly timed in recA mutants. We also find that more than 10% of recA mutant cells contain no DNA. These anucleate cells appear to arise from partitioning of all the DNA into one daughter cell and no DNA into the other daughter cell. Based on these and previously published results, we propose that RecA protein is required for equal partitioning of chromosomes into the two daughter cells.
Light‐induced carotenogenesis in Myxococcus xanthus: light‐dependent membrane sequestration of ECF sigma factor CarQ by anti‐sigma factor CarR
Authors: Gorham Hazel C. , McGowan Simon J. , Robson Paul R. H. , Hodgson David A.
Published: August 2010

Light‐induced carotenogenesis in Myxococcus xanthus is under the control of the carQRS operon. CarQ, a proposed extracytoplasmic (ECF) RNA polymerase sigma factor, is required for expression of the operon and the carC gene that encodes phytoene dehydrogenase. CarR, an inner membrane protein in Escherichia coli, is essential for carQRS promoter inactivation in the dark. CarS is required for the light‐dependent expression of the promoter of the carB gene cluster that encodes the rest of the structural genes for carotenogenesis. Regulation of carQRS is dependent on the stoichiometry of CarQ and CarR. Increasing the copy number of carQ over carR led to constitutive carotenogenesis, as did loss of translational coupling between carQ and carR. The severity of the constitutive phenotype depended on the distance between the uncoupled genes. When expressed in M. xanthus, a CarR:β‐galactosidase fusion protein disappeared in the light. We propose that anti‐sigma factor CarR sequesters CarQ to the membrane in the dark, but, in the light, loss of CarR leads to release of the sigma factor.
Novel mitochondrion‐related organelles in the anaerobic amoeba Mastigamoeba balamuthi
Authors: Gill Erin E. , Diaz‐Triviño Sara , Barberà Maria José , Silberman Jeffrey D. , Stechmann Alexandra , Gaston Daniel , Tamas Ivica , Roger Andrew J.
Published: August 2010

Unicellular eukaryotes that lack mitochondria typically contain related organelles such as hydrogenosomes or mitosomes. To characterize the evolutionary diversity of these organelles, we conducted an expressed sequence tag (EST) survey on the free‐living amoeba Mastigamoeba balamuthi, a relative of the human parasite Entamoeba histolytica. From 19 182 ESTs, we identified 21 putative mitochondrial proteins implicated in protein import, amino acid interconversion and carbohydrate metabolism, two components of the iron–sulphur cluster (Fe‐S) assembly apparatus as well as two enzymes characteristic of hydrogenosomes. By immunofluorescence microscopy and subcellular fractionation, we show that mitochondrial chaperonin 60 is targeted to small abundant organelles within Mastigamoeba. In transmission electron micrographs, we identified double‐membraned compartments that likely correspond to these mitochondrion‐derived organelles, The predicted organellar proteome of the Mastigamoeba organelle indicates a unique spectrum of functions that collectively have never been observed in mitochondrion‐related organelles. However, like Entamoeba, the Fe‐S cluster assembly proteins in Mastigamoeba were acquired by lateral gene transfer from ε‐proteobacteria and do not possess obvious organellar targeting peptides. These data indicate that the loss of classical aerobic mitochondrial functions and acquisition of anaerobic enzymes and Fe‐S cluster assembly proteins occurred in a free‐living member of the eukaryote super‐kingdom Amoebozoa.
The crystal structure of staphylococcal superantigen‐like protein 11 in complex with sialyl Lewis X reveals the mechanism for cell binding and immune inhibition
Authors: Chung Matthew C. , Wines Bruce D. , Baker Heather , Langley Ries J. , Baker Edward N. , Fraser John D.
Published: August 2010

