Play Key Roles (play + key_role)

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Selected Abstracts

Role of orexin in the regulation of glucose homeostasis

H. Tsuneki
Abstract Orexin-A (hypocretin-1) and orexin-B (hypocretin-2) are hypothalamic neuropeptides that play key roles in the regulation of wakefulness, feeding, reward, autonomic functions and energy homeostasis. To control these functions indispensable for survival, orexin-expressing neurones integrate peripheral metabolic signals, interact with many types of neurones in the brain and modulate their activities via the activation of orexin-1 receptor or orexin-2 receptor. In addition, a new functional role of orexin is emerging in the regulation of insulin and leptin sensitivities responsible for whole-body glucose metabolism. Recent evidence indicates that orexin efficiently protects against the development of peripheral insulin resistance induced by ageing or high-fat feeding in mice. In particular, the orexin receptor-2 signalling appears to confer resistance to diet-induced obesity and insulin insensitivity by improving leptin sensitivity. In fact, the expression of orexin gene is known to be down-regulated by hyperglycaemia in the rodent model of diabetes, such as ob/ob and db/db mice. Moreover, the levels of orexin receptor-2 mRNA have been shown to decline in the brain of mice along with ageing. These suggest that hyperglycaemia due to insulin insensitivity during ageing or by habitual consumption of a high-fat diet leads to the reduction in orexin expression in the hypothalamus, thereby further exacerbating peripheral insulin resistance. Therefore, orexin receptor controlling hypothalamic insulin/leptin actions may be a new target for possible future treatment of hyperglycaemia in patients with type 2 diabetes. [source]

Nanocrystalline non-planar carbons: Growth of carbon nanotubes and curled nanostructures

S. Orlanducci
Abstract We present a variety of non-planar graphitic nanostructures selectively generated in a modified Hot-Filament Chemical Vapour Deposition (HF-CVD) apparatus, using purpose-synthesized amorphous carbon nanoparticles or graphite powders as solid state precursor. The employed methodologies enable to successfully synthesize homogeneous and well organized deposits of single- and multi-walled carbon nanotubes, onion-like nanostructures, and nanotube bundles coated by nano-sized diamond grains. Variations in the morphological aspect of such non-planar graphite-based nanostructures are observed changing the experimental conditions: the solid state reactants, the filament and substrate temperatures, the catalyst concentration, and the atomic hydrogen flux over the substrate play key roles in the phenomenon. ( 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source]

Abundance and diversity of heterotrophic bacterial cells assimilating phosphate in the subtropical North Atlantic Ocean

Krista Longnecker
Summary Microorganisms play key roles in the cycles of carbon and nutrients in the ocean, and identifying the extent to which specific taxa contribute to these cycles will establish their ecological function. We examined the use of 33P-phosphate to identify heterotrophic bacteria actively involved in the cycling of phosphate, an essential inorganic nutrient. Seawater from the sub-tropical North Atlantic Ocean was incubated with 33P-phosphate and analysed by microautoradiography to determine the proportion and diversity of the bacterial community-assimilating phosphate. Complementary incubations using 3H-leucine and 3H-thymidine were also conducted. We found that a higher proportion of total heterotrophic bacterial cells in surface water samples assimilated phosphate compared with leucine or thymidine. Bacteria from all of the phylogenetic groups we identified by CARD-FISH were able to assimilate phosphate, although the abundances of cells within each group did not scale directly with the number found to assimilate phosphate. Furthermore, a significantly higher proportion of Alphaproteobacteria, Gammaproteobacteria and Cytophaga -like cells assimilated phosphate compared with leucine or thymidine. Our results suggest that a greater proportion of bacterial cells in surface waters are actively participating in the biogeochemical cycling of phosphorus, and possibly other elements, than is currently estimated through the use of 3H-leucine or 3H-thymidine. [source]

A molecular assessment of the iron stress response in the two phylogenetic clades of Trichodesmium

P. Dreux Chappell
Summary Trichodesmium spp. play key roles in global carbon and nitrogen budgets and thus defining what controls their productivity is important for understanding climate change. While iron availability has been shown to be an important chemical factor for controlling both growth and nitrogen fixation rates in Trichodesmium, all culture experiments to date have focused solely on representatives from one clade of Trichodesmium. Genomic sequence analysis determined that the Trichodesmium erythraeum (IMS101) genome contains many of the archetypical genes involved in the prokaryotic iron stress response. Focusing on three of these genes, isiB, idiA and feoB, we found that all three showed an iron stress response in axenic T. erythraeum (IMS101), and their sequences were well conserved across four species in our Trichodesmium culture collection [consisting of two T. erythraeum strains (IMS101 and GBRTRLI101), two Trichodesmium tenue strains (Z-1 and H9-4), Trichodesmium thiebautii and Trichodesmium spiralis]. With clade-specific quantitative PCR (qPCR) primers for one of these genes, isiB, we found that high isiB expression at low Fe levels corresponded to specific reductions in N2 fixation rates in both major phylogenetic clades of Trichodesmium (the T. erythraeum clade and T. tenue clade). With regard to the two clades, the most significant difference determined was temperature optima, while more subtle differences in growth, N2 fixation rate and gene expression responses to Fe stress were also observed. However the apparent conservation of the Fe stress response in the Trichodesmium genus suggests that it is an important adaptation for their niche in the oligotrophic ocean. [source]

The exopolysaccharide of Rhizobium sp.

