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1

Biochemical suppression of small molecule inhibitors: a new strategy to identify inhibitor targets and signaling pathway components  

PubMed Central

SUMMARY Identification of small molecule targets remains an important challenge for chemical genetics. We report a new approach for target identification and protein discovery based on functional suppression of chemical inhibition in vitro. We discovered pirl1, an inhibitor of actin assembly, in a screen conducted with cytoplasmic extracts. Pirl1 was used to partially inhibit actin assembly in the same assay and concentrated biochemical fractions of cytoplasmic extracts were added to find activities that suppressed pirl1 inhibition. Two activities were detected, separately purified, and identified as Arp2/3 complex and Cdc42/RhoGDI complex, both known regulators of actin assembly. We show that pirl1 directly inhibits activation of Cdc42/RhoGDI but that Arp2/3 complex represents a downstream suppressor. This work introduces a general method for using low micromolar chemical inhibitors to identify both inhibitor targets and other components of a signaling pathway.

Peterson, Jeffrey R.; Lebensohn, Andres M.; Pelish, Henry E.; Kirschner, Marc W.

2006-01-01

2

Jasmonate Biochemical Pathway  

NSDL National Science Digital Library

Plants possess an interrelated family of potent fatty acid-derived regulators—the jasmonates. These compounds, which play roles in both defense and development, are derived from tri-unsaturated fatty acids [α-linolenic acid (18:3) or 7Z,10Z,13Z-hexadecatrienoic acid (16:3)]. The lipoxygenase-catalyzed addition of molecular oxygen to α-linolenic acid initiates jasmonate synthesis by providing a 13-hydroperoxide substrate for the formation of an unstable allene oxide that is then subject to enzyme-guided cyclization to produce 12-oxo-phytodienoic acid (OPDA). OPDA has several fates, including esterification into plastid lipids or transformation into the 12-carbon co-regulator jasmonic acid (JA). JA, the best-characterized member of the family, regulates both male and female fertility (depending on the plant species) and is an important mediator of defense gene expression. JA is itself a substrate for further diverse modifications. Genetic dissection of the pathway is revealing how the different jasmonates modulate different physiological processes. Each new family member that is discovered provides another key to understanding the fine control of gene expression in immune responses, in the initiation and maintenance of long-distance signal transfer in response to wounding, in the regulation of fertility, and in the turnover, inactivation, and sequestration of jasmonates, among other processes. The Jasmonate Biochemical Pathway provides an overview of the growing jasmonate family, and new members will be included in future versions of the Connections Map.

Robin Liechti (University of Lausanne;Gene Expression Laboratory and Faculty of Biology and Medicine REV); Edward E. Farmer (University of Lausanne;Gene Expression Laboratory and Faculty of Biology and Medicine REV)

2006-02-14

3

Biochemical-Pathway Diversity in Archaebacteria.  

National Technical Information Service (NTIS)

A considerable extent of biochemical diversity exists in the metabolic pathways utilized in nature for the biosynthesis of aromatic amino acids and vitamin-like derivatives. The overall objective of this research is to evaluate the biochemical diversity w...

R. A. Jensen

1988-01-01

4

Mechanisms of Ovarian Cancer Metastasis: Biochemical Pathways  

PubMed Central

Ovarian cancer is the most lethal gynecologic malignancy. Despite advances in chemotherapy, the five-year survival rate of advanced ovarian cancer patients with peritoneal metastasis remains around 30%. The most significant prognostic factor is stage, and most patients present at an advanced stage with peritoneal dissemination. There is often no clearly identifiable precursor lesion; therefore, the events leading to metastatic disease are poorly understood. This article reviews metastatic suppressor genes, the epithelial-mesenchymal transition (EMT), and the tumor microenvironment as they relate to ovarian cancer metastasis. Additionally, novel chemotherapeutic agents targeting the metastasis-related biochemical pathways are discussed.

Nakayama, Kentaro; Nakayama, Naomi; Katagiri, Hiroshi; Miyazaki, Kohji

2012-01-01

5

Methods for integrated biochemical pathway analysis  

Microsoft Academic Search

The common goal for biological research is to develop models for the biological processes we seek to understand. Such models, in the form of biochemical pathway networks which describe the physical interactions between a living cell's genes, transcripts, proteins, and metabolites (\\

John Louis Van Hemert

2010-01-01

6

Biochemical pathways in seed oil synthesis.  

PubMed

Oil produced in plant seeds is utilized as a major source of calories for human nutrition, as feedstocks for non-food uses such as soaps and polymers, and can serve as a high-energy biofuel. The biochemical pathways leading to oil (triacylglycerol) synthesis in seeds involve multiple subcellular organelles, requiring extensive lipid trafficking. Phosphatidylcholine plays a central role in these pathways as a substrate for acyl modifications and likely as a carrier for the trafficking of acyl groups between organelles and membrane subdomains. Although much has been clarified regarding the enzymes and pathways responsible for acyl-group flux, there are still major gaps in our understanding. These include the identity of several key enzymes, how flux between alternative pathways is controlled and the specialized cell biology leading to biogenesis of oil bodies that store up to 80% of carbon in seeds. PMID:23529069

Bates, Philip D; Stymne, Sten; Ohlrogge, John

2013-06-01

7

Global Sensitivity Analysis of a Biochemical Pathway Model  

Microsoft Academic Search

Developing suitable dynamic models of biochemical pathways is a key issue in Systems Biology. Parameter identification is\\u000a therefore a critical aspect where the analysis of the model identifiability plays an important role. The study of model identifiability\\u000a aims to determine whether the unknown parameters can be uniquely estimated from the available experiments. As the number of\\u000a parameters and the complexity

Maria Rodriguez-fernandez; Julio R. Banga

2008-01-01

8

Identifying optimal models to represent biochemical systems.  

