Molecular identification and characterization of the Arabidopsis delta(3,5),delta(2,4)-dienoyl-coenzyme A isomerase, a peroxisomal enzyme participating in the beta-oxidation cycle of unsaturated fatty acids.

Degradation of unsaturated fatty acids through the peroxisomal beta-oxidation pathway requires the participation of auxiliary enzymes in addition to the enzymes of the core beta-oxidation cycle. The auxiliary enzyme delta(3,5),delta(2,4)-dienoyl-coenzyme A (CoA) isomerase has been well studied in yeast (Saccharomyces cerevisiae) and mammals, but no plant homolog had been identified and characterized at the biochemical or molecular level. A candidate gene (At5g43280) was identified in Arabidopsis (Arabidopsis thaliana) encoding a protein showing homology to the rat (Rattus norvegicus) delta(3,5),delta(2,4)-dienoyl-CoA isomerase, and possessing an enoyl-CoA hydratase/isomerase fingerprint as well as aspartic and glutamic residues shown to be important for catalytic activity of the mammalian enzyme. The protein, named AtDCI1, contains a peroxisome targeting sequence at the C terminus, and fusion of a fluorescent protein to AtDCI1 directed the chimeric protein to the peroxisome in onion (Allium cepa) cells. AtDCI1 expressed in Escherichia coli was shown to have delta(3,5),delta(2,4)-dienoyl-CoA isomerase activity in vitro. Furthermore, using the synthesis of polyhydroxyalkanoate in yeast peroxisomes as an analytical tool to study the beta-oxidation cycle, expression of AtDCI1 was shown to complement the yeast mutant deficient in the delta(3,5),delta(2,4)-dienoyl-CoA isomerase, thus showing that AtDCI1 is also appropriately targeted to the peroxisome in yeast and has delta(3,5),delta(2,4)-dienoyl-CoA isomerase activity in vivo. The AtDCI1 gene is expressed constitutively in several tissues, but expression is particularly induced during seed germination. Proteins showing high homology with AtDCI1 are found in gymnosperms as well as angiosperms belonging to the Monocotyledon or Dicotyledon classes.

A novel IFN-gamma regulated human melanoma associated antigen gp33-38 defined by monoclonal antibody Me14/D12. I. Identification and immunochemical characterization.

A novel melanoma-associated differentiation Ag whose surface expression can be enhanced or induced by IFN-gamma was identified by mAb Me14/D12. Testing of numerous tumor cell lines and tumor tissue sections showed that Me14/D12-defined Ag was present not only on melanoma but also on other tumor lines of neuroectodermal origin such as gliomas and neuroblastomas and on some lymphoblastic B cell lines, on monocytes and macrophages. Immunoprecipitation by mAb Me14/D12 of lysates from [35S]methionine-labeled melanoma cells analyzed by SDS-PAGE revealed two polypeptide chains of 33 and 38 KDa, both under reducing and nonreducing conditions. Cross-linking experiments indicated that the two chains were present at the cell surface as a dimeric structure. Two-dimensional gel electrophoresis showed that the two chains of 33 and 38 KDa had isoelectric points of 6.2 and 5.7, respectively. Treatment of the melanoma cells with tunicamycin, an inhibitor of N-linked glycosylation, resulted in a reduction of the Mr from 33 to 24 KDa and from 38 to 26 KDa. Peptide maps obtained after Staphylococcus aureus V8 protease digestion showed no shared peptides between the two chains. Although biochemical data indicate that Me14/D12 molecules do not correspond to any known MHC class II Ag, their dimeric structure, tissue distribution, and regulation of IFN-gamma suggest that they could represent a new member of the MHC class II family.