Brassica napus roots but contributes to root colonization, YAS34 is not necessary for biofilm formation on Arabidopsis thaliana
Summary Microbial exopolysaccharides (EPSs) play key roles in plant,microbe interactions, such as biofilm formation on plant roots and legume nodulation by rhizobia. Here, we focused on the function of an EPS produced by Rhizobium sp. YAS34 in the colonization and biofilm formation on non-legume plant roots (Arabidopsis thaliana and Brassica napus). Using random transposon mutagenesis, we isolated an EPS-deficient mutant of strain YAS34 impaired in a glycosyltransferase gene (gta). Wild type and mutant strains were tagged with a plasmid-born GFP and, for the first time, the EPS produced by the wild-type strain was seen in the rhizosphere using selective carbohydrate probing with a fluorescent lectin and confocal laser-scanning microscopy. We show for the fist time that Rhizobium forms biofilms on roots of non-legumes, independently of the EPS synthesis. When produced by strain YAS34 wild type, EPS is targeted at specific parts of the plant root system. Nutrient fluctuations, root exudates and bacterial growth phase can account for such a production pattern. The EPS synthesis in Rhizobium sp. YAS34 is not essential for biofilm formation on roots, but is critical to colonization of the basal part of the root system and increasing the stability of root-adhering soil. Thus, in Rhizobium sp. YAS34 and non-legume interactions, microbial EPS is implicated in root,soil interface, root colonization, but not in biofilm formation. [source]

Endoplasmic reticulum stress and the unfolded protein response are linked to synergistic IFN-, induction via X-box binding protein 1

Judith A. Smith Dr.
Abstract Type,I IFN are strongly induced upon engagement of certain pattern recognition receptors by microbial products, and play key roles in regulating innate and adaptive immunity. It has become apparent that the endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR), in addition to restoring ER homeostasis, also influences the expression of certain inflammatory cytokines. However, the extent to which UPR signaling regulates type,I IFN remains unclear. Here we show that cells undergoing a UPR respond to TLR4 and TLR3 ligands, and intracellular dsRNA, with log-fold greater IFN-, induction. This synergy is not dependent on autocrine type,I IFN signaling, but unexpectedly requires the UPR transcription factor X-box binding protein,1 (XBP-1). Synergistic IFN-, induction also occurs in HLA-B27/human ,2m-transgenic rat macrophages exhibiting a UPR as a consequence of HLA-B27 up-regulation, where it correlates with activation of XBP-1 splicing. Together these findings indicate that the cellular response to endogenous ,danger' that disrupts ER homeostasis is coupled to IFN-, induction by XBP-1, which has implications for the immune response and the pathogenesis of diseases involving the UPR. [source]

Redefining epithelial progenitor potential in the developing thymus

Abstract Cortical and medullary epithelium represent specialised cell types that play key roles in thymocyte development, including positive and negative selection of the T cell repertoire. While recent evidence shows that these epithelial lineages share a common embryonic origin, the phenotype and possible persistence of such progenitor cells in the thymus at later stages of development remain controversial. Through use of a panel of reagents including the putative progenitor marker Mts24, we set out to redefine the stages in the development of thymic epithelium. In the early embryonic day (E)12 thymus anlagen we find that almost all epithelial cells are uniformly positive for Mts24 expression. In addition, while the thymus at later stages of development was found to contain distinct Mts24+ and Mts24, epithelial subsets, thymus grafting experiments show that both Mts24+ and Mts24, epithelial subsets share the ability to form organised cortical and medullary thymic microenvironments that support T cell development, a function shown previously to be lost in the Mts24, cells by E15 when lower cell doses were used. Our data help to clarify stages in thymic epithelial development and provide important information in relation to currently used markers of epithelial progenitors. See accompanying commentary: [source]

A pseudosymmetric cell adhesion regulatory domain in the ,7 tail of the integrin ,4,7 that interacts with focal adhesion kinase and src