PubMed

Biochemical systems involving a high number of components with intricate interactions often lead to complex models containing a large number of parameters. Although a large model could describe in detail the mechanisms that underlie the system, its very large size may hinder us in understanding the key elements of the system. Also in terms of parameter identification, large models are often problematic. Therefore, a reduced model may be preferred to represent the system. Yet, in order to efficaciously replace the large model, the reduced model should have the same ability as the large model to produce reliable predictions for a broad set of testable experimental conditions. We present a novel method to extract an "optimal" reduced model from a large model to represent biochemical systems by combining a reduction method and a model discrimination method. The former assures that the reduced model contains only those components that are important to produce the dynamics observed in given experiments, whereas the latter ensures that the reduced model gives a good prediction for any feasible experimental conditions that are relevant to answer questions at hand. These two techniques are applied iteratively. The method reveals the biological core of a model mathematically, indicating the processes that are likely to be responsible for certain behavior. We demonstrate the algorithm on two realistic model examples. We show that in both cases the core is substantially smaller than the full model. PMID:24416170

Apri, Mochamad; de Gee, Maarten; van Mourik, Simon; Molenaar, Jaap

2014-01-01

9

Identifying dysregulated pathways in cancers from pathway interaction networks  

PubMed Central

Background Cancers, a group of multifactorial complex diseases, are generally caused by mutation of multiple genes or dysregulation of pathways. Identifying biomarkers that can characterize cancers would help to understand and diagnose cancers. Traditional computational methods that detect genes differentially expressed between cancer and normal samples fail to work due to small sample size and independent assumption among genes. On the other hand, genes work in concert to perform their functions. Therefore, it is expected that dysregulated pathways will serve as better biomarkers compared with single genes. Results In this paper, we propose a novel approach to identify dysregulated pathways in cancer based on a pathway interaction network. Our contribution is three-fold. Firstly, we present a new method to construct pathway interaction network based on gene expression, protein-protein interactions and cellular pathways. Secondly, the identification of dysregulated pathways in cancer is treated as a feature selection problem, which is biologically reasonable and easy to interpret. Thirdly, the dysregulated pathways are identified as subnetworks from the pathway interaction networks, where the subnetworks characterize very well the functional dependency or crosstalk between pathways. The benchmarking results on several distinct cancer datasets demonstrate that our method can obtain more reliable and accurate results compared with existing state of the art methods. Further functional analysis and independent literature evidence also confirm that our identified potential pathogenic pathways are biologically reasonable, indicating the effectiveness of our method. Conclusions Dysregulated pathways can serve as better biomarkers compared with single genes. In this work, by utilizing pathway interaction networks and gene expression data, we propose a novel approach that effectively identifies dysregulated pathways, which can not only be used as biomarkers to diagnose cancers but also serve as potential drug targets in the future.

2012-01-01

10

Biochemical-Pathway Diversity in Archaebacteria.  

National Technical Information Service (NTIS)

Extreme halophiles such as Halobacterium vallismortis possess a prehenate dehydratase enzyme which is subject to allosteric activation by hydrophobic amino acids. This example of cross-pathway regulation (termed metabolic interlock) is characteristic of m...

R. A. Jensen

1990-01-01

11

Using nanotopography and metabolomics to identify biochemical effectors of multipotency.  

PubMed

It is emerging that mesenchymal stem cell (MSC) metabolic activity may be a key regulator of multipotency. The metabolome represents a "snapshot" of the stem cell phenotype, and therefore metabolic profiling could, through a systems biology approach, offer and highlight critical biochemical pathways for investigation. To date, however, it has remained difficult to undertake unbiased experiments to study MSC multipotency in the absence of strategies to retain multipotency without recourse to soluble factors that can add artifact to experiments. Here we apply a nanotopographical systems approach linked to metabolomics to regulate plasticity and demonstrate rapid metabolite reorganization, allowing rational selection of key biochemical targets of self-renewal (ERK1/2, LDL, and Jnk). We then show that these signaling effectors regulate functional multipotency. PMID:23072705

Tsimbouri, P Monica; McMurray, Rebecca J; Burgess, Karl V; Alakpa, Enateri V; Reynolds, Paul M; Murawski, Kate; Kingham, Emmajayne; Oreffo, Richard O C; Gadegaard, Nikolaj; Dalby, Matthew J

2012-11-27

12

Lysosomal diseases: biochemical pathways and investigations.  

PubMed

This chapter summarizes our current knowledge of lysosomes and lysosomal proteins referring to recent reviews, general schemes for degradation of substrates, and various causes of lysosomal storage diseases (LSDs). It then discusses the main principles for laboratory diagnosis. Initial screening by study of accumulated substrates in urine is helpful for mucopolysaccharidoses and oligosaccharidoses. A majority of LSDs result from the deficient activity of one acid hydrolase (in some diseases, several). Establishment of the diagnosis in this group of disorders is based on the measurement of the particular enzymic activity. Pseudodeficiencies are a possible source of error. For defects in lysosomal membrane transporters such as cystinosin or sialin, study of substrate accumulation in readily available cells/fluids is still the method of choice. Demonstration of a metabolic block in living cells is rarely used today, except for Niemann-Pick C disease. For primary diagnosis of patients, molecular genetic testing is necessary when no functional tests exist (e.g., many ceroid lipofuscinoses, Danon disease) and it is the preferred strategy when functional tests are too elaborate. Genotyping patients already diagnosed by biochemical methods is, however, essential for genetic counseling in the family; it may also be useful for optimal management. PMID:23622390

Vanier, Marie T

2013-01-01

13

Using bioinformatic approaches to identify pathways targeted by human leukemogens.  

PubMed

We have applied bioinformatic approaches to identify pathways common to chemical leukemogens and to determine whether leukemogens could be distinguished from non-leukemogenic carcinogens. From all known and probable carcinogens classified by IARC and NTP, we identified 35 carcinogens that were associated with leukemia risk in human studies and 16 non-leukemogenic carcinogens. Using data on gene/protein targets available in the Comparative Toxicogenomics Database (CTD) for 29 of the leukemogens and 11 of the non-leukemogenic carcinogens, we analyzed for enrichment of all 250 human biochemical pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The top pathways targeted by the leukemogens included metabolism of xenobiotics by cytochrome P450, glutathione metabolism, neurotrophin signaling pathway, apoptosis, MAPK signaling, Toll-like receptor signaling and various cancer pathways. The 29 leukemogens formed 18 distinct clusters comprising 1 to 3 chemicals that did not correlate with known mechanism of action or with structural similarity as determined by 2D Tanimoto coefficients in the PubChem database. Unsupervised clustering and one-class support vector machines, based on the pathway data, were unable to distinguish the 29 leukemogens from 11 non-leukemogenic known and probable IARC carcinogens. However, using two-class random forests to estimate leukemogen and non-leukemogen patterns, we estimated a 76% chance of distinguishing a random leukemogen/non-leukemogen pair from each other. PMID:22851955