Identification and functional characterization of auxiliary enzymes of the β-oxidation in " Arabidopsis thaliana "

Abstract: The ß-oxidation is the universal pathway that allows living organisms to degrade fatty acids. leading to lipid homeostasis and carbon and energy recovery from the fatty acid molecules. This pathway is centred on four core enzymatic activities sufficient to degrade saturated fatty acids. Additional auxiliary enzymes of the ß-oxidation are necessary for the complete degradation of a larger array of molecules encompassing the unsaturated fatty acids. The main pathways of the ßoxidation of fatty acids have been investigated extensively and auxiliary enzymes are well-known in mammals and yeast. The comparison of the established ß-oxidation systems suggests that the activities that are required to proceed to the full degradation of unsaturated fatty acids are present regardless of the organism and rely on common active site templates. The precise identity of the plant enzymes was unknown. By homology searches in the genome of Arabidopsis thaliana, I identified genes. encoding for proteins that could be orthologous to the yeast or animal auxiliary enzymes Δ 3, Δ 2-enoyl-CoA isomerase, Δ 3,5, Δ 2,4 -dienoyl-CoA isomerase, and type 2 enoyl-CoA hydratase. I established that these genes are expressed in Arabidopsis and that their expression can be correlated to the expression of core ß-oxidation genes. Through the observation of chimeric fluorescent protein fusions, I demonstrated that the identified proteins are localized in the peroxisóme, the only organelle where the ß-oxidation occurs in plants. Enzymatic assays were performed with the partially purified enzymes to demonstrate that the identified enzymes can catalyze the same in vitro reactions as their non-plant orthologs. The activities in vivo of the plant enzymes were demonstrated by heterologous complementation of the corresponding yeast Saccharomyces cerevisiae mutants. The complementation was visualized using the artificial polyhydroxyalkanoate (PHA) production in yeast peroxisomes. The recombinant strains, expressing a Pseudomonas aeruginosa PHA synthase modified for a peroxisomal localization, produce this polymer that serves as a trap for the 3-hydroxyacyl-CoA intermediaries of the ßoxidation and that reflects qualitatively and quantitatively the array of molecules that are processed through the ß-oxidation. This complementation demonstrated the implication of the plant Δ 3, Δ 2-enoyl-CoA isomerases and Δ3,5, Δ2,4-dienoyl-CoA isomerase in the degradation of odd chain position unsaturated fatty acids. The presence of a monofunctional type 2 enoyl-CoA hydratase is a novel in eukaryotes. Downregulation of the corresponding gene expression in an Arabidopsis line, modified to produce PHA in the peroxisome, demonstrated thàt this enzyme participates in vivo to the conversion of the intermediate 3R-hydroxyacyl-CoA, generated by the metabolism of fatty acids with a cis (Z)-unsaturated bond on an even-numbered carbon, to the 2Eenoyl-CoA for further degradation through the core ß-oxidation cycle. Résumé: La ß-oxydation est une voie universelle de dégradation des acides gras qui permet aux organismes vivants d'assurer une homéostasie lipidique et de récupérer l'énergie et le carbone contenus dans les acides gras. Le coeur de cette voie est composé de quatre réactions enzymatiques suffisantes à la dégradation des acides gras saturés. La présence des enzymes auxiliaires de la ß-oxydation est nécessaire à la dégradation d'une gamme plus étendue de molécules comprenant les acides gras insaturés. Les voies principales de la ß-oxydation des acides gras ont été étudiées en détail et les enzymes auxiliaires sont déterminées chez les mammifères et la levure. La comparaison entre les systèmes de ß-oxydation connus suggère que les activités requises pour la dégradation complète des acides gras insaturés reposent sur la présence de site actifs similaires. L'identité précise des enzymes auxiliaires chez les plantes était inconnue. En cherchant par homologie dans le génome de la plante modèle Arabidopsis thaliana, j'ai identifié des gènes codant pour des protéines pouvant être orthologues aux enzymes auxiliaires Δ3 Δ2-enoyl-CoA isomérase, Δ 3,5 Δ 2,4-dienoyl-CoA isomérase et enoyl-CoA hydratase de type 2 d'origine fongique ou mammalienne. J'ai établi la corrélation de l'expression de ces gènes dans Arabidopsis avec celle de gènes des enzymes du coeur de la ß-oxydation. En observant des chimères de fusion avec des protéines fluorescentes, j'ai démontré que les protéines identifiées sont localisées dans le péroxysomes, le seul organelle où la ß-oxydation se déroule chez les plantes. Des essais enzymatiques ont été conduits avec ces enzymes partiellement purifiées pour démontrer que les enzymes identifiées sont capables de catalyser in vitro les mêmes réactions que leurs orthologues non végétaux. Les activités des enzymes végétales in vivo ont été .démontrées par complémentation hétérologue des mutants de délétion correspondants de levure Saccharomyces cerevisiae. La visualisation de la complémentation est rendue possible par la synthèse de polyhydroxyalcanoate (PHA) dans les péroxysomes de levure. Les souches recombinantes expriment la PHA synthase de Pseudomonas aeruginosa modifiée pour être localisée dans le péroxysome produisent ce polymère qui sert de piège pour les 3-hydroxyacylCoAs intermédiaires de la ß-oxydation et qui reflète qualitativement et quantitativement la gamme de molécules qui subit la ß-oxydation. Cette complémentation a permis de démontrer que les Δ3, Δ2-enoyl-CoA isomérases, et la Δ3.5, Δ2,4-dienoyl-CoA isomérase végétales sont impliquées dans la dégradation des acides gras insaturés en position impaire. L'enoyl-CoA hydratase de type 2 monofonctionelle est une enzyme nouvelle chez les eucaryotes. La sous-expression du gène correspondant dans une lignée d'Arabidopsis modifiée pour produite du PHA dans le péroxysome a permis de démontrer que cette enzyme participe in vivo à la dégradation des acides gras ayant une double liaison en conformation cis (Z) en position paire.

