bluuebunny/crossref_metadata_2025 huggingface dataset:
Genomes of the T4-related bacteriophages as windows on microbial genome evolution
Authors: Petrov Vasiliy M , Ratnayaka Swarnamala , Nolan James M , Miller Eric S , Karam Jim D
Published: October 2010

The T4-related bacteriophages are a group of bacterial viruses that share morphological similarities and genetic homologies with the well-studiedEscherichia coliphage T4, but that diverge from T4 and each other by a number of genetically determined characteristics including the bacterial hosts they infect, the sizes of their linear double-stranded (ds) DNA genomes and the predicted compositions of their proteomes. The genomes of about 40 of these phages have been sequenced and annotated over the last several years and are compared here in the context of the factors that have determined their diversity and the diversity of other microbial genomes in evolution. The genomes of the T4 relatives analyzed so far range in size between ~160,000 and ~250,000 base pairs (bp) and are mosaics of one another, consisting of clusters of homology between them that are interspersed with segments that vary considerably in genetic composition between the different phage lineages. Based on the known biological and biochemical properties of phage T4 and the proteins encoded by the T4 genome, the T4 relatives reviewed here are predicted to share a genetic core, or "Core Genome" that determines the structural design of their dsDNA chromosomes, their distinctive morphology and the process of their assembly into infectious agents (phage morphogenesis). The Core Genome appears to be the most ancient genetic component of this phage group and constitutes a mere 12-15% of the total protein encoding potential of the typical T4-related phage genome. The high degree of genetic heterogeneity that exists outside of this shared core suggests that horizontal DNA transfer involving many genetic sources has played a major role in diversification of the T4-related phages and their spread to a wide spectrum of bacterial species domains in evolution. We discuss some of the factors and pathways that might have shaped the evolution of these phages and point out several parallels between their diversity and the diversity generally observed within all groups of interrelated dsDNA microbial genomes in nature.
Dig1 protects against cell death provoked by glyphosate-based herbicides in human liver cell lines
Authors: Gasnier Céline , Benachour Nora , Clair Emilie , Travert Carine , Langlois Frédéric , Laurant Claire , Decroix-Laporte Cécile , Séralini Gilles-Eric
Published: October 2010

Background

Worldwide used pesticides containing different adjuvants like Roundup formulations, which are glyphosate-based herbicides, can provoke some in vivo toxicity and in human cells. These pesticides are commonly found in the environment, surface waters and as food residues of Roundup tolerant genetically modified plants. In order to know their effects on cells from liver, a major detoxification organ, we have studied their mechanism of action and possible protection by precise medicinal plant extracts called Dig1.

Methods

The cytotoxicity pathways of four formulations of glyphosate-based herbicides were studied using human hepatic cell lines HepG2 and Hep3B, known models to study xenobiotic effects. We monitored mitochondrial succinate dehydrogenase activity and caspases 3/7 for cell mortality and protection by Dig1, as well as cytochromes P450 1A1, 1A2, 3A4 and 2C9 and glutathione-S-transferase to approach the mechanism of actions.

Results

All the four Roundup formulations provoke liver cell death, with adjuvants having stronger effects than glyphosate alone. Hep3B are 3-5 times more sensitive over 48 h. Caspases 3/7 are greatly activated in HepG2 by Roundup at non-cytotoxic levels, and some apoptosis induction by Roundup is possible together with necrosis. CYP3A4 is specifically enhanced by Roundup at doses 400 times less than used in agriculture (2%). CYP1A2 is increased to a lesser extent together with glutathione-S-transferase (GST) down-regulation. Dig 1, non cytotoxic and not inducing caspases by itself, is able to prevent Roundup-induced cell death in a time-dependant manner with an important efficiency of up to 89%, within 48 h. In addition, we evidenced that it prevents Caspases 3/7 activation and CYP3A4 enhancement, and not GST reduction, but in turn it slightly inhibited CYP2C9 when added before Roundup.

Conclusion

Roundup is able to provoke intracellular disruption in hepatic cell lines at different levels, but a mixture of medicinal plant extracts Dig1 can protect to some extent human cell lines against this pollutants. All this system constitutes a tool for studying liver intoxication and detoxification.
Automated saccharification assay for determination of digestibility in plant materials
Authors: Gomez Leonardo D , Whitehead Caragh , Barakate Abdellah , Halpin Claire , McQueen-Mason Simon J
Published: October 2010

Background

Cell wall resistance represents the main barrier for the production of second generation biofuels. The deconstruction of lignocellulose can provide sugars for the production of fuels or other industrial products through fermentation. Understanding the biochemical basis of the recalcitrance of cell walls to digestion will allow development of more effective and cost efficient ways to produce sugars from biomass. One approach is to identify plant genes that play a role in biomass recalcitrance, using association genetics. Such an approach requires a robust and reliable high throughput (HT) assay for biomass digestibility, which can be used to screen the large numbers of samples involved in such studies.

Results

We developed a HT saccharification assay based on a robotic platform that can carry out in a 96-well plate format the enzymatic digestion and quantification of the released sugars. The handling of the biomass powder for weighing and formatting into 96 wells is performed by a robotic station, where the plant material is ground, delivered to the desired well in the plates and weighed with a precision of 0.1 mg. Once the plates are loaded, an automated liquid handling platform delivers an optional mild pretreatment (< 100°C) followed by enzymatic hydrolysis of the biomass. Aliquots from the hydrolysis are then analyzed for the release of reducing sugar equivalents. The same platform can be used for the comparative evaluation of different enzymes and enzyme cocktails. The sensitivity and reliability of the platform was evaluated by measuring the saccharification of stems from lignin modified tobacco plants, and the results of automated and manual analyses compared.