Abstract The ,7 integrins ,4,7 and ,E,7 play key roles in forming the gut-associated lymphoid tissue, and contribute to chronic inflammation. The ,4,7 integrin-mediated adhesion of activated lymphocytes is largely due to a transient increase in avidity from ligand-induced clustering of ,4,7 at the cell-surface. Here, we report that L and D enantiomers of a cell-permeable peptide YDRREY encompassing residues 735,740 of the cytoplasmic tail of the ,7 subunit inhibit the adhesion of T cells to ,7 integrin ligands. The YDRREY peptide abrogated mucosal addressin cell adhesion molecule-1-induced clustering of ,4,7 on the surface of activated T cells. A mutated form of the YDRREY peptide carrying either single or double conservative mutations at Tyr735Phe and Tyr740Phe was unable to inhibit T cell adhesion, suggesting that both tandem tyrosines are critical for activity. The YDRREY peptide was bound and phosphorylated by focal adhesion kinase and src, which may serve to sequester cytoskeletal proteins to the cytoplasmic domain of ,4,7. The quasi-palindromic sequence YDRREY within the ,7 cytoplasmic tail constitutes a cell adhesion regulatory domain that modulates the interaction of ,7-expressing leukocytes with their endothelial and epithelial ligands. Cell-permeable peptidomimetics based on this motif have utility as anti-inflammatory reagents for the treatment of chronic inflammatory disease. [source]

Design of Peptide Hydroxamate-Based Photoreactive Activity-Based Probes of Zinc-Dependent Metalloproteases

Paul P. Geurink
Abstract Metalloproteases (ADAMs, MMPs) are multidomain proteins that play key roles in extracellular matrix remodelling and degradation, in cell,cell and cell,matrix interactions and in the proteolytic liberation of membrane-anchored proforms of cytokines and growth factors, the so-called ectodomainshedding. In this work we describe the development ofphotoactivatable activity-based probes with which active metalloproteases can be visualised. Our probes are based on the succinyl hydroxamate motif and differ in the positioning of the trifluoromethylphenyldiazirine photoreactive group. We demonstrate that directing the photoactivatable group towards the S1, pocket yields activity-based probes more effective than the corresponding probe with the photoactivatable group directed towards the S2, pocket. [source]

Synthesis and Lithium Storage Properties of Co3O4 Nanosheet-Assembled Multishelled Hollow Spheres

Xi Wang
Abstract Single-, double-, and triple-shelled hollow spheres assembled by Co3O4 nanosheets are successfully synthesized through a novel method. The possible formation mechanism of these novel structures was investigated using powder X-ray diffraction, scanning and transmission electron microscopies, Fourier transform IR, X-ray photoelectron spectroscopy, and thermogravimetric analysis. Both poly(vinylpyrrolidone) (PVP) soft templates and the formation of cobalt glycolate play key roles in the formation of these novel multishelled hollow structures. When tested as the anode material in lithium-ion batteries (LIBs), these multishelled microspheres exhibit excellent cycling performance, good rate capacity, and enhanced lithium storage capacity. This superior cyclic stability and capacity result from the synergetic effect of small diffusion lengths in the nanosheet building blocks and sufficient void space to buffer the volume expansion. This facile strategy may be extended to synthesize other transition metal oxide materials with hollow multishelled micro-/nanostrucutures, which may find application in sensors and catalysts due to their unique structural features. [source]

Diel variation in surface and subsurface microbial activity along a gradient of drying in an Australian sand-bed stream

Cecile Claret
Summary 1. Microbes play key roles in nutrient transformation and organic matter mineralisation in the hyporheic zone but their short-term responses to diel variations in discharge and temperature are unknown. Rates of microbial esterase activity were hypothesised to vary vertically and along a gradient of moisture in a drying sand-bed stream where discharge fluctuated daily in response to evapotranspiration. 2. At ,fully saturated', ,moist' and ,dry' locations in three sites along a drying Australian sand-bed stream, microbial activity at three depths (surface, 10 and 30 cm) was assessed using fluorescein diacetate hydrolysis. Samples were collected in mid-summer in the late afternoon and again at dawn to assess diel variation in hydrolytic activity at each site and depth. Data loggers tracked diel variations in temperature at each depth. 3. Hydrolytic activity was up to 10-fold greater in the surface sediments in late afternoon than at dawn in all habitats, and was correlated with surface sediment temperature. Diel differences in activity were not detected at 10 cm, although daily thermal cycles were evident at this depth. Unexpectedly, activity was marginally higher at dawn at 30 cm in all habitats, perhaps reflecting lags in temperature at that depth. 4. Overall, microbial activity declined with depth, strongly correlated with vertical trends in total organic matter and concentrations of dissolved phosphorus. Particulate organic matter, probably buried during a flood 35 days earlier, appeared largely responsible for these vertical trends. On the other hand, there was little evidence for hydrological exchange between much of the hyporheic zone and the surface stream, implying that processes in the subsurface zone of this stream are effectively isolated during baseflow in mid-summer. 5. Diel cycles of wetting and drying in the moist habitats did not enhance esterase activity relative to the dry or fully saturated habitats. Sediment moisture was not correlated with microbial activity, and mats of senescent algae appeared to inhibit water loss from surface sediments in the moist habitat. In this sand-bed stream, local diel fluctuations in water level appear to have less influence on microbial activity and mineralisation of organic matter in the sediments than occasional floods that bury leaf litter and renew many hyporheic zone functions. Subreach-scale processes seem to be the major driving force of microbial processes and nutrient cycling in this sand-bed river. [source]