Thomas, Reuben; Phuong, Jimmy; McHale, Cliona M; Zhang, Luoping

2012-07-01

14

Identifying proteins controlling key disease signaling pathways  

PubMed Central

Motivation: Several types of studies, including genome-wide association studies and RNA interference screens, strive to link genes to diseases. Although these approaches have had some success, genetic variants are often only present in a small subset of the population, and screens are noisy with low overlap between experiments in different labs. Neither provides a mechanistic model explaining how identified genes impact the disease of interest or the dynamics of the pathways those genes regulate. Such mechanistic models could be used to accurately predict downstream effects of knocking down pathway members and allow comprehensive exploration of the effects of targeting pairs or higher-order combinations of genes. Results: We developed methods to model the activation of signaling and dynamic regulatory networks involved in disease progression. Our model, SDREM, integrates static and time series data to link proteins and the pathways they regulate in these networks. SDREM uses prior information about proteins’ likelihood of involvement in a disease (e.g. from screens) to improve the quality of the predicted signaling pathways. We used our algorithms to study the human immune response to H1N1 influenza infection. The resulting networks correctly identified many of the known pathways and transcriptional regulators of this disease. Furthermore, they accurately predict RNA interference effects and can be used to infer genetic interactions, greatly improving over other methods suggested for this task. Applying our method to the more pathogenic H5N1 influenza allowed us to identify several strain-specific targets of this infection. Availability: SDREM is available from http://sb.cs.cmu.edu/sdrem Contact: zivbj@cs.cmu.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Gitter, Anthony; Bar-Joseph, Ziv

2013-01-01

15

Genetic and Biochemical Analysis of the SLN1 Pathway in Saccharomyces cerevisiae  

PubMed Central

The histidine kinase based signal transduction pathway was first uncovered in bacteria and is a prominent form of regulation in prokaryotes. However, this type of signal transduction is not unique to prokaryotes; over the last decade two-component signal transduction pathways have been identified and characterized in diverse eukaryotes, from unicellular yeasts to multicellular land plants. A number of small but important differences have been noted in the architecture and function of eukaryotic pathways. Because of the powerful genetic approaches and facile molecular analysis associated with the yeast system, the SLN1 osmotic response pathway in S. cerevisiae is particularly useful as a eukaryotic pathway model. This chapter provides an overview of genetic and biochemical methods that have been important in elucidating the stimulus-response events that underlie this pathway and in understanding the details of a eukaryotic His-Asp phosphorelay.

West, Ann H.

2010-01-01

16

Deterministic modelling and stochastic simulation of biochemical pathways using MATLAB.  

PubMed

The analysis of complex biochemical networks is conducted in two popular conceptual frameworks for modelling. The deterministic approach requires the solution of ordinary differential equations (ODEs, reaction rate equations) with concentrations as continuous state variables. The stochastic approach involves the simulation of differential-difference equations (chemical master equations, CMEs) with probabilities as variables. This is to generate counts of molecules for chemical species as realisations of random variables drawn from the probability distribution described by the CMEs. Although there are numerous tools available, many of them free, the modelling and simulation environment MATLAB is widely used in the physical and engineering sciences. We describe a collection of MATLAB functions to construct and solve ODEs for deterministic simulation and to implement realisations of CMEs for stochastic simulation using advanced MATLAB coding (Release 14). The program was successfully applied to pathway models from the literature for both cases. The results were compared to implementations using alternative tools for dynamic modelling and simulation of biochemical networks. The aim is to provide a concise set of MATLAB functions that encourage the experimentation with systems biology models. All the script files are available from www.sbi.uni-rostock.de/ publications_matlab-paper.html. PMID:16986253

Ullah, M; Schmidt, H; Cho, K H; Wolkenhauer, O

2006-03-01

17

Classification and analysis of regulatory pathways using graph property, biochemical and physicochemical property, and functional property.  

PubMed

Given a regulatory pathway system consisting of a set of proteins, can we predict which pathway class it belongs to? Such a problem is closely related to the biological function of the pathway in cells and hence is quite fundamental and essential in systems biology and proteomics. This is also an extremely difficult and challenging problem due to its complexity. To address this problem, a novel approach was developed that can be used to predict query pathways among the following six functional categories: (i) "Metabolism", (ii) "Genetic Information Processing", (iii) "Environmental Information Processing", (iv) "Cellular Processes", (v) "Organismal Systems", and (vi) "Human Diseases". The prediction method was established trough the following procedures: (i) according to the general form of pseudo amino acid composition (PseAAC), each of the pathways concerned is formulated as a 5570-D (dimensional) vector; (ii) each of components in the 5570-D vector was derived by a series of feature extractions from the pathway system according to its graphic property, biochemical and physicochemical property, as well as functional property; (iii) the minimum redundancy maximum relevance (mRMR) method was adopted to operate the prediction. A cross-validation by the jackknife test on a benchmark dataset consisting of 146 regulatory pathways indicated that an overall success rate of 78.8% was achieved by our method in identifying query pathways among the above six classes, indicating the outcome is quite promising and encouraging. To the best of our knowledge, the current study represents the first effort in attempting to identity the type of a pathway system or its biological function. It is anticipated that our report may stimulate a series of follow-up investigations in this new and challenging area. PMID:21980418

Huang, Tao; Chen, Lei; Cai, Yu-Dong; Chou, Kuo-Chen

2011-01-01

18

Characterization of changes in gene expression and biochemical pathways at low levels of benzene exposure.  

PubMed

Benzene, a ubiquitous environmental pollutant, causes acute myeloid leukemia (AML). Recently, through transcriptome profiling of peripheral blood mononuclear cells (PBMC), we reported dose-dependent effects of benzene exposure on gene expression and biochemical pathways in 83 workers exposed across four airborne concentration ranges (from <1 ppm to >10 ppm) compared with 42 subjects with non-workplace ambient exposure levels. Here, we further characterize these dose-dependent effects with continuous benzene exposure in all 125 study subjects. We estimated air benzene exposure levels in the 42 environmentally-exposed subjects from their unmetabolized urinary benzene levels. We used a novel non-parametric, data-adaptive model selection method to estimate the change with dose in the expression of each gene. We describe non-parametric approaches to model pathway responses and used these to estimate the dose responses of the AML pathway and 4 other pathways of interest. The response patterns of majority of genes as captured by mean estimates of the first and second principal components of the dose-response for the five pathways and the profiles of 6 AML pathway response-representative genes (identified by clustering) exhibited similar apparent supra-linear responses. Responses at or below 0.1 ppm benzene were observed for altered expression of AML pathway genes and CYP2E1. Together, these data show that benzene alters disease-relevant pathways and genes in a dose-dependent manner, with effects apparent at doses as low as 100 ppb in air. Studies with extensive exposure assessment of subjects exposed in the low-dose range between 10 ppb and 1 ppm are needed to confirm these findings. PMID:24786086

Thomas, Reuben; Hubbard, Alan E; McHale, Cliona M; Zhang, Luoping; Rappaport, Stephen M; Lan, Qing; Rothman, Nathaniel; Vermeulen, Roel; Guyton, Kathryn Z; Jinot, Jennifer; Sonawane, Babasaheb R; Smith, Martyn T

2014-01-01

19

Characterization of Changes in Gene Expression and Biochemical Pathways at Low Levels of Benzene Exposure  