Direct three-dimensional myocardial strain tensor quantification and tracking using zHARP.

Images of myocardial strain can be used to diagnose heart disease, plan and monitor treatment, and to learn about cardiac structure and function. Three-dimensional (3D) strain is typically quantified using many magnetic resonance (MR) images obtained in two or three orthogonal planes. Problems with this approach include long scan times, image misregistration, and through-plane motion. This article presents a novel method for calculating cardiac 3D strain using a stack of two or more images acquired in only one orientation. The zHARP pulse sequence encodes in-plane motion using MR tagging and out-of-plane motion using phase encoding, and has been previously shown to be capable of computing 3D displacement within a single image plane. Here, data from two adjacent image planes are combined to yield a 3D strain tensor at each pixel; stacks of zHARP images can be used to derive stacked arrays of 3D strain tensors without imaging multiple orientations and without numerical interpolation. The performance and accuracy of the method is demonstrated in vitro on a phantom and in vivo in four healthy adult human subjects.

Identification and characterization of novel classes of macrophage migration inhibitory factor (MIF) inhibitors with distinct mechanisms of action.

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is considered an attractive therapeutic target in multiple inflammatory and autoimmune disorders. In addition to its known biologic activities, MIF can also function as a tautomerase. Several small molecules have been reported to be effective inhibitors of MIF tautomerase activity in vitro. Herein we employed a robust activity-based assay to identify different classes of novel inhibitors of the catalytic and biological activities of MIF. Several novel chemical classes of inhibitors of the catalytic activity of MIF with IC(50) values in the range of 0.2-15.5 microm were identified and validated. The interaction site and mechanism of action of these inhibitors were defined using structure-activity studies and a battery of biochemical and biophysical methods. MIF inhibitors emerging from these studies could be divided into three categories based on their mechanism of action: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro(1); 2) a novel class of catalytic site inhibitors; and finally 3) molecules that disrupt the trimeric structure of MIF. Importantly, all inhibitors demonstrated total inhibition of MIF-mediated glucocorticoid overriding and AKT phosphorylation, whereas ebselen, a trimer-disrupting inhibitor, additionally acted as a potent hyperagonist in MIF-mediated chemotactic migration. The identification of biologically active compounds with known toxicity, pharmacokinetic properties, and biological activities in vivo should accelerate the development of clinically relevant MIF inhibitors. Furthermore, the diversity of chemical structures and mechanisms of action of our inhibitors makes them ideal mechanistic probes for elucidating the structure-function relationships of MIF and to further determine the role of the oligomerization state and catalytic activity of MIF in regulating the function(s) of MIF in health and disease.