Polymorphisms in fatty acid-binding protein-3 (FABP3) , putative association with type 2 diabetes mellitus,,

HUMAN MUTATION, Issue 2 2003
Hyoung Doo Shin
Abstract Fatty acid-binding proteins (FABPs) play key roles as transport vehicles of fatty compounds throughout the cytoplasm. Human FABP3, one of the FABPs, is present in a wide variety of tissues with highest concentration in cardiac and skeletal muscle. In an effort to identify polymorphic markers in potential candidate genes for type 2 diabetes, we have sequenced the full gene of FABP3, including the ,1,500bp promoter region. Fourteen polymorphisms were identified in FABP3: two ins/dels, two STRs, and ten SNPs (two in promoter, nine in intron, two in 3'UTR, and one in the 3' end). Among identified polymorphisms, five common sites including c.-530underscore;-532delCTC, c.-345T>C, c.348+429(CA)9-18, c.246+1806G>C, and c.634+483delT were genotyped in subjects with type 2 diabetes mellitus and normal control (n=669). By logistic and multiple regression analysis, one insertion/deletion polymorphism in the 3' end (c.634+483delT) of FABP3 appeared to be weakly associated with increased risk of type 2 diabetes (OR=1.78,1.94, P=0.03,0.04) and waist/hip ratio (WHR) (P=0.03). 2003 Wiley-Liss, Inc. [source]

Nuclear factor-,B contributes to interleukin-4- and interferon-dependent polymeric immunoglobulin receptor expression in human intestinal epithelial cells

IMMUNOLOGY, Issue 1 2004
Laynez W. Ackermann
Summary Polymeric immunoglobulins (pIgs) that are present at mucosal surfaces play key roles in both the innate and adaptive immune responses. These pIgs are delivered to the mucosal surface via transcytosis across the epithelium, a process mediated by the polymeric immunoglobulin receptor (pIgR). Previous studies demonstrate that expression of the pIgR is regulated by multiple immunomodulatory factors including interleukin-4 (IL-4) and interferon-, (IFN-,). In studies using human intestinal epithelial cells (HT29), multiple inhibitors of the transcription factor nuclear factor-,B (NF-,B), including a dominant negative I,B,-serine mutant, inhibited both IL-4- and IFN-dependent increases in pIgR expression. Under identical conditions, NF-,B inhibitors had no effect on cytokine-dependent increases in expression of the transcription factor interferon regulatory factor-1. Over-expression of the I,B,-serine mutant also inhibited reporter gene expression in response to IL-4, TNF-,, IL-1,, and in some cases IFN-, using constructs with sequences from the pIgR promoter. Reduced levels of pIgR were observed even when inhibitors were added ,24 hr after cytokines suggesting that prolonged activation of NF-,B is required. Finally, reporter gene studies with NF-,B enhancer elements indicated that IFN-, alone and IL-4 in combination with other cytokines activated NF-,B in HT29 cells. Together, these studies provide additional insight into the signalling pathways that contribute to expression of the pIgR, a critical player in mucosal immunity. [source]

Synthesis of PbTe Nanoboxes Using a Solvothermal Technique,

Z. Wang
PbTe nanoboxes have been synthesized using a solvothermal route (see Figure). The experimental results show that both surfactant poly(ethylene glycol) and the volume ratio of ethanol to water play key roles in the formation of the nanoboxes. The contrast difference between the darker edges and lighter center indicate that the nanoboxes are hollow. The as-prepared PbTe nanoboxes are single crystals, and a possible formation mechanism is proposed. [source]

U.S. Policy toward Israel, Iraq, and Saudi Arabia: An Integrated Analysis, 1981,2004

Rachel Bzostek
This project is an integrated quantitative and qualitative examination of the influences on U.S. foreign policy toward a sample of Middle East states (Israel, Iraq, and Saudi Arabia) over the last quarter century. Examinations of general trends in the relationships between these dyads, regression analyses, and brief case studies look at a number of factors contributing to the construction of these relationships, what these relationships look like, and how they have changed over time. The findings show that both policy reciprocation and U.S. executive play key roles in determining U.S. foreign policy outcomes. Also discussed is the utility of a broad-based research approach including the integration of qualitative and quantitative work, the examination of individual-level, state-level, and structure-level influences in an inclusive framework, as well as the taking interactive trends over time and the various degrees of conflict within these trends ("high,""low," and "middle") into account. [source]