Conclusions

The automated assay systems are sensitive, robust and reliable. The system can reliably detect differences in the saccharification of plant tissues, and is able to process large number of samples with a minimum amount of human intervention. The automated system uncovered significant increases in the digestibility of certain lignin modified lines in a manner compatible with known effects of lignin modification on cell wall properties. We conclude that this automated assay platform is of sufficient sensitivity and reliability to undertake the screening of the large populations of plants necessary for mutant identification and genetic association studies.
Analysis of the expression profile of Dickkopf-1 gene in human glioma and the association with tumor malignancy
Authors: Zhou Youxin , Liu Fang , Xu Qinian , Wang Xiuyun
Published: October 2010

Background

Gliomas represent the most common primary malignant brain tumors, yet little is known about the molecular pathogenesis of these tumors. The highly-regulated Wnt signal transduction pathway is essential for normal developmental processes, and defects in the pathway are closely linked to oncogenesis. Dickkopf-1 (DKK-1) is a secreted protein that acts as a potent inhibitor of the Wnt pathway. The aim of this study was to examine the expression profile of DKK-1 gene in human glioma and its association with tumor malignancy.

Methods

We determined the expression levels of DKK-1 transcript and protein in 12 glioblastoma cell lines, medulloblastoma cells, low-grade glioma cells, and human astrocyte cells by semiquantitative RT-PCR and ELISA. A total of 47 tumor biopsy specimens and 11 normal brain tissue samples from patients with cerebral trauma internal decompression were embedded in paraffin blocks and used for immunostaining. Twenty-six primary tumors and 7 corresponding brain samples were stored in liquid nitrogen and used for RT-PCR. We further examined serologic concentrations and cerebral fluid levels of DKK-1 in patients with tumors.

Results

DKK-1 could only be detected in 12 human glioblastoma cell lines, not in a panel of other tumor and normal cell lines. The difference between glioma patients and healthy individuals was significant. Kendall's tau-c association analysis also revealed the increased DKK-1 protein expression in tumor tissues of higher pathologic classification. The levels of cerebral fluid DKK-1 protein were significantly higher in glioma patients than in healthy donors or in neuronal benign tumor patients, suggesting that the DKK-1 molecule in cerebral fluids can be applicable to detect the presence of glioma and be developed as a novel prognostic treatment.

Conclusion

The Wnt antagonist DKK-1 gene may have important roles in glioma tumorigenesis and act as a novel biomarker in human malignant glioblastoma.
Gum Guar fiber associated with fructose reduces serum triacylglycerol but did not improve the glucose tolerance in rats
Authors: Motoyama Caio SM , Pinto Mônica JS , Lira Fabio S , Ribeiro Eliane B , do Nascimento Claudia MO , Oyama Lila M
Published: October 2010

Introduction

The increased intake of dietary fructose can be associated with alterations on energy homeostasis and lipid/carbohydrate metabolism, such as insulin resistance and dislipidemia. On the other hand, the ingestion of soluble fiber gum guar could improve benefic mechanism on glucose tolerance and lipids profile.

Objective

The aim of the present study were to investigate the effects of the supplemental feeding partially hydrolyzed gum guar on glucose and lipid homeostasis, in rats fed with fructose solution.

Methods

The study was performed on thirty day-old male Wistar rats randomly assigned into four groups: control(C) or treated with fructose (F-20%), fiber (FB-5%), or fructose plus fiber (F-20% + FB-5% = FF) solution for 30 days on glucose tolerance (OGTT), triacylglycerol concentration in the liver by chloroform/methanol method, glucose, triacylglycerol and total cholesterol serum concentration by assayed by enzymatic colorimetric method, insulin receptor (IR) concentration in the liver by Western Blotting.

Results

The total body weight gain was not different between groups; in regards of total caloric intake, in the F group was significantly higher and in the FB group was lower than other groups. The triacylglycerol concentration in the liver of FF group was significantly higher than F group, the triacylglycerol concentration in the serum was higher the F group compared with other groups. The OGTT reveal impaired on glucose tolerance in the F, FB, FF compared with C. The IR concentration in the liver was lower in the F, FB, FF compared with C, no significant difference was observed between groups for IR concentration in the gastrocnemius muscle. No significant difference was observed between groups for carcass fat content and serum total cholesterol.

Conclusion

Fructose induced important alterations on glucose tolerance and lipid metabolism, despite of fiber showed reversion of part this alterations. The association fructose plus fiber to seem decrease insulin receptor concentration in the liver, with consequent impair on glucose tolerance.
MicroR159 regulation of most conserved targets in Arabidopsis has negligible phenotypic effects
Authors: Allen Robert S , Li Junyan , Alonso-Peral Maria M , White Rosemary G , Gubler Frank , Millar Anthony A
Published: October 2010

Background

A current challenge of microRNA (miRNA) research is the identification of biologically relevant miRNA:target gene relationships. In plants, high miRNA:target gene complementarity has enabled accurate target predictions, and slicing of target mRNAs has facilitated target validation through rapid amplification of 5' cDNA ends (5'-RACE) analysis. Together, these approaches have identified more than 20 targets potentially regulated by the deeply conserved miR159 family in Arabidopsis, including eight MYB genes with highly conserved miR159 target sites. However, genetic analysis has revealed the functional specificity of the major family members, miR159a and miR159b is limited to only two targets, MYB33 and MYB65. Here, we examine the functional role of miR159 regulation for the other potential MYB target genes.