Staphylococcus aureus is a major pathogen that produces a family of 14 staphylococcal superantigen‐like (SSL) proteins, which are structurally similar to superantigens but do not stimulate T cells. SSL11 is one member of the family that is found in all staphylococcal strains. Recombinant SSL11 bound to granulocytes and monocytes through a sialic acid‐dependent mechanism and was rapidly internalized. SSL11 also bound to sialic acid‐containing glycoproteins, such as the Fc receptor for IgA (FcαRI) and P‐selectin glycoprotein ligand‐1 (PSGL‐1), and inhibited neutrophil attachment to a P‐selectin‐coated surface. Biosensor analysis of two SSL11 alleles binding to sialyl Lewis X [sLex– Neu5Acα2‐3Galβ1‐4(Fuc1‐3)GlcNAc] coupled to bovine serum albumin gave dissociation constants of 0.7 and 7 μm respectively. Binding of SSL11 to a glycan array revealed specificity for glycans containing the trisaccharide sialyllactosamine (sLacNac – Neu5Acα2‐3Galβ1‐4GlcNAc). A 1.6 Å resolution crystal structure of SSL11 complexed with sLex revealed a discrete binding site in the C‐terminal β‐grasp domain, with predominant interactions with the sialic acid and galactose residues. A single amino acid mutation in the carbohydrate binding site abolished all SSL11 binding. Thus, SSL11 is a staphylococcal protein that targets myeloid cells by binding sialyllactosamine‐containing glycoproteins.
A gene cluster that regulates both heterocyst differentiation and pattern formation in Anabaena sp. strain PCC 7120
Authors: Zhang Wei , Du Ye , Khudyakov Ivan , Fan Qing , Gao Hong , Ning Degang , Wolk C. Peter , Xu Xudong
Published: August 2010

Wild‐type Anabaena sp. strain PCC 7120, a filamentous nitrogen‐fixing cyanobacterium, produces single heterocysts at semi‐regular intervals. asr0100 (patU5) and alr0101 (patU3) are homologous to the 5′ and 3′ portions of patU of Nostoc punctiforme. alr0099 (hetZ) overlaps the 5′ end of patU5. hetZ, patU5 and patU3 were all upregulated, or expressed specifically, in proheterocysts and heterocysts. Mutants of hetZ showed delayed or no heterocyst differentiation. In contrast, a patU3 mutation produced a multiple contiguous heterocyst (Mch) phenotype and restored the formation of otherwise lost intercalary heterocysts in a patA background. Decreasing the expression of patU3 greatly increased the frequency of heterocysts in a mini‐patS strain. Two promoter regions and two principal, corresponding transcripts were detected in the hetZ‐patU5‐patU3 region. Transcription of hetZ was upregulated in a hetZ mutant and downregulated in a patU3 mutant. When mutants hetZ::C.K2 and hetZ::Tn5‐1087b were nitrogen‐deprived, PhetC‐gfp was very weakly expressed, and in hetZ::Tn5‐1087b, PhetR‐gfp was relatively strongly expressed in cells that had neither a regular pattern nor altered morphology. We conclude that the hetZ‐patU5‐patU3 cluster plays an important role in co‐ordination of heterocyst differentiation and pattern formation. The presence of homologous clusters in filamentous genera without heterocysts is suggestive of a more general role.
Beta toxin is essential for the intestinal virulence of Clostridium perfringens type C disease isolate CN3685 in a rabbit ileal loop model
Authors: Sayeed Sameera , Uzal Francisco A. , Fisher Derek J. , Saputo Juliann , Vidal Jorge E. , Chen Yue , Gupta Phalguni , Rood Julian I. , McClane Bruce A.
Published: August 2010

Clostridium perfringens type C isolates, which cause enteritis necroticans in humans and enteritis and enterotoxaemias of domestic animals, typically produce (at minimum) beta toxin (CPB), alpha toxin (CPA) and perfringolysin O (PFO) during log‐phase growth. To assist development of improved vaccines and therapeutics, we evaluated the contribution of these three toxins to the intestinal virulence of type C disease isolate CN3685. Similar to natural type C infection, log‐phase vegetative cultures of wild‐type CN3685 caused haemorrhagic necrotizing enteritis in rabbit ileal loops. When isogenic toxin null mutants were prepared using TargeTron® technology, even a double cpa/pfoA null mutant of CN3685 remained virulent in ileal loops. However, two independent cpb null mutants were completely attenuated for virulence in this animal model. Complementation of a cpb mutant restored its CPB production and intestinal virulence. Additionally, pre‐incubation of wild‐type CN3685 with a CPB‐neutralizing monoclonal antibody rendered the strain avirulent for causing intestinal pathology. Finally, highly purified CPB reproduced the intestinal damage of wild‐type CN3685 and that damage was prevented by pre‐incubating purified CPB with a CPB monoclonal antibody. These results indicate that CPB is both required and sufficient for CN3685‐induced enteric pathology, supporting a key role for this toxin in type C intestinal pathogenesis.
Identification and functional analysis of novel (p)ppGpp synthetase genes in Bacillus subtilis
Authors: Nanamiya Hideaki , Kasai Koji , Nozawa Akira , Yun Choong‐Soo , Narisawa Takakuni , Murakami Kana , Natori Yousuke , Kawamura Fujio , Tozawa Yuzuru
Published: August 2010