Non-conventional signal transduction by type 1 interferons: The NF-,B pathway

Ziyun Du
Abstract Type I interferons (IFNs) regulate diverse cellular functions by modulating the expression of IFN-stimulated genes (ISGs) through the activation of the well established signal transduction pathway of the Janus Kinase (JAK) and signal transducers and activators of transcription (STAT) proteins. Although the JAK,STAT signal transduction pathway is critical in mediating IFN's antiviral and antiproliferative activities, other signaling pathways are activated by IFNs and regulate cellular response to IFN. The NF-,B transcription factor regulates the expression of genes involved in cell survival and immune responses. We have identified a novel IFN mediated signal pathway that leads to NF-,B activation and demonstrate that a subset of ISGs that play key roles in cellular response to IFN is regulated by NF-,B. This review focuses on the IFN-induced NF-,B activation pathway and the role of NF-,B in ISG expression, antiviral activity and apoptosis, and the therapeutic application of IFN in cancer and infectious disease. J. Cell. Biochem. 102: 1087,1094, 2007. 2007 Wiley-Liss, Inc. [source]

Exploring climatic and biotic controls on Holocene vegetation change in Fennoscandia

Paul A. Miller
Summary 1We investigated the potential drivers of Holocene vegetation changes recorded at four Scandinavian pollen sites, two in Sweden and two in Finland, at a time when they were largely free of anthropogenic influence. 2We used the generalized dynamic vegetation model LPJ-GUESS forced with climate anomaly output from an atmospheric general circulation model to simulate tree species dynamics from 10 000 years ago to the present. The model results were compared to high-resolution pollen accumulation rates gathered at the sites. 3Our results indicate that both the observed northern distributional limits of temperate trees, and the limits of Pinus sylvestris and Alnus incana at the tree line, are a result of millennial variations in summer and winter temperatures. The simulation of several distinct trends in species occurrence observed in the pollen record indicates that a time lag due to the slow spreading of species need not be invoked for most species. 4Sensitivity studies indicate that competition, natural disturbance and the magnitude of interannual variability play key roles in determining the biomass, establishment and even the presence of species near their bioclimatic limits. However, neither disturbance due to fire nor limits on establishment due to drought were likely to have been major determinants of the observed trends on the timescales considered. 5We were unable to limit the modelled occurrence of Picea abies at the study sites to the periods at which it was observed in the pollen records, indicating that we have still not completely understood the driving or limiting factors for Holocene changes in Picea abies abundance. 6Synthesis. This study shows that by combining quantitative vegetation reconstructions with a modern, process-based dynamic vegetation model, we may gain new insights into the potential biotic and abiotic drivers of Holocene vegetation dynamics, and their relative importance. This knowledge will be crucial in enabling us to assess more confidently the response of northern European vegetation to future climate change. [source]

Geographic diversification strategy and the implications of global market integration in table grapes

Angela M. Krueger
The geographic diversification mode for U.S. agribusinesses to establish an international presence is examined, using the example of table grapes. This study extends the analytical work on geographic diversification strategy in a firm-level application that considers how longer marketing seasons might affect early-season premium prices. The method draws on market integration tests from the industrial organization literature. The extent of market integration is examined using a probabilistic measure. Then, a simulation of profit incorporates the probability that markets are integrated. Tests on the market for table grapes indicate high probability that markets for domestic grapes and imports from Chile are not integrated (0.81 and 0.91). Long distances and the lack of overlap in production seasons play key roles in this finding. The simulation that makes operational the findings of limited integration suggests that geographic diversification is more profitable and of lower risk than production in California alone. [JEL Classification: Q130, Q170, F140] 2002 Wiley Periodicals, Inc. [source]

The Actin Cytoskeleton and Signaling Network during Pollen Tube Tip Growth

Ying Fu
The organization and dynamics of the actin cytoskeleton play key roles in many aspects of plant cell development. The actin cytoskeleton responds to internal developmental cues and environmental signals and is involved in cell division, subcellular organelle movement, cell polarity and polar cell growth. The tip-growing pollen tubes provide an ideal model system to investigate fundamental mechanisms of underlying polarized cell growth. In this system, most signaling cascades required for tip growth, such as Ca2+ -, small GTPases- and lipid-mediated signaling have been found to be involved in transmitting signals to a large group of actin-binding proteins. These actin-binding proteins subsequently regulate the structure of the actin network, as well as the rapid turnover of actin filaments (F-actin), thereby eventually controlling tip growth. The actin cytoskeleton acts as an integrator in which multiple signaling pathways converge, providing a general growth and regulatory mechanism that applies not only for tip growth but also for polarized diffuse growth in plants. [source]

Roles of cell adhesion molecules nectin and nectin-like molecule-5 in the regulation of cell movement and proliferation