Results

For these target genes, functional analysis failed to identify miR159 regulation that resulted in any major phenotypic impact, either at the morphological or molecular level. This appears to be mainly due to the quiescent nature of the remaining family member, MIR159c. Although its expression overlaps in a temporal and spatial cell-specific manner with a subset of these targets in anthers, the abundance of miR159c is extremely low and concomitantly a mir159c mutant displays no anther defects. Examination of potential miR159c targets with conserved miR159 binding sites found neither their spatial or temporal expression domains appeared miR159 regulated, despite the detection of miR159-guided cleavage products by 5'-RACE. Moreover, expression of a miR159-resistant target (mMYB101) resulted predominantly in plants that are indistinguishable from wild type. Plants that displayed altered morphological phenotypes were found to be ectopically expressing the mMYB101 transgene, and hence were misrepresentative of the in vivo functional role of miR159.

Conclusions

This study presents a novel explanation for a paradox common to plant and animal miRNA systems, where among many potential miRNA-target relationships usually only a few appear physiologically relevant. The identification of a quiescent miR159c:target gene regulatory module in anthers provides a likely rationale for the presence of conserved miR159 binding sites in many targets for which miR159 regulation has no obvious functional role. Remnants from the demise of such modules may lead to an overestimation of miRNA regulatory complexity when investigated using bioinformatic, 5'-RACE or transgenic approaches.
Cancer-related ectopic expression of the bone-related transcription factor RUNX2 in non-osseous metastatic tumor cells is linked to cell proliferation and motility
Authors: Leong David T , Lim Joleen , Goh Xuewei , Pratap Jitesh , Pereira Barry P , Kwok Hui Si , Nathan Saminathan Suresh , Dobson Jason R , Lian Jane B , Ito Yoshiaki , Voorhoeve P Mathijs , Stein Gary S , Salto-Tellez Manuel , Cool Simon M , van Wijnen Andre J
Published: October 2010

Introduction

Metastatic breast cancer cells frequently and ectopically express the transcription factor RUNX2, which normally attenuates proliferation and promotes maturation of osteoblasts. RUNX2 expression is inversely regulated with respect to cell growth in osteoblasts and deregulated in osteosarcoma cells.

Methods

Here, we addressed whether the functional relationship between cell growth and RUNX2 gene expression is maintained in breast cancer cells. We also investigated whether the aberrant expression of RUNX2 is linked to phenotypic parameters that could provide a selective advantage to cells during breast cancer progression.

Results

We find that, similar to its regulation in osteoblasts, RUNX2 expression in MDA-MB-231 breast adenocarcinoma cells is enhanced upon growth factor deprivation, as well as upon deactivation of the mitogen-dependent MEK-Erk pathway or EGFR signaling. Reduction of RUNX2 levels by RNAi has only marginal effects on cell growth and expression of proliferation markers in MDA-MB-231 breast cancer cells. Thus, RUNX2 is not a critical regulator of cell proliferation in this cell type. However, siRNA depletion of RUNX2 in MDA-MB-231 cells reduces cell motility, while forced exogenous expression of RUNX2 in MCF7 cells increases cell motility.

Conclusions

Our results support the emerging concept that the osteogenic transcription factor RUNX2 functions as a metastasis-related oncoprotein in non-osseous cancer cells.
Circulating retinol binding protein 4 in critically ill patients before specific treatment: prognostic impact and correlation with organ function, metabolism and inflammation
Authors: Koch Alexander , Weiskirchen Ralf , Sanson Edouard , Zimmermann Henning W , Voigt Sebastian , Dückers Hanna , Trautwein Christian , Tacke Frank
Published: October 2010

Introduction

Hyperglycemia and insulin resistance are well-known features of critical illness and impact the mortality rate, especially in sepsis. Retinol binding protein 4 (RBP4) promotes insulin resistance in mice and is systemically elevated in patients with obesity and type 2 diabetes. We investigated the potential role of RBP4 in critically ill patients.

Methods

We conducted a prospective single-center study of serum RBP4 concentrations in critically ill patients. One hundred twenty-three patients (85 with sepsis, 38 without sepsis) were studied at admission to a medical intensive care unit (ICU) before initiation of specific intensive care treatment measures and compared to 42 healthy nondiabetic controls. Clinical data, various laboratory parameters and metabolic and endocrine functions were assessed. Patients were followed for approximately 3 years.

Results

Serum RBP4 was significantly reduced in ICU patients, independently of sepsis, as compared to healthy controls (P < 0.001). Patients with liver cirrhosis as the primary underlying diagnosis for ICU admission had significantly lower RBP4 levels as compared with other ICU patients. Accordingly, in all ICU patients, serum RBP4 closely correlated with liver function and increased with renal failure. No significant differences of serum RBP4 concentrations in septic patients with pulmonary or other origins of sepsis or nonseptic patients could be revealed. Acute phase proteins were inversely correlated with RBP4 in sepsis patients. RBP4 did not differ between patients with or without obesity or preexisting diabetes. However, serum RBP4 levels correlated with endogenous insulin secretion (C-peptide) and insulin resistance (HOMA index). Low serum RBP4 upon admission was an adverse predictor of short-term survival in the ICU, but was not associated with overall survival during long-term follow-up.