Rapid Biodiversity Assessment and Monitoring Method for Highly Diverse Benthic Communities: A Case Study of Mediterranean Coralligenous Outcrops

Increasing anthropogenic pressures urge enhanced knowledge and understanding of the current state of marine biodiversity. This baseline information is pivotal to explore present trends, detect future modifications and propose adequate management actions for marine ecosystems. Coralligenous outcrops are a highly diverse and structurally complex deep-water habitat faced with major threats in the Mediterranean Sea. Despite its ecological, aesthetic and economic value, coralligenous biodiversity patterns are still poorly understood. There is currently no single sampling method that has been demonstrated to be sufficiently representative to ensure adequate community assessment and monitoring in this habitat. Therefore, we propose a rapid non-destructive protocol for biodiversity assessment and monitoring of coralligenous outcrops providing good estimates of its structure and species composition, based on photographic sampling and the determination of presence/absence of macrobenthic species. We used an extensive photographic survey, covering several spatial scales (100s of m to 100s of km) within the NW Mediterranean and including 2 different coralligenous assemblages: Paramuricea clavata (PCA) and Corallium rubrum assemblage (CRA). This approach allowed us to determine the minimal sampling area for each assemblage (5000 cm² for PCA and 2500 cm²for CRA). In addition, we conclude that 3 replicates provide an optimal sampling effort in order to maximize the species number and to assess the main biodiversity patterns of studied assemblages in variability studies requiring replicates. We contend that the proposed sampling approach provides a valuable tool for management and conservation planning, monitoring and research programs focused on coralligenous outcrops, potentially also applicable in other benthic ecosystems

Rapid genotypic change and plasticity in arbuscular mycorrhizal fungi is caused by a host shift and enhanced by segregation.

Arbuscular mycorrhizal fungi (AMF) are among the most abundant symbionts of plants, improving plant productivity and diversity. They are thought to mostly grow vegetatively, a trait assumed to limit adaptability. However, AMF can also harbor genetically different nuclei (nucleotypes). It has been shown that one AMF can produce genotypically novel offspring with proportions of different nucleotypes. We hypothesized that (1) AMF respond rapidly to a change of environment (plant host) through changes in the frequency of nucleotypes; (2) genotypically novel offspring exhibit different genetic responses to environmental change than the parent; and (3) genotypically novel offspring exhibit a wide range of phenotypic plasticity to a change of environment. We subjected AMF parents and offspring to a host shift. We observed rapid and large genotypic changes in all AMF lines that were not random. Genotypic and phenotypic responses were different among offspring and their parents. Even though growing vegetatively, AMF offspring display a broad range of genotypic and phenotypic changes in response to host shift. We conclude that AMF have the ability to rapidly produce variable progeny, increasing their probability to produce offspring with different fitness than their parents and, consequently, their potential adaptability to new environmental conditions. Such genotypic and phenotypic flexibility could be a fast alternative to sexual reproduction and is likely to be a key to the ecological success of AMF.

Identification and staining of distinct populations of secretory organelles in astrocytes.

Increasing evidence indicates that astrocytes, the most abundant glial cell type in the brain, respond to an elevation in cytoplasmic calcium concentration ([Ca(2+)]i) by releasing chemical transmitters (also called gliotransmitters) via regulated exocytosis of heterogeneous classes of organelles. By this process, astrocytes exert modulatory influences on neighboring cells and are thought to participate in the control of synaptic circuits and cerebral blood flow. Studying the properties of exocytosis in astrocytes is a challenge, because the cell biological basis of this process is incompletely defined. Astrocytic exocytosis involves multiple populations of secretory vesicles, including synaptic-like microvesicles (SLMVs), dense-core granules (DCGs), and lysosomes. Here we summarize the available information for identifying individual populations of secretory organelles in astrocytes, including DCGs, SLMVs, and lysosomes, and present experimental procedures for specifically staining such populations.