PubMed Central

Benzene, a ubiquitous environmental pollutant, causes acute myeloid leukemia (AML). Recently, through transcriptome profiling of peripheral blood mononuclear cells (PBMC), we reported dose-dependent effects of benzene exposure on gene expression and biochemical pathways in 83 workers exposed across four airborne concentration ranges (from <1 ppm to >10 ppm) compared with 42 subjects with non-workplace ambient exposure levels. Here, we further characterize these dose-dependent effects with continuous benzene exposure in all 125 study subjects. We estimated air benzene exposure levels in the 42 environmentally-exposed subjects from their unmetabolized urinary benzene levels. We used a novel non-parametric, data-adaptive model selection method to estimate the change with dose in the expression of each gene. We describe non-parametric approaches to model pathway responses and used these to estimate the dose responses of the AML pathway and 4 other pathways of interest. The response patterns of majority of genes as captured by mean estimates of the first and second principal components of the dose-response for the five pathways and the profiles of 6 AML pathway response-representative genes (identified by clustering) exhibited similar apparent supra-linear responses. Responses at or below 0.1 ppm benzene were observed for altered expression of AML pathway genes and CYP2E1. Together, these data show that benzene alters disease-relevant pathways and genes in a dose-dependent manner, with effects apparent at doses as low as 100 ppb in air. Studies with extensive exposure assessment of subjects exposed in the low-dose range between 10 ppb and 1 ppm are needed to confirm these findings.

Thomas, Reuben; Hubbard, Alan E.; McHale, Cliona M.; Zhang, Luoping; Rappaport, Stephen M.; Lan, Qing; Rothman, Nathaniel; Vermeulen, Roel; Guyton, Kathryn Z.; Jinot, Jennifer; Sonawane, Babasaheb R.; Smith, Martyn T.

2014-01-01

20

A Computational Model for the Identification of Biochemical Pathways in the Krebs Cycle  

SciTech Connect

We have applied an algorithmic methodology which provably decomposes any complex network into a complete family of principal subcircuits to study the minimal circuits that describe the Krebs cycle. Every operational behavior that the network is capable of exhibiting can be represented by some combination of these principal subcircuits and this computational decomposition is linearly efficient. We have developed a computational model that can be applied to biochemical reaction systems which accurately renders pathways of such reactions via directed hypergraphs (Petri nets). We have applied the model to the citric acid cycle (Krebs cycle). The Krebs cycle, which oxidizes the acetyl group of acetyl CoA to CO2 and reduces NAD and FAD to NADH and FADH2 is a complex interacting set of nine subreaction networks. The Krebs cycle was selected because of its familiarity to the biological community and because it exhibits enough complexity to be interesting in order to introduce this novel analytic approach. This study validates the algorithmic methodology for the identification of significant biochemical signaling subcircuits, based solely upon the mathematical model and not upon prior biological knowledge. The utility of the algebraic-combinatorial model for identifying the complete set of biochemical subcircuits as a data set is demonstrated for this important metabolic process.

Oliveira, Joseph S.; Bailey, Colin G.; Jones-Oliveira, Janet B.; Dixon, David A.; Gull, Dean W.; Chandler, Mary L.

2003-03-01

21

Identifying Escherichia Species with Biochemical Test Kits and Standard Bacteriological Tests.  

National Technical Information Service (NTIS)

Two commercially available biochemical test identification systems were evaluated for their ability to accurately identify species of the genus Escherichia. Three separate laboratories participated in the study. The test kits did not always correctly iden...

E. W. Rice M. J. Allen T. C. Covert J. Lanegwis J. Standridge

1993-01-01

22

UT Southwestern team identifies tumor-specific pathway:  

Cancer.gov

A research team led by UT Southwestern Medical Center scientists has identified an atypical metabolic pathway unique to some tumors, possibly providing a future target for drugs that could reduce or halt the spread of cancer.

23

A Biochemical Screen for Identification of Small-Molecule Regulators of the Wnt Pathway Using Xenopus Egg Extracts  

PubMed Central

Misregulation of the Wnt pathway has been shown to be responsible for a variety of human diseases, most notably cancers. Screens for inhibitors of this pathway have been performed almost exclusively using cultured mammalian cells or with purified proteins. We have previously developed a biochemical assay using Xenopus egg extracts to recapitulate key cytoplasmic events in the Wnt pathway. Using this biochemical system, we show that a recombinant form of the Wnt coreceptor, LRP6, regulates the stability of two key components of the Wnt pathway (?-catenin and Axin) in opposing fashion. We have now fused ?-catenin and Axin to firefly and Renilla luciferase, respectively, and demonstrate that the fusion proteins behave similarly as their wild-type counterparts. Using this dual luciferase readout, we adapted the Xenopus extracts system for high-throughput screening. Results from these screens demonstrate signal distribution curves that reflect the complexity of the library screened. Of several compounds identified as cytoplasmic modulators of the Wnt pathway, one was further validated as a bona fide inhibitor of the Wnt pathway in cultured mammalian cells and Xenopus embryos. We show that other embryonic pathways may be amendable to screening for inhibitors/modulators in Xenopus egg extracts.

Thorne, Curtis A.; Lafleur, Bonnie; Lewis, Michelle; Hanson, Alison J.; Jernigan, Kristin K.; Weaver, David C.; Huppert, Kari A.; Chen, Tony W.; Wichadiit, Chonlarat; Cselenyi, Christopher S.; Tahinci, Emilios; Meyers, Kelly C.; Waskow, Emily; Orton, Darren; Salic, Adrian; Lee, Laura A.; Robbins, David J.; Huppert, Stacey S.; Lee, Ethan

2013-01-01

24

A systems biology approach identifies the biochemical mechanisms regulating monoterpenoid essential oil composition in peppermint  

PubMed Central

The integration of mathematical modeling and experimental testing is emerging as a powerful approach for improving our understanding of the regulation of metabolic pathways. In this study, we report on the development of a kinetic mathematical model that accurately simulates the developmental patterns of monoterpenoid essential oil accumulation in peppermint (Mentha × piperita). This model was then used to evaluate the biochemical processes underlying experimentally determined changes in the monoterpene pathway under low ambient-light intensities, which led to an accumulation of the branchpoint intermediate (+)-pulegone and the side product (+)-menthofuran. Our simulations indicated that the environmentally regulated changes in monoterpene profiles could only be explained when, in addition to effects on biosynthetic enzyme activities, as yet unidentified inhibitory effects of (+)-menthofuran on the branchpoint enzyme pulegone reductase (PR) were assumed. Subsequent in vitro analyses with recombinant protein confirmed that (+)-menthofuran acts as a weak competitive inhibitor of PR (Ki = 300 ?M). To evaluate whether the intracellular concentration of (+)-menthofuran was high enough for PR inhibition in vivo, we isolated essential oil-synthesizing secretory cells from peppermint leaves and subjected them to steam distillations. When peppermint plants were grown under low-light conditions, (+)-menthofuran was selectively retained in secretory cells and accumulated to very high levels (up to 20 mM), whereas under regular growth conditions, (+)-menthofuran levels remained very low (<400 ?M). These results illustrate the utility of iterative cycles of mathematical modeling and experimental testing to elucidate the mechanisms controlling flux through metabolic pathways.