Summary In response to chemoattractants, migrating cells form protrusions, such as lamellipodia and filopodia, and structures, such as ruffles over lamellipodia, focal complexes and focal adhesions at leading edges. The formation of these leading edge structures is essential for directional cell movement. Nectin-like molecule-5 (Necl-5) interacts in cis with PDGF receptor and integrin ,v,3, and enhances the activation of signalling molecules associated with these transmembrane proteins, which results in the formation of leading edge structures and enhancement of directional cell movement. When migrating cells come into contact with each other, cell,cell adhesion is initiated, resulting in reduced cell velocity. Necl-5 first interacts in trans with nectin-3. This interaction is transient and induces down-regulation of Necl-5 expression at the cell surface, resulting in reduced cell movement. Cell proliferation is also suppressed by the down-regulation of Necl-5, because the inhibitory effect of Necl-5 on Sprouty2, a negative regulator of the Ras signalling, is diminished. PDGF receptor and integrin ,v,3, which have interacted with Necl-5, then form a complex with nectin, which initiates cell,cell adhesion and recruits cadherin to the nectin-based cell,cell adhesion sites to form stable adherens junctions. The formation of adherens junctions stops cell movement, in part through inactivation of integrin ,v,3 caused by the trans -interaction of nectin. Thus, nectin and Necl-5 play key roles in the regulation of cell movement and proliferation. [source]

MukB colocalizes with the oriC region and is required for organization of the two Escherichia coli chromosome arms into separate cell halves

Olessia Danilova
Summary The circular Escherichia coli chromosome is organized by bidirectional replication into two equal left and right arms (replichores). Each arm occupies a separate cell half, with the origin of replication (oriC) at mid-cell. E. coli MukBEF belongs to the ubiquitous family of SMC protein complexes that play key roles in chromosome organization and processing. In mukBEF mutants, viability is restricted to low temperature with production of anucleate cells, reflecting chromosome segregation defects. We show that in mukB mutant cells, the two chromosome arms do not separate into distinct cell halves, but extend from pole to pole with the oriC region located at the old pole. Mutations in topA, encoding topoisomerase I, do not suppress the aberrant positioning of chromosomal loci in mukB cells, despite suppressing the temperature-sensitivity and production of anucleate cells. Furthermore, we show that MukB and the oriC region generally colocalize throughout the cell cycle, even when oriC localization is aberrant. We propose that MukBEF initiates the normal bidirectional organization of the chromosome from the oriC region. [source]

Proteomic analysis of Aspergillus nidulans cultured under hypoxic conditions

Motoyuki Shimizu
Abstract The fungus Aspergillus nidulans reduces nitrate to ammonium and simultaneously oxidizes ethanol to acetate to generate ATP under hypoxic conditions in a mechanism called ammonia fermentation (Takasaki, K. et al.. J. Biol. Chem. 2004, 279, 12414,12420). To elucidate the mechanism, the fungus was cultured under normoxic and hypoxic (ammonia fermenting) conditions, intracellular proteins were resolved by 2-DE, and 332 protein spots were identified using MALDI MS after tryptic digestion. Alcohol and aldehyde dehydrogenases that play key roles in oxidizing ethanol were produced at the basal level under hypoxic conditions but were obviously provoked by ethanol under normoxic conditions. Enzymes involved in gluconeogenesis, as well as the tricarboxylic and glyoxylate cycles, were downregulated. These results indicate that the mechanism of fungal energy conservation is altered under hypoxic conditions. The results also showed that proteins in the pentose phosphate pathway as well as the metabolism of both nucleotide and thiamine were upregulated under hypoxic conditions. Levels of xanthine and hypoxanthine, deamination products of guanine and adenine were increased in DNA from hypoxic cells, indicating an association between hypoxia and intracellular DNA base damage. This study is the first proteomic comparison of the hypoxic responses of A. nidulans. [source]

Application of thin-layer chromatography/infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry to structural analysis of bacteria-binding glycosphingolipids selected by affinity detection

Anne Msken
Glycosphingolipids (GSLs) play key roles in the manifestation of infectious diseases as attachment sites for pathogens. The thin-layer chromatography (TLC) overlay assay represents one of the most powerful approaches for the detection of GSL receptors of microorganisms. Here we report on the direct structural characterization of microbial GSL receptors by employment of the TLC overlay assay combined with infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry (IR-MALDI-o-TOF-MS). The procedure includes TLC separation of GSL mixtures, overlay of the chromatogram with GSL-specific bacteria, detection of bound microbes with primary antibodies against bacterial surface proteins and appropriate alkaline phosphatase labeled secondary antibodies, and in situ MS analysis of bacteria-specific GSL receptors. The combined method works on microgram scale of GSL mixtures and is advantageous in that it omits laborious and time-consuming GSL extraction from the silica gel layer. This technique was successfully applied to the compositional analysis of globo-series neutral GSLs recognized by P-fimbriated Escherichia coli bacteria, which were used as model microorganisms for infection of the human urinary tract. Thus, direct TLC/IR-MALDI-o-TOF-MS adds a novel facet to this fast and sensitive method offering a wide range of applications for the investigation of carbohydrate-specific pathogens involved in human infectious diseases. Copyright 2010 John Wiley & Sons, Ltd. [source]