Highway/Railroad Grade Crossings Identification and Signing, HR-254, 1987

In 1982, Iowa's crossing warning identification system and signage at rail crossings were outdated, inconsistent and inadequate. Iowa's railroad system had been reduced and reorganized during the 1970's and many of the surviving railroad companies were unable to install new signs or devote staff to updating information. The preliminary engineering part of this project improved the information inventory about each crossing, provided for installation of identification tags and resulted in a comprehensive list of posts and signs eligible for replacement. The sign installation portion of this project resulted in erection of nearly 10,000 new crossbuck signs and 10,000 advance warning signs with high intensity reflectorization. In addition, new posts and multiple track signs were replaced where appropriate. Increased visibility of crossings for the motoring public has resulted from proper sign placement and use of high intensity reflectorization. The tagging has provided a consistent correct identification of crossings for accident reporting. The computer inventory of information about the crossings is now correct and provides for informed decision making to administrators of Federal and State crossing safety funds.

Identification and prospective validation of clinically relevant chronic obstructive pulmonary disease (COPD) subtypes.

Background Chronic obstructive pulmonary disease (COPD) is increasingly considered a heterogeneous condition. It was hypothesised that COPD, as currently defined, includes different clinically relevant subtypes. Methods To identify and validate COPD subtypes, 342 subjects hospitalised for the first time because of a COPD exacerbation were recruited. Three months after discharge, when clinically stable, symptoms and quality of life, lung function, exercise capacity, nutritional status, biomarkers of systemic and bronchial inflammation, sputum microbiology, CT of the thorax and echocardiography were assessed. COPD groups were identified by partitioning cluster analysis and validated prospectively against cause-specific hospitalisations and all-cause mortality during a 4 year follow-up. Results Three COPD groups were identified: group 1 (n ¼ 126, 67 years) was characterised by severe airflow limitation (postbronchodilator forced expiratory volume in 1 s (FEV 1 ) 38% predicted) and worse performance in most of the respiratory domains of the disease; group 2 (n ¼ 125, 69 years) showed milder airflow limitation (FEV 1 63% predicted); and group 3 (n ¼ 91, 67 years) combined a similarly milder airflow limitation (FEV 1 58% predicted) with a high proportion of obesity, cardiovascular disorders, iabetes and systemic inflammation. During follow-up, group 1 had more frequent hospitalisations due to COPD (HR 3.28, p < 0.001) and higher all-cause mortality (HR 2.36, p ¼ 0.018) than the other two groups, whereas group 3 had more admissions due to cardiovascular disease (HR 2.87, p ¼ 0.014). Conclusions In patients with COPD recruited at their first hospitalisation, three different COPD subtypes were identified and prospectively validated:"severe respiratory COPD","moderate respiratory COPD", and"systemic COPD'

Identification and characterization of pathogenic Pestalotiopsis species to pecan tree in Brazil

The objective of this work was to characterize and cluster isolates of Pestalotiopsis species and to identify those that are pathogenic to pecan, based on morphological and molecular characters. Pestalotiopsis spp. isolates were identified by sequencing the internal transcribed spacer (ITS) and β?tubulin regions. Identification methods were compared to indicate the key morphological characters for species characterization. Thirteen isolates were used for the pathogenicity tests. Morphological characterization was performed using the following variables: mycelial growth rate, sporulation, colony pigmentation, and conidial length and width. Ten pathogenic isolates were identified, three as -tubulin regions. Identification methods were compared to indicate the key morphological characters for species characterization. Thirteen isolates were used for the pathogenicity tests. Morphological characterization was performed using the following variables: mycelial growth rate, sporulation, colony pigmentation, and conidial length and width. Ten pathogenic isolates were identified, three as Pestalotiopsis clavispora and three as P. cocculi. The other isolates remained as an undefined species. The morphological characters were efficient for an initial separation of the isolates, which were grouped according to differences at species level, mainly colony diameter, which was identified as an important morphological describer. Beta-tubulin gene sequencing was less informative than the ITS region sequencing for species identification.