Rios-Estepa, Rigoberto; Turner, Glenn W.; Lee, James M.; Croteau, Rodney B.; Lange, B. Markus

2008-01-01

25

Galactosemia: A strategy to identify new biochemical phenotypes and molecular genotypes  

SciTech Connect

We describe a stratagem for identifying new mutations in the galactose-1-phosphate uridyl transferase (GALT) gene. GALT enzyme activity and isoforms were defined in erythrocytes from probands and their first-degree relatives. If the biochemical phenotypes segregated in an autosomal recesssive pattern, we screened for common mutations by using multiplex PCR and restriction endonuclease digestions. If common mutant alleles were not present, the 11 exons of the GALT gene were amplified by PCR, and variations from the normal nucleotide sequences were identified by SSCP. The suspected region(s) was then analyzed by direct DNA sequencing. We identified 86 mutant GALT alleles that reduced erythrocyte GALT activity. Seventy-five of these GALT genomes had abnormal SSCP patterns, of which 41 were sequenced, yielding 12 new and 21 previously reported, rare mutations. Among the novel group of 12 new mutations, an unusual biochemical phenotype was found in a family whose newborn proband has classical galactosemia. He had inherited two mutations in cis (N314D-E204K) from his father, whose GALT activity was near normal, and an additional GALT mutation in the splice-acceptor site of intron C (IVSC) from his mother. The substitution of a positively charged E204K mutation created a unique isoform-banding pattern. An asymptomatic sister`s GALT genes carries three mutations (E203K-N314D/N314D) with eight distinct isoform bands. Surprisingly, her erythrocytes have normal GALT activity. We conclude that the synergism of pedigree, biochemical, SSCP, and direct GALT gene analyses is an efficient protocol for identifying new mutations and speculate that E203K and N314D codon changes produce intra-allelic complementation when in cis. 40 refs., 4 figs., 3 tabs.

Elsas, L.J.; Langley, S.; Steele, E.; Evinger, J.; Brown, A.; Singh, R.; Fernhoff, P.; Hjelm, L.N.; Dembure, P.P.; Fridovich-Keil, J.L. [Emory Univ. School of Medicine, Atlanta, GA (United States)

1995-03-01

26

Automated Network Analysis Identifies Core Pathways in Glioblastoma  

PubMed Central

Background Glioblastoma multiforme (GBM) is the most common and aggressive type of brain tumor in humans and the first cancer with comprehensive genomic profiles mapped by The Cancer Genome Atlas (TCGA) project. A central challenge in large-scale genome projects, such as the TCGA GBM project, is the ability to distinguish cancer-causing “driver” mutations from passively selected “passenger” mutations. Principal Findings In contrast to a purely frequency based approach to identifying driver mutations in cancer, we propose an automated network-based approach for identifying candidate oncogenic processes and driver genes. The approach is based on the hypothesis that cellular networks contain functional modules, and that tumors target specific modules critical to their growth. Key elements in the approach include combined analysis of sequence mutations and DNA copy number alterations; use of a unified molecular interaction network consisting of both protein-protein interactions and signaling pathways; and identification and statistical assessment of network modules, i.e. cohesive groups of genes of interest with a higher density of interactions within groups than between groups. Conclusions We confirm and extend the observation that GBM alterations tend to occur within specific functional modules, in spite of considerable patient-to-patient variation, and that two of the largest modules involve signaling via p53, Rb, PI3K and receptor protein kinases. We also identify new candidate drivers in GBM, including AGAP2/CENTG1, a putative oncogene and an activator of the PI3K pathway; and, three additional significantly altered modules, including one involved in microtubule organization. To facilitate the application of our network-based approach to additional cancer types, we make the method freely available as part of a software tool called NetBox.

Cerami, Ethan; Demir, Emek; Schultz, Nikolaus; Taylor, Barry S.; Sander, Chris

2010-01-01

27

Identifying frankincense impact by biochemical analysis and histological examination on rats  

PubMed Central

Frankincense (Gum Olibanum), made from resins of Burseraceae family, grows in Somalia, India and Yemen. Many years ago the oldest doctors used this plant for treatment of many diseases. This study identifies frankincense impact by biochemical analysis and histological examination on rats. In this study, forty male Wister Albino rats weighing 70–100 g were maintained in clean cages. The rats were divided into 2 groups, each group contained 20 rats. Frankincense extract was prepared by heating distilled water (400 ml) to 80 °C and soaking 20 g of herbs for about 60 min. After cooking at room temperature the dose was given orally through special drinking bottles daily. The first group acted as control drinking water. The second group served as treated group and was given frankincense in the drinking water during the whole duration of the experiment. After 15 and 30 days of treatment, the rats were anesthetized with ether, and blood was collected from the livers and kidneys; some biochemical analyses were performed including aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and non-bilirubin, urea, uric acid, and creatinine. Rats were killed by cervical decapitation of livers and kidneys. Each group was divided into 2 parts. The first part was used for the determination of glutathione (GSH), glucose 6 phosphate dehydrogenase (G6PDH), xanthine oxidase (XO), malonyldealdehyde (MDA), nitric oxide (NO), and xanthine oxidase (XO). The second part of livers and kidneys was kept in formalin solution (10%) and stained by Hematoxylin and Eosin (H & E), to be used for histological examination. I demonstrated in the biochemical analysis in the serum, tissue and histological examination, different impact between group (B) and group (A), and that frankincense is not absolutely safe and that precautions must be taken during it’s us as a traditional medicine and that increase the awareness with safety and health hazards of many other traditional medicine is critically needed.

Yousef, Jehad M.

2010-01-01

28

Method to assemble and integrate biochemical pathways into the chloroplast genome of Chlamydomonas reinhardtii.  