Bacterial alarmone (p)ppGpp, is a global regulator responsible for the stringent control. Two homologous (p)ppGpp synthetases, RelA and SpoT, have been identified and characterized in Escherichia coli, whereas Gram‐positive bacteria such as Bacillus subtilis have been thought to possess only a single RelA‐SpoT enzyme. We have now identified two genes, yjbM and ywaC, in B. subtilis that encode a novel type of alarmone synthetase. The predicted products of these genes are relatively small proteins (∼25 kDa) that correspond to the (p)ppGpp synthetase domain of RelA‐SpoT family members. A database survey revealed that genes homologous to yjbM and ywaC are conserved in certain bacteria belonging to Firmicutes or Actinobacteria phyla but not in other phyla such as Proteobacteria. We designated the proteins as small alarmone synthetases (SASs) to distinguish them from RelA‐SpoT proteins. The (p)ppGpp synthetase function of YjbM and YwaC was confirmed by genetic complementation analysis and by in vitro assay of enzyme activity. Molecular genetic analysis also revealed that ywaC is induced by alkaline shock, resulting in the transient accumulation of ppGpp. The SAS proteins thus likely function in the biosynthesis of alarmone with a mode of action distinct from that of RelA‐SpoT homologues.
Mapping a common interaction site used by Plasmodium falciparum Duffy binding‐like domains to bind diverse host receptors
Authors: Howell Dasein P.‐G. , Levin Emily A. , Springer Amy L. , Kraemer Susan M. , Phippard David J. , Schief William R. , Smith Joseph D.
Published: August 2010

The Duffy binding‐like (DBL) domain is a key adhesive module in Plasmodium falciparum, present in both erythrocyte invasion ligands (EBLs) and the large and diverse P. falciparum erythrocyte membrane protein 1 (PfEMP1) family of cytoadherence receptors. DBL domains bind a variety of different host receptors, including intercellular adhesion molecule 1 (ICAM‐1), a receptor interaction that may have a role in infected erythrocyte binding to cerebral blood vessels and cerebral malaria. In this study, we expressed the nearly full complement of DBLβ‐C2 domains from the IT4/25/5 (IT4) parasite isolate and showed that ICAM‐1‐binding domains (DBLβ‐C2ICAM‐1) were confined to group B and group C PfEMP1 proteins and were not present in group A, suggesting that ICAM‐1 selection pressure differs between PfEMP1 groups. To further dissect the molecular determinants of binding, we modelled a DBLβ‐C2ICAM‐1 domain on a solved DBL structure and created alanine substitution mutants in two DBLβ‐C2ICAM‐1 domains. This analysis indicates that the DBLβ‐C2::ICAM‐1 interaction maps to the equivalent glycan binding region of EBLs, and suggests a general model for how DBL domains evolve under dual selection for host receptor binding and immune evasion.
The Bacteroides fragilis transcriptome response to oxygen and H2O2: the role of OxyR and its effect on survival and virulence
Authors: Sund Christian J. , Rocha Edson R. , Tzinabos Arthur O. , Wells W. Greg , Gee Jason M. , Reott Michael A. , O'Rourke Dorcas P. , Smith C. Jeffrey
Published: August 2010

The intestinal anaerobic symbiont, Bacteroides fragilis, is highly aerotolerant and resistant to H2O2. Analysis of the transcriptome showed that expression of 45% of the genome was significantly affected by oxidative stress. The gene expression patterns suggested that exposure to oxidative stress induced an acute response to rapidly minimize the immediate effects of reactive oxygen species, then upon extended exposure a broad metabolic response was induced. This metabolic response induced genes encoding enzymes that can supply reducing power for detoxification and restore energy‐generating capacity. An integral aspect of the metabolic response was downregulation of genes related to translation and biosynthesis which correlated with decreased growth and entry into a stationary phase‐like growth state. Examination of oxyR mutants showed that they were impaired for the acute response and they induced the expanded metabolic response with only minimal exposure to stress. The oxyR mutants were more sensitive to oxidants in vitro and in vivo they were attenuated in an intra‐abdominal abscess infection model. Aerotolerance and resistance to oxidative stress are physiological adaptations of B. fragilis to its environment that enhance survival in extra‐intestinal sites and promote opportunistic infections.
Bistability and biofilm formation in Bacillus subtilis
Authors: Chai Yunrong , Chu Frances , Kolter Roberto , Losick Richard
Published: August 2010