Conclusions

Serum RBP4 concentrations are significantly reduced in critically ill patients. The strong associations with hepatic and renal function, insulin resistance and acute mortality collectively suggest a role of RBP4 in the pathogenesis of critical illness, possibly as a negative acute phase reactant, and allow a proposition as a potential novel biomarker for ICU patients.
Reading with the Grain: A New World in Literary Criticism
Authors: Bewes Timothy
Published: November 2010

Taking issue with recent interventions on critical reading that appear caught between demolishing and reestablishing topographical modes of literary analysis, this article reexamines the approach of “symptomatic reading” as developed in Louis Althusser's reading of Karl Marx. Following Althusser, it offers a model for reading “generously” that is specific to a world in which the dominant forms of literary and cultural engagement have emerged alongside the novel as a form. The article revisits Walter Benjamin's “Theses on the Philosophy of History,” as well as Alain Badiou's proposition of a “subtractive” reading, Paul Ricoeur's method of “recollection,” and Gilles Deleuze's notion of the “image of thought” to argue that under current historical conditions the most pressing injunction is not to read “against the grain” but to read with it and that, furthermore, in so doing we remain faithful to the spirit of Benjamin's injunction to “brush history against the grain.”
Regulation of hematopoietic cell clusters in the placental niche through SCF/Kit signaling in embryonic mouse
Authors: Sasaki Tatsuya , Mizuochi Chiyo , Horio Yuka , Nakao Kazuki , Akashi Koichi , Sugiyama Daisuke
Published: October 2010

Hematopoietic stem cells (HSCs) emerge from and expand in the mouse placenta at mid-gestation. To determine their compartment of origin and define extrinsic signals governing their commitment to this lineage, we identified hematopoietic cell (HC) clusters in mouse placenta, defined as cells expressing the embryonic HSC markers CD31, CD34 and Kit, by immunohistochemistry. HC clusters were first observed in the placenta at 9.5 days post coitum (dpc). To determine their origin, we tagged the allantoic region with CM-DiI at 8.25 dpc, prior to placenta formation, and cultured embryos in a whole embryo culture (WEC) system. CM-DiI-positive HC clusters were observed 42 hours later. To determine how clusters are extrinsically regulated, we isolated niche cells using laser capture micro-dissection and assayed them for expression of genes encoding hematopoietic cytokines. Among a panel of candidates assayed, only stem cell factor (SCF) was expressed in niche cells. To define niche cells, endothelial and mesenchymal cells were sorted by flow cytometry from dissociated placenta and hematopoietic cytokine gene expression was investigated. The endothelial cell compartment predominantly expressed SCF mRNA and protein. To determine whether SCF/Kit signaling regulates placental HC cluster proliferation, we injected anti-Kit neutralizing antibody into 10.25 dpc embryos and assayed cultured embryos for expression of hematopoietic transcription factors. Runx1, Myb and Gata2 were downregulated in the placental HC cluster fraction relative to controls. These observations demonstrate that placental HC clusters originate from the allantois and are regulated by endothelial niche cells through SCF/Kit signaling.
Clonal analyses reveal roles of organ founding stem cells, melanocyte stem cells and melanoblasts in establishment, growth and regeneration of the adult zebrafish fin
Authors: Tu Shu , Johnson Stephen L.
Published: October 2010

In vertebrates, the adult form emerges from the embryo by mobilization of precursors or adult stem cells. What different cell types these precursors give rise to, how many precursors establish the tissue or organ, and how they divide to establish and maintain the adult form remain largely unknown. We use the pigment pattern of the adult zebrafish fin, with a variety of clonal and lineage analyses, to address these issues. Early embryonic labeling with lineage-marker-bearing transposons shows that all classes of fin melanocytes (ontogenetic, regeneration and kit-independent melanocytes) and xanthophores arise from the same melanocyte-producing founding stem cells (mFSCs), whereas iridophores arise from distinct precursors. Additionally, these experiments show that, on average, six and nine mFSCs colonize the caudal and anal fin primordia, and daughters of different mFSCs always intercalate to form the adult pattern. Labeled clones are arrayed along the proximal-distal axis of the fin, and melanocyte time-of-differentiation lineage assays show that although most of the pigment pattern growth is at the distal edge of the fin, significant growth also occurs proximally. This suggests that leading edge melanocyte stem cells (MSCs) divide both asymmetrically to generate new melanocytes, and symmetrically to expand the MSCs and leave quiescent MSCs in their wake. Clonal labeling in adult stages confirms this and reveals different contributions of MSCs and transient melanoblasts during growth. These analyses build a comprehensive picture for how MSCs are established and grow to form the pigment stripes of the adult zebrafish fins.
Towards improved animal models of neonatal white matter injury associated with cerebral palsy
Authors: Silbereis John C. , Huang Eric J. , Back Stephen A. , Rowitch David H.
Published: October 2010