PubMed

Recombinant protein expression in the chloroplasts of green algae has recently become more routine; however, the heterologous expression of multiple proteins or complete biosynthetic pathways remains a significant challenge. Here, we show that a modified DNA Assembler approach can be used to rapidly assemble multiple-gene biosynthetic pathways in yeast and then integrate these assembled pathways at a site-specific location in the chloroplast genome of the microalgal species Chlamydomonas reinhardtii. As a proof of concept, this method was used to successfully integrate and functionally express up to three reporter proteins (AphA6, AadA, and GFP) in the chloroplast of C. reinhardtii. An analysis of the relative gene expression of the engineered strains showed significant differences in the mRNA expression levels of the reporter genes and thus highlights the importance of proper promoter/untranslated region selection when constructing a target pathway. This new method represents a useful genetic tool in the construction and integration of complex biochemical pathways into the chloroplast genome of microalgae and should aid current efforts to engineer algae for biofuels production and other desirable natural products. PMID:22674415

Noor-Mohammadi, Samaneh; Pourmir, Azadeh; Johannes, Tyler W

2012-11-01

29

Synthesis of Substrates and Biochemical Probes for Study of the Peptidoglycan Biosynthetic Pathway  

PubMed Central

Several widely used antibiotics such as ?-lactams, glycopeptides, and lipoglycopeptides exhibit their activity by interfering with peptidoglycan biosynthesis. Ever-increasing resistance to these and other agents has placed a renewed emphasis on the need to understand the reactions in this bio-synthetic pathway at the molecular level. While efficient access to many of the biosynthetic enzymes has been gained, the isolation of their natural substrates has proven difficult. Chemical synthesis has provided valuable tools to circumvent this problem and has allowed convenient access to several key intermediates and analogs thereof. Recent advances in the synthesis of the late-stage intermediates, including the Park nucleotide, lipid I, lipid II, fragments of the bacterial cell wall, along with other biochemical probes are reviewed. A brief discussion on the use of these substrates in study of this important biosynthetic pathway is also included.

Narayan, Radha S.

2007-01-01

30

Extended CADLIVE: a novel graphical notation for design of biochemical network maps and computational pathway analysis.  

PubMed

Biochemical network maps are helpful for understanding the mechanism of how a collection of biochemical reactions generate particular functions within a cell. We developed a new and computationally feasible notation that enables drawing a wide resolution map from the domain-level reactions to phenomenological events and implemented it as the extended GUI network constructor of CADLIVE (Computer-Aided Design of LIVing systEms). The new notation presents 'Domain expansion' for proteins and RNAs, 'Virtual reaction and nodes' that are responsible for illustrating domain-based interaction and 'InnerLink' that links real complex nodes to virtual nodes to illustrate the exact components of the real complex. A modular box is also presented that packs related reactions as a module or a subnetwork, which gives CADLIVE a capability to draw biochemical maps in a hierarchical modular architecture. Furthermore, we developed a pathway search module for virtual knockout mutants as a built-in application of CADLIVE. This module analyzes gene function in the same way as molecular genetics, which simulates a change in mutant phenotypes or confirms the validity of the network map. The extended CADLIVE with the newly proposed notation is demonstrated to be feasible for computational simulation and analysis. PMID:17940089

Kurata, Hiroyuki; Inoue, Kentaro; Maeda, Kazuhiro; Masaki, Koichi; Shimokawa, Yuki; Zhao, Quanyu

2007-01-01

31

A probabilistic approach to identify putative drug targets in biochemical networks  

PubMed Central

Network-based drug design holds great promise in clinical research as a way to overcome the limitations of traditional approaches in the development of drugs with high efficacy and low toxicity. This novel strategy aims to study how a biochemical network as a whole, rather than its individual components, responds to specific perturbations in different physiological conditions. Proteins exerting little control over normal cells and larger control over altered cells may be considered as good candidates for drug targets. The application of network-based drug design would greatly benefit from using an explicit computational model describing the dynamics of the system under investigation. However, creating a fully characterized kinetic model is not an easy task, even for relatively small networks, as it is still significantly hampered by the lack of data about kinetic mechanisms and parameters values. Here, we propose a Monte Carlo approach to identify the differences between flux control profiles of a metabolic network in different physiological states, when information about the kinetics of the system is partially or totally missing. Based on experimentally accessible information on metabolic phenotypes, we develop a novel method to determine probabilistic differences in the flux control coefficients between the two observable phenotypes. Knowledge of how differences in flux control are distributed among the different enzymatic steps is exploited to identify points of fragility in one of the phenotypes. Using a prototypical cancerous phenotype as an example, we demonstrate how our approach can assist researchers in developing compounds with high efficacy and low toxicity.

Murabito, Ettore; Smallbone, Kieran; Swinton, Jonathan; Westerhoff, Hans V.; Steuer, Ralf

2011-01-01

32

WinBEST-KIT: Windows-based biochemical reaction simulator for metabolic pathways.  

PubMed

We have implemented an efficient, user-friendly biochemical reaction simulator called Web-based BEST-KIT (Biochemical Engineering System analyzing Tool-KIT) for analyzing large-scale nonlinear networks such as metabolic pathways. Users can easily design and analyze an arbitrary reaction scheme through the Internet and an efficient graphical user interface without considering the mathematical equations. The reaction scheme can include several reaction types, which are represented by both the mass action law (mass balance) and approximated velocity functions of enzyme kinetics at steady state, such as Michaelis-Menten, Hill cooperative, Competitive inhibition. However, since all modules in Web-based BEST-KIT have been developed in Java applet style, users cannot optionally make use of original mathematical equations in addition to the prepared equations. In the present study, we have developed a new version of BEST-KIT (for Microsoft Windows called WinBEST-KIT) to allow users to define original mathematical equations and to customize these equations very easily as user-defined reaction symbols. The following powerful system-analytical methods are prepared for system analysis: time-course calculation, parameter scanning, estimation of the values of unknown kinetic parameters based on experimentally observed time-course data of reactants, dynamic response of reactants against virtual external perturbations, and real-time simulation (Virtual Dry Lab). PMID:16960966

Sekiguchi, Tatsuya; Okamoto, Masahiro

2006-06-01

33

Pathways-Driven Sparse Regression Identifies Pathways and Genes Associated with High-Density Lipoprotein Cholesterol in Two Asian Cohorts  

PubMed Central

Standard approaches to data analysis in genome-wide association studies (GWAS) ignore any potential functional relationships between gene variants. In contrast gene pathways analysis uses prior information on functional structure within the genome to identify pathways associated with a trait of interest. In a second step, important single nucleotide polymorphisms (SNPs) or genes may be identified within associated pathways. The pathways approach is motivated by the fact that genes do not act alone, but instead have effects that are likely to be mediated through their interaction in gene pathways. Where this is the case, pathways approaches may reveal aspects of a trait's genetic architecture that would otherwise be missed when considering SNPs in isolation. Most pathways methods begin by testing SNPs one at a time, and so fail to capitalise on the potential advantages inherent in a multi-SNP, joint modelling approach. Here, we describe a dual-level, sparse regression model for the simultaneous identification of pathways and genes associated with a quantitative trait. Our method takes account of various factors specific to the joint modelling of pathways with genome-wide data, including widespread correlation between genetic predictors, and the fact that variants may overlap multiple pathways. We use a resampling strategy that exploits finite sample variability to provide robust rankings for pathways and genes. We test our method through simulation, and use it to perform pathways-driven gene selection in a search for pathways and genes associated with variation in serum high-density lipoprotein cholesterol levels in two separate GWAS cohorts of Asian adults. By comparing results from both cohorts we identify a number of candidate pathways including those associated with cardiomyopathy, and T cell receptor and PPAR signalling. Highlighted genes include those associated with the L-type calcium channel, adenylate cyclase, integrin, laminin, MAPK signalling and immune function.