Biofilms of Bacillus subtilis consist of long chains of cells that are held together in bundles by an extracellular matrix of exopolysaccharide and the protein TasA. The exopolysaccharide is produced by enzymes encoded by the epsA‐O operon and the gene encoding TasA is located in the yqxM‐sipW‐tasA operon. Both operons are under the control of the repressor SinR. Derepression is mediated by the antirepressor SinI, which binds to SinR with a 1:1 stoichiometry. Paradoxically, in medium promoting derepression of the matrix operons, the overall concentration of SinR in the culture greatly exceeded that of SinI. We show that under biofilm‐promoting conditions sinI, which is under the control of the response regulator Spo0A, was expressed only in a small subpopulation of cells, whereas sinR was expressed in almost all cells. Activation of Spo0A is known to be subject to a bistable switch, and we infer that SinI reaches levels sufficient to trigger matrix production only in the subpopulation of cells in which Spo0A is active. Additionally, evidence suggests that sinI is expressed at intermediate, but not low or high, levels of Spo0A activity, which may explain why certain nutritional conditions are more effective in promoting biofilm formation than others.
Acquisition of a bacterial RumA‐type tRNA(uracil‐54, C5)‐methyltransferase by Archaea through an ancient horizontal gene transfer
Authors: Urbonavičius Jaunius , Auxilien Sylvie , Walbott Hélène , Trachana Kalliopi , Golinelli‐Pimpaneau Béatrice , Brochier‐Armanet Céline , Grosjean Henri
Published: August 2010

The Pyrococcus abyssi genome displays two genes possibly coding for S‐adenosyl‐l‐methionine‐dependent RNA(uracil, C5)‐methyltransferases (PAB0719 and PAB0760). Their amino acid sequences are more closely related to Escherichia coli RumA catalysing the formation of 5‐methyluridine (m5U)‐1939 in 23S rRNA than to E. coli TrmA (tRNA methyltransferase
A) methylating uridine‐54 in tRNA. Comparative genomic and phylogenetic analyses show that homologues of PAB0719 and PAB0760 occur only in a few Archaea, these genes having been acquired via a single horizontal gene transfer from a bacterial donor to the common ancestor of Thermococcales and Nanoarchaea. This transfer event was followed by a duplication event in Thermococcales leading to two closely related genes. None of the gene products of the two P. abyssi paralogues catalyses in vitro the formation of m5U in a P. abyssi rRNA fragment homologous to the bacterial RumA substrate. Instead, PAB0719 enzyme (renamed PabTrmU54) displays an identical specificity to TrmA, as it catalyses the in vitro formation of m5U‐54 in tRNA. Thus, during evolution, at least one of the two P. abyssi RumA‐type enzymes has changed of target specificity. This functional shift probably occurred in an ancestor of all Thermococcales. This study also provides new evidence in favour of a close relationship between Thermococcales and Nanoarchaea.
Binding of Dr adhesins of Escherichia coli to carcinoembryonic antigen triggers receptor dissociation
Authors: Korotkova Natalia , Yang Yi , Le Trong Isolde , Cota Ernesto , Demeler Borries , Marchant Jan , Thomas Wendy E. , Stenkamp Ronald E. , Moseley Steve L. , Matthews Steve
Published: August 2010