Newborn neurological injuries are the leading cause of intellectual and motor disabilities that are associated with cerebral palsy. Cerebral white matter injury is a common feature in hypoxic-ischemic encephalopathy (HIE), which affects full-term infants, and in periventricular leukomalacia (PVL), which affects preterm infants. This article discusses recent efforts to model neonatal white matter injury using mammalian systems. We emphasize that a comprehensive understanding of oligodendrocyte development and physiology is crucial for obtaining new insights into the pathobiology of HIE and PVL as well as for the generation of more sophisticated and faithful animal models.
The AP-1 family member FOS blocks transcriptional activity of the nuclear receptor steroidogenic factor 1
Authors: Sirianni Rosa , Nogueira Edson , Bassett Mary H. , Carr Bruce R. , Suzuki Takashi , Pezzi Vincenzo , Andò Sebastiano , Rainey William E.
Published: October 2010

Steroid production in the adrenal zona glomerulosa is under the control of angiotensin II (Ang II), which, upon binding to its receptor, activates protein kinase C (PKC) within these cells. PKC is a potent inhibitor of the steroidogenic enzyme CYP17. We have demonstrated that, in the ovary, PKC activates expression of FOS, a member of the AP-1 family, and increased expression of this gene is linked to CYP17 downregulation. However, the pathway and the molecular mechanism responsible for the inhibitory effect of PKC on CYP17 expression are not defined. Herein, we demonstrated that Ang II inhibited CYP17 through PKC and ERK1/2-activated FOS and that blocking FOS expression decreased PKC-mediated inhibition. Although CYP17 transcription was activated by the nuclear receptor SF-1, expression of FOS resulted in a decrease in SF-1-mediated gene transcription. FOS physically interacted with the hinge region of SF-1 and modulated its transactivity, thus preventing binding of cofactors such as SRC1 and CBP, which were necessary to fully activate CYP17 transcription. Collectively, these results indicate a new regulatory mechanism for SF-1 transcriptional activity that might influence adrenal zone-specific expression of CYP17, a mechanism that can potentially be applied to other steroidogenic tissues.
Competition between SOCS36E and Drk modulates Sevenless receptor tyrosine kinase activity
Authors: Almudi Isabel , Corominas Montserrat , Serras Florenci
Published: October 2010

Modulation of signalling pathways can trigger different cellular responses, including differences in cell fate. This modulation can be achieved by controlling the pathway activity with great precision to ensure robustness and reproducibility of the specification of cell fate. The development of the photoreceptor R7 in the Drosophila melanogaster retina has become a model in which to investigate the control of cell signalling. During R7 specification, a burst of Ras small GTPase (Ras) and mitogen-activated protein kinase (MAPK) controlled by Sevenless receptor tyrosine kinase (Sev) is required. Several cells in each ommatidium express sev. However, the spatiotemporal expression of the boss ligand and the action of negative regulators of the Sev pathway will restrict the R7 fate to a single cell. The Drosophila suppressor of cytokine signalling 36E (SOCS36E) protein contains an SH2 domain and acts as a Sev signalling attenuator. By contrast, downstream of receptor kinase (Drk), the fly homolog of the mammalian Grb2 adaptor protein, which also contains an SH2 domain, acts as a positive activator of the pathway. Here, we apply the Förster resonance energy transfer (FRET) assay to transfected Drosophila S2 cells and demonstrate that Sev binds directly to either the suppressor protein SOCS36E or the adaptor protein Drk. We propose a mechanistic model in which the competition between these two proteins for binding to the same docking site results in either attenuation of the Sev transduction in cells that should not develop R7 photoreceptors or amplification of the Ras–MAPK signal only in the R7 precursor.
Specific proteolytic cleavage of agrin regulates maturation of the neuromuscular junction
Authors: Bolliger Marc F. , Zurlinden Andreas , Lüscher Daniel , Bütikofer Lukas , Shakhova Olga , Francolini Maura , Kozlov Serguei V. , Cinelli Paolo , Stephan Alexander , Kistler Andreas D. , Rülicke Thomas , Pelczar Pawel , Ledermann Birgit , Fumagalli Guido , Gloor Sergio M. , Kunz Beat , Sonderegger Peter
Published: October 2010

During the initial stage of neuromuscular junction (NMJ) formation, nerve-derived agrin cooperates with muscle-autonomous mechanisms in the organization and stabilization of a plaque-like postsynaptic specialization at the site of nerve–muscle contact. Subsequent NMJ maturation to the characteristic pretzel-like appearance requires extensive structural reorganization. We found that the progress of plaque-to-pretzel maturation is regulated by agrin. Excessive cleavage of agrin via transgenic overexpression of an agrin-cleaving protease, neurotrypsin, in motoneurons resulted in excessive reorganizational activity of the NMJs, leading to rapid dispersal of the synaptic specialization. By contrast, expression of cleavage-resistant agrin in motoneurons slowed down NMJ remodeling and delayed NMJ maturation. Neurotrypsin, which is the sole agrin-cleaving protease in the CNS, was excluded as the physiological agrin-cleaving protease at the NMJ, because NMJ maturation was normal in neurotrypsin-deficient mice. Together, our analyses characterize agrin cleavage at its proteolytic α- and β-sites by an as-yet-unspecified protease as a regulatory access for relieving the agrin-dependent constraint on endplate reorganization during NMJ maturation.
Specific phosphorylation of Ser458 of A-type lamins inLMNA-associated myopathy patients
Authors: Mitsuhashi Hiroaki , Hayashi Yukiko K. , Matsuda Chie , Noguchi Satoru , Wakatsuki Shuji , Araki Toshiyuki , Nishino Ichizo
Published: October 2010