Silver, Matt; Chen, Peng; Li, Ruoying; Cheng, Ching-Yu; Wong, Tien-Yin; Tai, E-Shyong; Teo, Yik-Ying; Montana, Giovanni

2013-01-01

34

Computationally identifying virulence factors based on KEGG pathways.  

PubMed

Virulence factors are molecules that play very important roles in enhancing the pathogen's capability in causing diseases. Many efforts were made to investigate the mechanism of virulence factors using in silico methods. In this study, we present a novel computational method to predict virulence factors by integrating protein-protein interactions in a STRING database and biological pathways in the KEGG. Three specific species were studied according to their records in the VFDB. They are Campylobacter jejuni NCTC 11168, Escherichia coli O6?:?K15?:?H31 536 (UPEC) and Pseudomonas aeruginosa PAO1. The prediction accuracies reached were 0.9467, 0.9575 and 0.9180, respectively. Metabolism pathways, flagellar assembly and chemotaxis may be of importance for virulence based on the analysis of the optimal feature sets we obtained. We hope this can provide some insight and guidance for related research. PMID:23519087

Cui, Weiren; Chen, Lei; Huang, Tao; Gao, Qian; Jiang, Min; Zhang, Ning; Zheng, Lulu; Feng, Kaiyan; Cai, Yudong; Wang, Hongwei

2013-06-01

35

Identifying dysfunctional crosstalk of pathways in various regions of Alzheimer's disease brains  

PubMed Central

Background Alzheimer's disease (AD) is a major neurodegenerative disorder leading to amnesia, cognitive impairment and dementia in the elderly. Usually this type of lesions results from dysfunctional protein cooperations in the biological pathways. In addition, AD progression is known to occur in different brain regions with particular features. Thus identification and analysis of crosstalk among dysregulated pathways as well as identification of their clusters in various diseased brain regions are expected to provide deep insights into the pathogenetic mechanism. Results Here we propose a network-based systems biology approach to detect the crosstalks among AD related pathways, as well as their dysfunctions in the six brain regions of AD patients. Through constructing a network of pathways, the relationships among AD pathway and its neighbor pathways are systematically investigated and visually presented by their intersections. We found that the significance degree of pathways related to the fatal disorders and the pathway overlapping strength can indicate the impacts of these neighbored pathways to AD development. Furthermore, the crosstalks among pathways reveal some evidence that the neighbor pathways of AD pathway closely cooperate and play important tasks in the AD progression. Conclusions Our study identifies the common and distinct features of the dysfunctional crosstalk of pathways in various AD brain regions. The global pathway crosstalk network and the clusters of relevant pathways of AD provide evidence of cooperativity among pathways for potential pathogenesis of the neuron complex disease.

2010-01-01

36

Pathway-Based Association Analyses Identified TRAIL Pathway for Osteoporotic Fractures  

Microsoft Academic Search

IntroductionHip OF carries the highest morbidity and mortality. Previous studies revealed that individual genes\\/loci in the Tumor Necrosis Factor (TNF) -Related Apoptosis-Inducing Ligand (TRAIL) pathway were associated with bone metabolism. This study aims to verify the potential association between hip OF and TRAIL pathway.MethodsUsing genome-wide genotype data from Affymetrix 500 K SNP arrays, we performed novel pathway-based association analyses for

Yin-Ping Zhang; Yao-Zhong Liu; Yan Guo; Xiao-Gang Liu; Xiang-Hong Xu; Yan-Fang Guo; Yuan Chen; Feng Zhang; Feng Pan; Xue-Zhen Zhu; Hong-Wen Deng

2011-01-01

37

Identifiability and inference of pathway motifs by epistasis analysis  

NASA Astrophysics Data System (ADS)

The accuracy of genetic network inference is limited by the assumptions used to determine if one hypothetical model is better than another in explaining experimental observations. Most previous work on epistasis analysis--in which one attempts to infer pathway relationships by determining equivalences among traits following mutations--has been based on Boolean or linear models. Here, we delineate the ultimate limits of epistasis-based inference by systematically surveying all two-gene network motifs and use symbolic algebra with arbitrary regulation functions to examine trait equivalences. Our analysis divides the motifs into equivalence classes, where different genetic perturbations result in indistinguishable experimental outcomes. We demonstrate that this partitioning can reveal important information about network architecture, and show, using simulated data, that it greatly improves the accuracy of genetic network inference methods. Because of the minimal assumptions involved, equivalence partitioning has broad applicability for gene network inference.

Phenix, Hilary; Perkins, Theodore; Kærn, Mads

2013-06-01

38

Identifiability and inference of pathway motifs by epistasis analysis.  

PubMed

The accuracy of genetic network inference is limited by the assumptions used to determine if one hypothetical model is better than another in explaining experimental observations. Most previous work on epistasis analysis-in which one attempts to infer pathway relationships by determining equivalences among traits following mutations-has been based on Boolean or linear models. Here, we delineate the ultimate limits of epistasis-based inference by systematically surveying all two-gene network motifs and use symbolic algebra with arbitrary regulation functions to examine trait equivalences. Our analysis divides the motifs into equivalence classes, where different genetic perturbations result in indistinguishable experimental outcomes. We demonstrate that this partitioning can reveal important information about network architecture, and show, using simulated data, that it greatly improves the accuracy of genetic network inference methods. Because of the minimal assumptions involved, equivalence partitioning has broad applicability for gene network inference. PMID:23822501

Phenix, Hilary; Perkins, Theodore; Kærn, Mads

2013-06-01

39

Aldehyde Dehydrogenases in Arabidopsis thaliana: Biochemical Requirements, Metabolic Pathways, and Functional Analysis  

PubMed Central

Aldehyde dehydrogenases (ALDHs) are a family of enzymes which catalyze the oxidation of reactive aldehydes to their corresponding carboxylic acids. Here we summarize molecular genetic and biochemical analyses of selected Arabidopsis ALDH genes. Aldehyde molecules are very reactive and are involved in many metabolic processes but when they accumulate in excess they become toxic. Thus activity of aldehyde dehydrogenases is important in regulating the homeostasis of aldehydes. Overexpression of some ALDH genes demonstrated an improved abiotic stress tolerance. Despite the fact that several reports are available describing a role for specific ALDHs, their precise physiological roles are often still unclear. Therefore a number of genetic and biochemical tools have been generated to address the function with an emphasis on stress-related ALDHs. ALDHs exert their functions in different cellular compartments and often in a developmental and tissue specific manner. To investigate substrate specificity, catalytic efficiencies have been determined using a range of substrates varying in carbon chain length and degree of carbon oxidation. Mutational approaches identified amino acid residues critical for coenzyme usage and enzyme activities.