Carcinoembryonic antigen (CEA)‐related cell adhesion molecules (CEACAMs) are host receptors for the Dr family of adhesins of Escherichia coli. To define the mechanism for binding of Dr adhesins to CEACAM receptors, we carried out structural studies on the N‐terminal domain of CEA and its complex with the Dr adhesin. The crystal structure of CEA reveals a dimer similar to other dimers formed by receptors with IgV‐like domains. The structure of the CEA/Dr adhesin complex is proposed based on NMR spectroscopy and mutagenesis data in combination with biochemical characterization. The Dr adhesin/CEA interface overlaps appreciably with the region responsible for CEA dimerization. Binding kinetics, mutational analysis and spectroscopic examination of CEA dimers suggest that Dr adhesins can dissociate CEA dimers prior to the binding of monomeric forms. Our conclusions include a plausible mechanism for how E. coli, and perhaps other bacterial and viral pathogens, exploit CEACAMs. The present structure of the complex provides a powerful tool for the design of novel inhibitory strategies to treat E. coli infections.
Genomic rearrangements and transcriptional analysis of the spliced leader‐associated retrotransposon in RNA interference‐deficient Trypanosoma brucei
Authors: Patrick Kristin L. , Luz Paula M. , Ruan Jia‐peng , Shi Huafang , Ullu Elisabetta , Tschudi Christian
Published: August 2010

The Trypanosoma brucei genome is colonized by the site‐specific non‐LTR retrotransposon SLACS, or spliced leader‐associated conserved sequence, which integrates exclusively into the spliced leader (SL) RNA genes. Although there is evidence that the RNA interference (RNAi) machinery regulates SLACS transcript levels, we do not know whether RNAi deficiency affects the genomic stability of SLACS, nor do we understand the mechanism of SLACS transcription. Here, we report that prolonged culturing of RNAi‐deficient T. brucei cells, but not wild‐type cells, results in genomic rearrangements of SLACS. Furthermore, two populations of SLACS transcripts persist in RNAi‐deficient cells: a full‐length transcript of approximately 7 kb and a heterogeneous population of small SLACS transcripts ranging in size from 450 to 550 nt. We provide evidence that SLACS transcription initiates at the +1 of the interrupted SL RNA gene and proceeds into the 5′ UTR and open reading frame 1 (ORF1). This transcription is carried out by an RNA polymerase with α‐amanitin sensitivity reminiscent of SL RNA synthesis and is dependent on the SL RNA promoter. Additionally, we show that both sense and antisense small SLACS transcripts originate from ORF1 and that they are associated with proteins in vivo. We speculate that the small SLACS transcripts serve as substrates for the production of siRNAs to regulate SLACS expression.
Acetylation of histone H4K4 is cell cycle regulated and mediated by HAT3 in Trypanosoma brucei
Authors: Siegel T. Nicolai , Kawahara Taemi , DeGrasse Jeffrey A. , Janzen Christian J. , Horn David , Cross George A. M.
Published: August 2010

Post‐translational histone modifications have been studied intensively in several eukaryotes. It has been proposed that these modifications constitute a ‘histone code’ that specifies epigenetic information for transcription regulation. With a limited number of histone‐modifying enzymes, implying less redundancy, Trypanosoma brucei represents an excellent system in which to investigate the function of individual histone modifications and histone‐modifying enzymes. In this study, we characterized the acetylation of lysine 4 of histone H4 (H4K4), the most abundant acetylation site in T. brucei histones. Because of the large sequence divergence of T. brucei histones, we generated highly specific antibodies to acetylated and unmodified H4K4. Immunofluorescence microscopy and Western blots with sorted cells revealed a strong enrichment of unmodified H4K4 in S phase and suggested a G1/G0‐specific masking of the site, owing to non‐covalently binding factors. Finally, we showed that histone acetyltransferase 3 (HAT3) is responsible for H4K4 acetylation and that treatment of cells with the protein synthesis inhibitor cycloheximide led to an almost instantaneous loss of unmodified H4K4 sites. As HAT3 is located inside the nucleus, our findings suggest that newly synthesized histone H4 with an unmodified K4 is imported rapidly into the nucleus, where it is acetylated, possibly irreversibly.
A small protein from the bop–brp intergenic region of Halobacterium salinarum contains a zinc finger motif and regulates bop and crtB1 transcription
Authors: Tarasov Valery Y. , Besir Hüseyin , Schwaiger Rita , Klee Kathrin , Furtwängler Katarina , Pfeiffer Friedhelm , Oesterhelt Dieter
Published: August 2010