Mutations in LMNA, which encodes A-type nuclear lamins, cause various human diseases, including myopathy, cardiomyopathy, lipodystrophy and progeria syndrome. To date, little is known about how mutations in a single gene cause a wide variety of diseases. Here, by characterizing an antibody that specifically recognizes the phosphorylation of Ser458 of A-type lamins, we uncover findings that might contribute to our understanding of laminopathies. This antibody only reacts with nuclei in muscle biopsies from myopathy patients with mutations in the Ig-fold motif of A-type lamins. Ser458 phosphorylation is not seen in muscles from control patients or patients with any other neuromuscular diseases. In vitro analysis confirmed that only lamin A mutants associated with myopathy induce phosphorylation of Ser458, whereas lipodystrophy- or progeria-associated mutants do not. We also found that Akt1 directly phosphorylates Ser458 of lamin A with myopathy-related mutations in vitro. These results suggest that Ser458 phosphorylation of A-type lamins correlates with striated muscle laminopathies; this might be useful for the early diagnosis of LMNA-associated myopathies. We propose that disease-specific phosphorylation of A-type lamins by Akt1 contributes to myopathy caused by LMNA mutations.
Transcriptional regulation of ferritin and antioxidant genes by HIPK2 under genotoxic stress
Authors: Hailemariam Kiros , Iwasaki Kenta , Huang Bo-Wen , Sakamoto Kensuke , Tsuji Yoshiaki
Published: October 2010

ATF1 (activating transcription factor 1), a stimulus-induced CREB family transcription factor, plays important roles in cell survival and proliferation. Phosphorylation of ATF1 at Ser63 by PKA (cAMP-dependent protein kinase) and related kinases was the only known post-translational regulatory mechanism of ATF1. Here, we found that HIPK2 (homeodomain-interacting protein kinase 2), a DNA-damage-responsive nuclear kinase, is a new ATF1 kinase that phosphorylates Ser198 but not Ser63. ATF1 phosphorylation by HIPK2 activated ATF1 transcription function in the GAL4-reporter system. ATF1 is a transcriptional repressor of ferritin H, the major intracellular iron storage gene, through an ARE (antioxidant-responsive element). HIPK2 overrode the ATF1-mediated ARE repression in a kinase-activity-dependent manner in HepG2 cells. Furthermore, DNA-damage-inducing agents doxorubicin, etoposide and sodium arsenite induced ferritin H mRNA expression in HIPK2+/+ MEF cells, whereas it was significantly impaired in HIPK2−/− MEF cells. Induction of other ARE-regulated detoxification genes such as NQO1 (NADPH quinone oxidoreductase 1), GST (glutathione S-transferase) and HO1 (heme oxygenase 1) by genotoxic stress was also decreased in HIPK2-deficient cells. Taken together, these results suggest that HIPK2 is a new ATF1 kinase involved in the regulation of ferritin H and other antioxidant detoxification genes in genotoxic stress conditions.
The AP-1 clathrin adaptor facilitates cilium formation and functions with RAB-8 in C. elegans ciliary membrane transport
Authors: Kaplan Oktay I. , Molla-Herman Anahi , Cevik Sebiha , Ghossoub Rania , Kida Katarzyna , Kimura Yoshishige , Jenkins Paul , Martens Jeffrey R. , Setou Mitsutoshi , Benmerah Alexandre , Blacque Oliver E.
Published: October 2010

Clathrin adaptor (AP) complexes facilitate membrane trafficking between subcellular compartments. One such compartment is the cilium, whose dysfunction underlies disorders classified as ciliopathies. Although AP-1mu subunit (UNC-101) is linked to cilium formation and targeting of transmembrane proteins (ODR-10) to nematode sensory cilia at distal dendrite tips, these functions remain poorly understood. Here, using Caenorhabditis elegans sensory neurons and mammalian cell culture models, we find conservation of AP-1 function in facilitating cilium morphology, positioning and orientation, and microtubule stability and acetylation. These defects appear to be independent of IFT, because AP-1-depleted cells possess normal IFT protein localisation and motility. By contrast, disruption of chc-1 (clathrin) or rab-8 phenocopies unc-101 worms, preventing ODR-10 vesicle formation and causing misrouting of ODR-10 to all plasma membrane destinations. Finally, ODR-10 colocalises with RAB-8 in cell soma and they cotranslocate along dendrites, whereas ODR-10 and UNC-101 signals do not overlap. Together, these data implicate conserved roles for metazoan AP-1 in facilitating cilium structure and function, and suggest cooperation with RAB-8 to coordinate distinct early steps in neuronal ciliary membrane sorting and trafficking.
Cdc14p resets the competency of replication licensing by dephosphorylating multiple initiation proteins during mitotic exit in budding yeast
Authors: Zhai Yuanliang , Yung Philip Y. K. , Huo Lin , Liang Chun
Published: October 2010