Synaptic organization of complex ganglion cells in rabbit retina: Type and arrangement of inputs to directionally selective and local-edge-detector cells

Edward V. Famiglietti
Abstract The type and topographic distribution of synaptic inputs to a directionally selective (DS) rabbit retinal ganglion cell (GC) were examined and were compared with those received by two other complex GC types. The percentage of cone bipolar cell (BC) input, presumably an index of sustained responses and simple receptive field properties, is much higher than expected for complex GCs in reference to previous reports in other species: approximately 20% for the type 1 bistratified ON,OFF DS GC and for a multistratified GC, and approximately 40% for the small-tufted local-edge-detector GC. Consistent with a previous study (Famiglietti [1991] J. Comp. Neurol. 309:40,70), no ultrastructural evidence is found for inhibitory synapses from starburst amacrine cells to the ON,OFF DS GC. The density of inputs to the ON,OFF DS GC is high and rather evenly distributed over the dendritic tree. Clustering of inputs brings excitatory and inhibitory inputs into proximity, but the strict on-path condition of more proximal inhibitory inputs, favoring shunting inhibition, is not satisfied. Prominent BC input and its regional variation suggest that BCs play key roles in DS neural circuitry, both pre- and postsynaptic to the ON,OFF DS GC, according to a bilayer model (Famiglietti [1993] Invest. Ophthalmol. Vis. Sci. 34:S985). Asymmetry of inhibitory amacrine cell input may signify a region on the preferred side of the receptive field, the inhibition-free zone (Barlow and Levick [1965] J. Physiol. (Lond.) 178:477,504), supporting a role for postsynaptic integration in the DS mechanism. Prominent BC input to the local-edge-detector, often without accompanying amacrine cell input, indicates presynaptic integration in forming its trigger feature. J. Comp. Neurol. 484:357,391, 2005. 2005 Wiley-Liss, Inc. [source]

Metallomics and chemical speciation: towards a better understanding of metal-induced stress in plants

M.A.Z. Arruda
Abstract Most metal ions are toxic to plants, even at low concentrations, despite the fact that some are essential for growth and play key roles in metabolism. The majority of metals induce the formation of reactive oxygen species, which require the synthesis of additional antoxidant compounds and enzymes for their removal. New techniques that have greatly improved the identification, localisation and quantification of metals within plant tissues have led to the science of metallomics. This advancement in knowledge should eventually allow the characterisation of plants used in the process of phytoremediation of soils contaminated with toxic metals. [source]

High-density genotyping of STAT4 reveals multiple haplotypic associations with systemic lupus erythematosus in different racial groups

Bahram Namjou
Objective Systemic lupus erythematosus (SLE) is the prototypical systemic autoimmune disorder, with complex etiology and a strong genetic component. Recently, gene products involved in the interferon pathway have been under intense investigation in terms of the pathogenesis of SLE. STAT-1 and STAT-4 are transcription factors that play key roles in the interferon and Th1 signaling pathways, making them attractive candidates for involvement in SLE susceptibility. Methods Fifty-six single-nucleotide polymorphisms (SNPs) across STAT1 and STAT4 on chromosome 2 were genotyped using the Illumina platform, as part of an extensive association study in a large collection of 9,923 lupus patients and control subjects from different racial groups. DNA samples were obtained from the peripheral blood of patients with SLE and control subjects. Principal components analyses and population-based case,control association analyses were performed, and the P values, false discovery rate q values, and odds ratios with 95% confidence intervals were calculated. Results We observed strong genetic associations with SLE and multiple SNPs located within STAT4 in different ethnic groups (Fisher's combined P = 7.02 10,25). In addition to strongly confirming the previously reported association in the third intronic region of this gene, we identified additional haplotypic association across STAT4 and, in particular, a common risk haplotype that is found in multiple racial groups. In contrast, only a relatively weak suggestive association was observed with STAT1, probably due to its proximity to STAT4. Conclusion Our findings indicate that STAT4 is likely to be a crucial component in SLE pathogenesis in multiple racial groups. Knowledge of the functional effects of this association, when they are revealed, might improve our understanding of the disease and provide new therapeutic targets. [source]

The ratio of circulating osteoprotegerin to RANKL in early rheumatoid arthritis predicts later joint destruction