Bacteriorhodopsin, the photosynthetic protein of Halobacterium salinarum, is optimally expressed under anaerobic growth conditions. We identified Brz (OE3104F, bacteriorhodopsin‐regulating zinc finger protein), a new regulator of the bop gene. It is a small protein with a zinc finger motif, encoded directly upstream of the bop gene in the same orientation. Deletion of the brz gene caused a large decrease of bop mRNA levels as shown by Northern blot and microarray analysis. A similar effect was obtained by site‐directed mutagenesis of Cys and His residues in the zinc finger motif, indicating the importance of this motif for the function of the protein. In silico analysis of the genomes from H. salinarum and other archaea revealed a large family of similar small zinc finger motif proteins, some of which may also be involved in transcription regulation of their adjacent genes.
Molecular basis of gene regulation by the THI‐box riboswitch
Authors: Ontiveros‐Palacios Nancy , Smith Angela M. , Grundy Frank J. , Soberon Mario , Henkin Tina M. , Miranda‐Ríos Juan
Published: August 2010

Riboswitches are genetic control elements located mainly within the 5′ untranslated regions of messenger RNAs. These RNA elements undergo conformational changes that modulate gene expression upon binding of regulatory signals including vitamins, amino acids, nucleobases and uncharged tRNA. The thiamin pyrophosphate (TPP)‐binding riboswitch (THI‐box) is found in all three kingdoms of life and can regulate gene expression at the levels of premature termination of transcription, initiation of translation and mRNA splicing. The THI‐box is composed of two parallel stacked helices bound by another helix in a three‐way junction. We performed an in vivo expression analysis of mutants with substitutions in conserved bases located at the interior and terminal loops of the Escherichia coli thiM THI‐box, which is translationally regulated, and observed two different phenotypic classes. One class exhibited high expression during growth in the presence or absence of thiamin, while the second class exhibited low expression regardless of the presence of thiamin. Accessibility of the Shine‐Dalgarno region of the RNA following the addition of TPP was monitored by means of an oligonucleotide‐dependent RNase H cleavage assay, and binding of 30S ribosomal subunits. These studies showed that high‐ and low‐expression mutant RNAs are locked in the non‐repressive and repressive conformations respectively.
Post‐transcriptional cross‐talk between pro‐ and anti‐colonization pili biosynthesis systems in Vibrio cholerae
Authors: Hsiao Ansel , Toscano Kristin , Zhu Jun
Published: August 2010

The pathogen Vibrio cholerae modulates the expression of many genes in order to transition from its environmental reservoir to its niche in the human host. Among these are genes encoding two related Type IV pili, the mannose‐sensitive haemagglutinin (MSHA) pilus, which aids V. cholerae persistence in aquatic environments but causes clearance of bacteria by host immune defences, and the toxin co‐regulated pilus (TCP) required for colonization. These antagonistic effects are resolved transcriptionally by the regulator ToxT, which represses msh genes while activating tcp genes during infection. We show that these two pili systems are also intertwined post‐transcriptionally through the ToxT‐regulated pre‐pilin peptidase TcpJ. We found that the major MSHA pilin, MshA, was degraded in V. cholerae in a TcpJ‐dependent fashion. In a heterologous Escherichia coli system, TcpJ can recognize both MshA and its cognate substrate, the TCP subunit TcpA, but that processing by TcpJ causes the degradation of MshA. Through site‐directed mutagenesis and chimeric pilin analysis, we show that this process targets a combination of MshA N‐terminal motifs and depends on the proteolytic activity of TcpJ. Moreover, overexpression of tcpJ partially restored the ability of bacteria unable to transcriptionally downregulate msh genes to colonize infant mice. These findings describe co‐ordinated proteolysis as a regulatory mechanism in V. cholerae and illustrate this organism's adaptability in the face of dramatic environmental changes.
The Streptomyces coelicolor GlnR regulon: identification of new GlnR targets and evidence for a central role of GlnR in nitrogen metabolism in actinomycetes
Authors: Tiffert Yvonne , Supra Petra , Wurm Reinhild , Wohlleben Wolfgang , Wagner Rolf , Reuther Jens
Published: August 2010