In eukaryotes, replication licensing is achieved through sequential loading of several replication-initiation proteins onto replication origins to form pre-replicative complexes (pre-RCs), and unscheduled replication licensing is prevented by cyclin-dependent kinases (CDKs) through inhibitory phosphorylations of multiple initiation proteins. It is known that CDK inactivation during mitotic exit promotes pre-RC formation for the next cell cycle. However, whether the removal of the inhibitory phosphorylations on the initiation proteins is essential and the identity of the acting phosphatase(s) remain unknown. Here, we show that cell division cycle protein 14 (Cdc14p) dephosphorylates replication-initiation proteins Orc2p, Orc6p, Cdc6p and Mcm3p to restore their competence for pre-RC assembly in the budding yeast Saccharomyces cerevisiae. Cells without functional Cdc14p fail to dephosphorylate initiation proteins and to form pre-RCs – even when CDK activities are suppressed – and cannot replicate DNA in mitotic rereplication systems, whereas pulsed ectopic expression of Cdc14p in mitotic cells results in efficient pre-RC assembly and DNA rereplication. Furthermore, Cdc14p becomes dispensable for DNA rereplication in mitotic cells with combined non-phosphorylatable and/or phosphorylation-insensitive alleles of the initiation proteins. These data unravel the essential role of Cdc14p in replication licensing, beyond its established functions in mitotic exit, providing new insight into the intricate regulation of DNA replication through the interplay of CDKs and the Cdc14p phosphatase.
A balance between circular and linear forms of the dengue virus genome is crucial for viral replication
Authors: Villordo Sergio M. , Alvarez Diego E. , Gamarnik Andrea V.
Published: October 2010

The plasticity of viral plus strand RNA genomes is fundamental for the multiple functions of these molecules. Local and long-range RNA–RNA interactions provide the scaffold for interacting proteins of the translation, replication, and encapsidation machinery. Using dengue virus as a model, we investigated the relevance of the interplay between two alternative conformations of the viral genome during replication. Flaviviruses require long-range RNA–RNA interactions and genome cyclization for RNA synthesis. Here, we define a sequence present in the viral 3′UTR that overlaps two mutually exclusive structures. This sequence can form an extended duplex by long-range 5′-3′ interactions in the circular conformation of the RNA or fold locally into a small hairpin (sHP) in the linear form of the genome. A mutational analysis of the sHP structure revealed an absolute requirement of this element for viral viability, suggesting the need of a linear conformation of the genome. Viral RNA replication showed high vulnerability to changes that alter the balance between circular and linear forms of the RNA. Mutations that shift the equilibrium toward the circular or the linear conformation of the genome spontaneously revert to sequences with different mutations that tend to restore the relative stability of the two competing structures. We propose a model in which the viral genome exists in at least two alternative conformations and the balance between these two states is critical for infectivity.
Mechanism of N-methylation by the tRNA m1G37 methyltransferase Trm5
Authors: Christian Thomas , Lahoud Georges , Liu Cuiping , Hoffmann Katherine , Perona John J. , Hou Ya-Ming
Published: October 2010

Trm5 is a eukaryal and archaeal tRNA methyltransferase that catalyzes methyl transfer from S-adenosylmethionine (AdoMet) to the N1 position of G37 directly 3′ to the anticodon. While the biological role of m1G37 in enhancing translational fidelity is well established, the catalytic mechanism of Trm5 has remained obscure. To address the mechanism of Trm5 and more broadly the mechanism of N-methylation to nucleobases, we examined the pH-activity profile of an archaeal Trm5 enzyme, and performed structure-guided mutational analysis. The data reveal a marked dependence of enzyme-catalyzed methyl transfer on hydrogen ion equilibria: the single-turnover rate constant for methylation increases by one order of magnitude from pH 6.0 to reach a plateau at pH 7.0. This suggests a mechanism involving proton transfer from G37 as the key element in catalysis. Consideration of the kinetic data in light of the Trm5–tRNA–AdoMet ternary cocrystal structure, determined in a precatalytic conformation, suggests that proton transfer is associated with an induced fit rearrangement of the complex that precedes formation of the reactive configuration in the active site. Key roles for the conserved R145 side chain in stabilizing a proposed oxyanion at G37-O6, and for E185 as a general base to accept the proton from G37-N1, are suggested based on the mutational analysis.
Characterization of purified human Bact spliceosomal complexes reveals compositional and morphological changes during spliceosome activation and first step catalysis
Authors: Bessonov Sergey , Anokhina Maria , Krasauskas Andrius , Golas Monika M. , Sander Bjoern , Will Cindy L. , Urlaub Henning , Stark Holger , Lührmann Reinhard
Published: October 2010

To better understand the compositional and structural dynamics of the human spliceosome during its activation, we set out to isolate spliceosomal complexes formed after precatalytic B but prior to catalytically active C complexes. By shortening the polypyrimidine tract of the PM5 pre-mRNA, which lacks a 3′ splice site and 3′ exon, we stalled spliceosome assembly at the activation stage. We subsequently affinity purified human Bact complexes under the same conditions previously used to isolate B and C complexes, and analyzed their protein composition by mass spectrometry. A comparison of the protein composition of these complexes allowed a fine dissection of compositional changes during the B to Bact and Bact to C transitions, and comparisons with the Saccharomyces cerevisiae Bact complex revealed that the compositional dynamics of the spliceosome during activation are largely conserved between lower and higher eukaryotes. Human SF3b155 and CDC5L were shown to be phosphorylated specifically during the B to Bact and Bact to C transition, respectively, suggesting these modifications function at these stages of splicing. The two-dimensional structure of the human Bact complex was determined by electron microscopy, and a comparison with the B complex revealed that the morphology of the human spliceosome changes significantly during its activation. The overall architecture of the human and S. cerevisiae Bact complex is similar, suggesting that many of the higher order interactions among spliceosomal components, as well as their dynamics, are also largely conserved.
Towards a typology of cross-media news consumption: a qualitativequantitative synthesis
Authors: Schrder Kim Christian , Kobbernagel Christian
Published: October 2010