How Social Dominance Orientation affects union participation: The role of union identification and perceived union instrumentality

Bridging social dominance theory and labour studies, this field study investigated the mechanisms underpinning the relationship between rejection of group-based domination and participation in union activities. Respondents (N = 135) were members of a public sector union in California, that is, a hierarchy-attenuating institution. Results revealed that union identification mediated the negative relationship between social dominance orientation and active union participation. Moreover, the mediational effect of union identification was moderated by perceived union instrumentality (i.e. outcome- and process-based benefits afforded by the union), indicating that the relationship between union identification and participation was stronger among those union members who consider that the union affects workplace justice. The findings reveal the importance of both identity-based and instrumental motivations underlying union participation. The novelty of applying social dominance theory to union behaviour is underscored.

Identification and characterization of a resistance gene analog (RGA) from the Caricaceae Dumort family

The majority of cloned resistance (R) genes characterized so far contain a nucleotide-binding site (NBS) and a leucine-rich repeat (LRR) domain, where highly conserved motifs are found. Resistance genes analogs (RGAs) are genetic markers obtained by a PCR-based strategy using degenerated oligonucleotide primers drawn from these highly conserved "motifs". This strategy has the advantage of the high degree of structural and amino acid sequence conservation that is observed in R genes. The objective of the present study was to search for RGAs in Carica papaya L. and Vasconcellea cauliflora Jacq. A. DC. Out of three combinations of primers tested, only one resulted in amplification. The amplified product was cloned in pCR2.1TOPO and than sequenced using M13 forward and reverse primers. Forty-eight clones were sequenced from each species. The 96 sequences generated for each species were cleaned of vector sequences and clustered using CAP3 assembler. From the GENEBANK, one RGA was identified in C. papaya showing a BlastX e-value of 2x10-61 to the gb|AAP45165.1| putative disease resistant protein RGA3 (Solanum bulbocastanum). To the extent of our knowledge this is the first report of a RGA in the Caricaceae Dumort family. Preliminary structural studies were performed to further characterize this putative NBS-LRR type protein. Efforts to search for other RGAs in papaya should continue, mostly to provide basis for the development of transgenic papaya with resistance to diseases.

Identification and mutational analyses of phosphorylation sites of the calcineurin-binding protein CbpA and the identification of domains required for calcineurin binding in Aspergillus fumigatus.

Calcineurin is a key protein phosphatase required for hyphal growth and virulence in Aspergillus fumigatus, making it an attractive antifungal target. However, currently available calcineurin inhibitors, FK506 and cyclosporine A, are immunosuppressive, limiting usage in the treatment of patients with invasive aspergillosis. Therefore, the identification of endogenous inhibitors of calcineurin belonging to the calcipressin family is an important parallel strategy. We previously identified the gene cbpA as the A. fumigatus calcipressin member and showed its involvement in hyphal growth and calcium homeostasis. However, the mechanism of its activation/inhibition through phosphorylation and its interaction with calcineurin remains unknown. Here we show that A. fumigatus CbpA is phosphorylated at three distinct domains, including the conserved SP repeat motif (phosphorylated domain-I; PD-I), a filamentous fungal-specific domain (PD-II), and the C-terminal CIC motif (Calcipressin Inhibitor of Calcineurin; PD-III). While mutation of three phosphorylated residues (Ser208, Ser217, Ser223) in the PD-II did not affect CbpA function in vivo, mutation of the two phosphorylated serines (Ser156, Ser160) in the SP repeat motif caused reduced hyphal growth and sensitivity to oxidative stress. Mutational analysis in the key domains in calcineurin A (CnaA) and proteomic interaction studies confirmed the requirement of PxIxIT motif-binding residues (352-NIR-354) and the calcineurin B (CnaB)-binding helix residue (V371) for the binding of CbpA to CnaA. Additionally, while the calmodulin-binding residues (442-RVF-444) did not affect CbpA binding to CnaA, three mutations (T359P, H361L, and L365S) clustered between the CnaA catalytic and the CnaB-binding helix were also required for CbpA binding. This is the first study to analyze the phosphorylation status of calcipressin in filamentous fungi and identify the domains required for binding to calcineurin.