Streptomyces coelicolor GlnR is a global regulator that controls genes involved in nitrogen metabolism. By genomic screening 10 new GlnR targets were identified, including enzymes for ammonium assimilation (glnII, gdhA), nitrite reduction (nirB), urea cleavage (ureA) and a number of biochemically uncharacterized proteins (SCO0255, SCO0888, SCO2195, SCO2400, SCO2404, SCO7155). For the GlnR regulon, a GlnR binding site which comprises the sequence gTnAc‐n6‐GaAAc‐n6‐GtnAC‐n6‐GAAAc‐n6 has been found. Reverse transcription analysis of S. coelicolor and the S. coelicolor glnR mutant revealed that GlnR activates or represses the expression of its target genes. Furthermore, glnR expression itself was shown to be nitrogen‐dependent. Physiological studies of S. coelicolor and the S. coelicolor glnR mutant with ammonium and nitrate as the sole nitrogen source revealed that GlnR is not only involved in ammonium assimilation but also in ammonium supply. blast analysis demonstrated that GlnR‐homologous proteins are present in different actinomycetes containing the glnA gene with the conserved GlnR binding site. By DNA binding studies, it was furthermore demonstrated that S. coelicolor GlnR is able to interact with these glnA upstream regions. We therefore suggest that GlnR‐mediated regulation is not restricted to Streptomyces but constitutes a regulon conserved in many actinomycetes.
Preferential localization of the endocytic internalization machinery to hyphal tips underlies polarization of the actin cytoskeleton in Aspergillus nidulans
Authors: Araujo‐Bazán Lidia , Peñalva Miguel A. , Espeso Eduardo A.
Published: August 2010

AbpA, SlaB and AmpA, three demonstrated components of the endocytic internalization machinery, are strongly polarized in Aspergillus nidulans hyphae, forming a ring that embraces the hyphal tip, leaving an area of exclusion at the apex. AbpA, a prototypic endocytic internalization marker, localizes to highly motile and transient (average half life, 24 ± 5 s) peripheral punctate structures overlapping with actin patches, which also predominate in the tip. SlaB also localizes to peripheral patches, but these are markedly more abundant and cortical than those of AbpA. In contrast to its polarized distribution in hyphae, endocytic patches show random distribution during the isotropic growth phase preceding polarity establishment, but polarize as soon as a germtube primordium emerges from the swelled conidiospore. Thus, while endocytosis can occur along the hyphae, the apical predominance and the spatial organization of actin patches and of the above endocytic machinery proteins as a slightly subapical ring strongly suggests that tight spatial coupling of apical secretion and subapical compensatory endocytosis underlies hyphal growth. In agreement, the phenotype of a null slaB allele indicates that endocytosis is essential.
The gapped xylem mutant identifies a common regulatory step in secondary cell wall deposition
Authors: Turner Simon R. , Hall Madeleine
Published: August 2010

The phenotype of the novel gapped xylem (gpx) mutant is described. gpx plants exhibit gaps in the xylem in positions where xylem elements would normally be located. These gaps are not part of the transpiration stream and result in gpx plants having fewer functional xylem elements. The gaps are due to the absence of a secondary cell wall in developing xylem elements, resulting in complete degradation of these elements during cell death, and illustrate the importance of the secondary cell wall in retaining a functional xylem element following programmed cell death. Consequently the gpx phenotype suggests that the processes of secondary cell wall formation and cell death are independently regulated in developing xylem. gpx plants also exhibit a highly irregular pattern of secondary cell wall thickening in interfascicular cells, with some cells apparently undergoing little or no secondary cell wall deposition. Secondary cell wall deposition in plants involves the co‐ordinate regulation of several complex metabolic pathways. The gpx mutant identifies a key step involved in regulating the deposition of secondary cell wall material in both xylem and interfascicular cells, and suggests that a common regulatory step controls secondary cell wall formation in these diverse cell types. The gpx mutant offers a unique opportunity to elucidate the mechanism by which the complex processes involved in secondary cell wall formation are co‐ordinately regulated.
A member of a novel Arabidopsis thaliana gene family of candidate Mg2+ ion transporters complements a yeast mitochondrial group II intron‐splicing mutant
Authors: Schock Irene , Gregan Juraj , Steinhauser Siegfried , Schweyen Rudolf , Brennicke Axel , Knoop Volker
Published: August 2010