P. P. Geusens
Objective Rheumatoid arthritis (RA) is a chronic inflammatory disease that may result in debilitating joint deformities with destruction of bone and cartilage. Inflammation is still considered the pivotal inducer of both components of joint damage. Results of recent animal studies suggested a prominent contribution of osteoclastic bone resorption that could be dissociated from inflammation. RANKL and its natural decoy receptor, osteoprotegerin (OPG), play key roles in osteoclast activation. In a group of patients with early RA not treated with disease-modifying drugs, we tested the hypothesis that osteoclast activation, reflected by the serum OPG:RANKL ratio at baseline, is negatively associated with progression of bone damage, independent of inflammation. Methods OPG and RANKL levels, together with a parameter of inflammation (first-year time-averaged erythrocyte sedimentation rate [tESR]), were measured in 92 patients with newly diagnosed early active RA who were participants in a randomized study. The tESR and the OPG:RANKL ratio were evaluated for the ability to predict 5-year radiographic progression of joint damage. Results The first-year tESR and the OPG:RANKL ratio, as measured at baseline, independently predicted 5-year radiographic progression of joint damage (both P , 0.001). Progression of radiographic damage was greatest in patients with a high tESR and a low OPG:RANKL ratio and was lowest in patients with a low tESR and a high OPG:RANKL ratio. Conclusion This study in patients with early untreated RA is the first to confirm the findings in animal models of arthritis, that radiographic progression of the bone component of joint destruction is dependent on both inflammation (tESR) and osteoclast activation (the OPG:RANKL ratio). [source]

Modulation of activity by Arg407: structure of a fungal ,-1,2-mannosidase in complex with a substrate analogue

Yuri D. Lobsanov
Class I ,-mannosidases (glycoside hydrolase family GH47) play key roles in the maturation of N-glycans and the ER-associated degradation of unfolded glycoproteins. The 1.95, resolution structure of a fungal ,-1,2-mannosidase in complex with the substrate analogue methyl-,- d -lyxopyranosyl-(1,,2)-,- d -mannopyranoside (LM) shows the intact disaccharide spanning the ,1/+1 subsites, with the d -lyxoside ring in the ,1 subsite in the 1C4 chair conformation, and provides insight into the mechanism of catalysis. The absence of the C5, hydroxymethyl group on the d -lyxoside moiety results in the side chain of Arg407 adopting two alternative conformations: the minor one interacting with Asp375 and the major one interacting with both the d -lyxoside and the catalytic base Glu409, thus disrupting its function. Chemical modification of Asp375 has previously been shown to inactivate the enzyme. Taken together, the data suggest that Arg407, which belongs to the conserved sequence motif RPExxE, may act to modulate the activity of the enzyme. The proposed mechanism for modulating the activity is potentially a general mechanism for this superfamily. [source]

Expression, purification, crystallization and preliminary X-ray diffraction analysis of thioredoxin Trx1 from Saccharomyces cerevisiae

Yaru Zhang
Thioredoxins play key roles in the cellular response to oxidative stress. Three isoforms of thioredoxin have been identified in Saccharomyces cerevisiae: two that are cytosolic (Trx1 and Trx2) and one that is mitochondrial (Trx3). In the present work, the cytosolic form Trx1 was cloned, expressed, purified and crystallized. Crystals were obtained by the hanging-drop vapour-diffusion method. A data set was collected from a single crystal to 1.7, resolution. The crystal belongs to space group P212121, with unit-cell parameters a = 32.29, b = 46.59, c = 64.20,, , = , = , = 90. [source]

Rosiglitazone via upregulation of Akt/eNOS pathways attenuates dysfunction of endothelial progenitor cells, induced by advanced glycation end products

Chun Liang
Background and purpose:, Advanced glycation end products (AGEs) and endothelial progenitor cells (EPCs) play key roles in pathogenesis of diabetes-related vascular complications. AGEs can induce dysfunction in EPCs. The peroxisome proliferator-activated receptor-gamma (PPAR,) agonists are widely used in the treatment of type 2 diabetes, and it remains unknown if they could attenuate EPC dysfunction induced by AGEs. Experimental approach:, EPCs isolated from healthy adults were cultured with various concentrations of AGEs (0, 50, 100 and 200 mgL,1) with or without rosiglitazone (10 nM), antibody for the receptors for AGE-human serum albumin (anti-receptor for advanced glycation end products (RAGE); 50 gmL,1), phosphatidylinositol-3-kinase (PI3K) inhibitor (LY294002, 5 M), nitric oxide (NO) synthase inhibitor (L-NG -nitro-arginine methyl ester (L-NAME), 100 M) or sodium nitroprusside (SNP, 25 M). Proliferation, apoptosis, cell adhesion, migration and NO production in EPCs were assessed, and expressions of endothelial NO synthase (eNOS) and Akt were determined. Key results:, Number, proliferation/migration capacities, eNOS and Akt phosphorylation as well as NO synthesized by EPCs were increased by rosiglitazone and reduced by AGEs. AGEs promoted while rosiglitazone reduced EPC apoptosis. The AGE-induced effects were significantly ameliorated by pre-incubation with rosiglitazone, RAGE antibody and SNP. The beneficial effects of rosiglitazone could be blocked by pretreatment with L-NAME and LY294002. Conclusions and implications:, The PPAR, agonist rosiglitazone increased EPC function and attenuated EPC dysfunction induced by AGEs via upregulating the Akt-eNOS signal pathways of EPCs. [source]