The article presents the first version of a methodologically innovative typology of people's use and experience of news, across different media platforms. Theoretically, the article is based on the modernized version of Jrgen Habermas's theory of the public sphere, which is sometimes
labelled theory of cultural citizenship, or civic agency. We observe the citizen-consumers' selection from the available news media through the theoretical lens of perceived worthwhileness, which consists of seven dimensions that aggregate to condition an individual's portfolio of news media
in everyday life. The empirical investigation uses an integrated qualitative-quantitative method, resulting in a typology of cross-media news consumption with seven user types, which is compared with the Pew 2008 study of news consumption in the United States.
The views of the news: The role of political newspapers in a changing media landscape
Authors: Hjarvard Stig
Published: October 2010

Our understanding of newspapers, for obvious reasons, is to a large degree based on the fact that they report on the news. However, there are limits to the informational news paradigm if we are to understand the political and social role of newspapers. Newspapers are also written, published
and read because they carry political viewpoints and arguments and, in a wider sense, interpretations of the social and cultural world. As such, individual newspapers and their respective readerships constitute circuits of shared beliefs and opinion formation. According to professional journalistic
norms, news and views must be separated, but in actual practice the borderline may not always be clear, and in the minds of some readers it may not even be desirable. Due to a changing media system in Denmark, the newspaper market has become differentiated and two different types of newspapers
have emerged: a political press, with mixed commercial and publicist objectives, and a non-political press, with a clear commercial objective. Empirically, the analysis and discussion are based on a quantitative content analysis of Danish national dailies and surveys of newspaper readers.
Global DNA methylation: comparison of enzymatic- and non-enzymatic-based methods
Authors: Rocha Monica S. , Castro Rita , Rivera Isabel , Kok Robert M. , Smulders Yvo M. , Jakobs Cornelis , de Almeida Isabel Tavares , Blom Henk J.
Published: October 2010

Background: The most frequently used methods for meas-uring global DNA methylation are based on two different principles: the use of methylation-sensitive restriction endonucleases followed by analysis of the obtained fragments, or the hydrolysis of genomic DNA followed by specific detection and quantification of the 5-methylcytosine content. We aimed to compare two different methods for evaluation of global DNA methylation: the cytosine extension assay after enzymatic digestion of DNA (Cyt-Ext), and a recently described method using liquid chromatography-electrospray ionization-tandem mass spectrometry after DNA hydrolysis (LC-MS/MS).

Methods: Both approaches were applied to evaluate global DNA methylation in leukocyte DNA from 96 healthy subjects. Calf thymus and pBR322 DNAs were used as hyper- and hypo-methylated references, respectively.

Results: Using the Cyt-Ext method, the DNA from healthy individuals showed radiolabel incorporation of 11,312± 1600 Dpm/μg DNA, while the LC-MS/MS method showed 4.55±0.1% methylation. Results are shown as mean±SD. The analysis of hypo- and hyper-methylated references showed that both methods are practical for discriminating different levels of methylation.

Conclusions: Cyt-Ext and LC-MS/MS are viable methods in evaluating global DNA methylation status. However, the LC-MS/MS assay allows absolute quantification and displays far superior intra-day precision. Therefore, we consider the later approach to be better for use in global DNA methylation studies.

Clin Chem Lab Med 2010;48:1793–8.
Abstraction-based Efficiency in the Lexicon
Authors: Cutler Anne
Published: November 2010

Listeners learn from their past experience of listening to spoken words, and use this learning to maximise the efficiency of future word recognition. This paper summarises evidence that the facilitatory effects of drawing on past experience are mediated by abstraction, enabling learning to be generalised across new words and new listening situations. Phoneme category retuning, which allows adaptation to speaker-specific articulatory characteristics, is generalised on the basis of relatively brief experience to words previously unheard from that speaker. Abstract knowledge of prosodic regularities is applied to recognition even of novel words for which these regularities were violated. Prosodic word-boundary regularities drive segmentation of speech into words independently of the membership of the lexical candidate set resulting from the segmentation operation. Each of these different cases illustrates how abstraction from past listening experience has contributed to the efficiency of lexical recognition.
Zur Übersetzung der skandinavischen Pseudokoordination
Authors: Hesse Andrea
Published: November 2010

Pseudocoordination is a specific grammatical construction in Scandinavian (i. e. Danish, Norwegian, Swedish and Faroese), combining local (and several other) verbs as auxiliaries with another finite verbal phrase as in Swedish Emil sitter och täljer ‘(lit.) Emil sit-PRS and carve-PRS → Emil is carving’. The construction is partially grammaticalized: on the one hand it is an aspectual marker, referring to an ongoing action or process; on the other hand it still contains some lexical background information about the subject referent's sitting or standing or the like. This paper investigates how German and Icelandic translators deal with pseudocoordination, since neither language has equivalents with this special set of semantics. In addition, both target languages differ in their possibilities to indicate aspectual meanings. Based on four Scandinavian novels and their translations, the article discusses translation strategies in isolated cases and their effect on text cohesion. It will be shown that the translators often do not focus on the construction's aspectuality, but on its lexical contents. As a result, Icelandic translations repeatedly adapt the pseudocoordination as an interference, while German translations prefer a more complex local construction.