Autocatalytic activity of some group II introns has been demonstrated in vitro, but helper functions such as the yeast MRS2 protein are essential for splicing in vivo. In our search for such helper factors in plants, we pursued the cloning of two Arabidopsis thaliana homologues, atmrs2‐1 and atmrs2‐2. Atmrs2‐1, but not atmrs2‐2, complements the yeast deletion mutant of mrs2, and this is congruent with the prediction of two adjacent transmembrane stretches in AtMRS2‐1 and yeast MRS2 but not in AtMRS2‐2. This complementation depends on fusion of the native yeast mitochondrial import sequence to atmrs2‐1. A differing, non‐mitochondrial, cellular targeting in Arabidopsis is supported by the analysis of green fluorescent protein fusion constructs after transient transformation into plant protoplasts. Further members of what now appears to be a family of 10 mrs2 homologues are identified in the Arabidopsis genome. Similarity searches with the psi‐blast algorithm in the protein database fail to identify homologues of this novel gene family in any eukaryotes other than yeasts, but do identify its distant relatedness to the corA group of bacterial magnesium transporters. In line with this observation, intramitochondrial magnesium concentrations are indeed restored to wild‐type levels in the yeast mutant on complementation with atmrs2‐1.
Application of high‐performance liquid chromatography with photodiode array detection to the metabolic profiling of plant isoprenoids
Authors: Fraser Paul D. , Pinto M. Elisabete S. , Holloway Daniel E. , Bramley Peter M.
Published: August 2010

The application of high‐performance liquid chromatography (HPLC) using a C30 reverse‐phase stationary matrix has enabled the simultaneous separation of carotenes, xanthophylls, ubiquinones, tocopherols and plastoquinones in a single chromatogram. Continuous photodiode array (PDA) detection ensured identification and quantification of compounds upon elution. Applications of the method to the characterization of transgenic and mutant tomato varieties with altered isoprenoid content, biochemical screening of Arabidopsis thaliana, and elucidation of the modes of action of bleaching herbicides are described to illustrate the versatility of the procedure.
Chloroplast signalling in the light induction of nuclear HSP70 genes requires the accumulation of chlorophyll precursors and their accessibility to cytoplasm/nucleus
Authors: Kropat Janette , Oster Ulrike , Rüdiger Wolfhart , Beck Christoph F.
Published: August 2010

Chlorophyll precursors Mg‐protoporphyrin IX and its monomethylester are candidates for plastid‐derived molecules involved in light signalling from the chloroplast to the nucleus. The pool sizes of these two Mg2+‐containing porphyrins and of protoporphyrin IX transiently increased upon a shift of Chlamydomonas cultures from dark to light. This increase coincided with the accumulation of mRNAs encoded by the nuclear genes HSP70A and HSP70B. Analysis of a mutant (brs‐1), previously shown to be defective in the light induction of these genes, revealed high levels of protoporphyrin IX but no light‐induced increase in the levels of Mg2+‐containing porphyrins. Inhibitors of cytoplasmic protein synthesis prevented both the light‐induced rise in pool levels and induction of the HSP70 genes. Similarly, pre‐gametes, intermediates of sexual differentiation, lacked both responses to light. The block in light induction of the HSP70 genes in inhibitor‐treated cells and in pre‐gametes could be circumvented by the exogenous addition of Mg‐protoporphyrin IX in the dark. This suggests an essential role for light‐induced Mg‐protoporphyrin IX accumulation in this chloroplast‐to‐nucleus signalling pathway. However, accumulation of this porphyrin in the dark – presumably in the chloroplast – did not result in induction. A second crucial role for light in this signalling pathway is postulated which makes this plastidic compound accessible to the cytoplasm/nucleus where the downstream signalling pathway may be activated.
Plant sucrose‐H+ symporters mediate the transport of vitamin H
Authors: Ludwig Andreas , Stolz Jürgen , Sauer Norbert
Published: August 2010

A cDNA coding for a vitamin H (biotin) transport protein from Arabidopsis was identified by genetic complementation of a biotin uptake‐deficient yeast mutant. Vitamin H transport by this protein was sensitive to the SH‐group inhibitor p‐chloromercuribenzene sulfonic acid (PCMBS) and to the uncoupler carbonyl cyanide‐m‐chlorophenylhydrazone (CCCP), suggesting an energy‐dependent biotin‐H+ symport mechanism. The transport activity could contribute to the so‐far uncharacterized plant sucrose‐H+ symporter AtSUC5 which mediates the energy‐dependent transport of biotin and sucrose, and restores growth of the biotin transport‐deficient yeast mutant on medium with low biotin concentrations. Functional comparison of the AtSUC5 transporter with previously characterized plant sucrose or monosaccharide transporters revealed that biotin transport may be a general and specific property of all plant sucrose transporters (sucrose/biotin‐H+ symporters). This first report on a transporter with dual substrate specificity for two structurally unrelated molecules has a major impact on general thinking concerning the specificity of membrane transporters. The physiological relevance of this finding is discussed.