Stiti, Naim; Missihoun, Tagnon D.; Kotchoni, Simeon O.; Kirch, Hans-Hubert; Bartels, Dorothea

2011-01-01

40

A method for integrating and ranking the evidence for biochemical pathways by mining reactions from text  

PubMed Central

Motivation: To create, verify and maintain pathway models, curators must discover and assess knowledge distributed over the vast body of biological literature. Methods supporting these tasks must understand both the pathway model representations and the natural language in the literature. These methods should identify and order documents by relevance to any given pathway reaction. No existing system has addressed all aspects of this challenge. Method: We present novel methods for associating pathway model reactions with relevant publications. Our approach extracts the reactions directly from the models and then turns them into queries for three text mining-based MEDLINE literature search systems. These queries are executed, and the resulting documents are combined and ranked according to their relevance to the reactions of interest. We manually annotate document-reaction pairs with the relevance of the document to the reaction and use this annotation to study several ranking methods, using various heuristic and machine-learning approaches. Results: Our evaluation shows that the annotated document-reaction pairs can be used to create a rule-based document ranking system, and that machine learning can be used to rank documents by their relevance to pathway reactions. We find that a Support Vector Machine-based system outperforms several baselines and matches the performance of the rule-based system. The success of the query extraction and ranking methods are used to update our existing pathway search system, PathText. Availability: An online demonstration of PathText 2 and the annotated corpus are available for research purposes at http://www.nactem.ac.uk/pathtext2/. Contact: makoto.miwa@manchester.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online.

Miwa, Makoto; Ohta, Tomoko; Rak, Rafal; Rowley, Andrew; Kell, Douglas B.; Pyysalo, Sampo; Ananiadou, Sophia

2013-01-01

41

Biological Pathway-Based Genome-Wide Association Analysis Identified the Vasoactive Intestinal Peptide (VIP) Pathway Important for Obesity  

PubMed Central

Recent genome-wide association (GWA) studies have identified a number of novel genes/variants predisposing to obesity. However, most GWA studies have focused on individual single-nucleotide polymorphism (SNPs)/genes with a strong statistical association with a phenotypic trait without considering potential biological interplay of the tested genes. In this study, we performed biological pathway-based GWA analysis for BMI and body fat mass. We used individual level genotype data generated from 1,000 unrelated US whites that were genotyped for ~500,000 SNPs. Statistical analysis of pathways was performed using a modification of the Gene Set Enrichment Algorithm. A total of 963 pathways extracted from the BioCarta, Kyoto Encyclopedia of Genes and Genomes (KEGG), Ambion GeneAssist, and Gene Ontology (GO) databases were analyzed. Among all of the pathways analyzed, the vasoactive intestinal peptide (VIP) pathway was most strongly associated with fat mass (nominal P = 0.0009) and was the third most strongly associated pathway with BMI (nominal P = 0.0006). After multiple testing correction, the VIP pathway achieved false-discovery rate (FDR) q values of 0.042 and 0.120 for fat mass and BMI, respectively. Our study is the first to demonstrate that the VIP pathway may play an important role in development of obesity. The study also highlights the importance of pathway-based GWA analysis in identification of additional genes/variants for complex human diseases.

Liu, Yong-Jun; Guo, Yan-Fang; Zhang, Li-Shu; Pei, Yu-Fang; Yu, Na; Yu, Ping; Papasian, Christopher J; Deng, Hong-Wen

2010-01-01

42

Characterization of the biochemical-pathway of uranium (VI) reduction in facultative anaerobic bacteria.  

PubMed

Cultures of U(VI) reducing bacteria sourced from abandoned uranium mine tailing dam were evaluated for their ability to reduce U(VI) to U(IV). The species in the cultures reduced U(VI) in solutions with initial U(VI) concentration up to 400mgL(-)(1) under a near neutral pH of 6.5. The electron flow pathway and fate of reduced species was also analysed in the individual species in order to evaluate the potential for control and optimisation of the reduction potential at the biochemical level. The results showed that U(VI) reduction in live cells was completely blocked by the NADH-dehydrogenase inhibitor, rotenone (C23H22O6), and thioredoxin inhibitor, cadmium chloride (CdCl2), showing that U(VI) reduction involves the electron flow through NADH-dehydrogenase, a primary electron donor to the electron transport respiratory (ETR) system. Mass balance analysis of uranium species aided by visual and electron microscopy suggest that most U(VI) reduction occurred on the cell surface of the isolated species. This finding indicates the possibility of easy uranium recovery for beneficial use through biological remediation. Should the U(VI) be reduced inside the cell, recovery would require complete disruption of the cells and therefore would be difficult. The study contributes new knowledge on the underlying mechanisms in the U(VI) reduction in facultative anaerobes. PMID:25065785

Mtimunye, Phalazane J; Chirwa, Evans M N

2014-10-01

43

Biochemical evidence for an alternate pathway in N-linked glycoprotein biosynthesis  

PubMed Central

Asparagine-linked glycosylation is a complex protein modification conserved among all three domains of life. Herein we report the in vitro analysis of N-linked glycosylation from the methanogenic archaeon Methanococcus voltae. Using a suite of synthetic and semisynthetic substrates, we show that AglK initiates N-linked glycosylation in M. voltae through the formation of ?-linked dolichyl monophosphate N-acetylglucosamine (Dol-P-GlcNAc), which contrasts with the polyprenyl-diphosphate intermediates that feature in both eukaryotes and bacteria. Intriguingly, AglK exhibits high sequence homology to dolichyl-phosphate ?-glucosyltransferases, including Alg5 in eukaryotes, suggesting a common evolutionary origin. The combined action of the first two enzymes, AglK and AglC, afforded an ?-linked Dol-P-glycan that serves as a competent substrate for the archaeal oligosaccharyl transferase AglB. These studies provide the first biochemical evidence revealing that despite the apparent similarity of the overall pathways, there are actually two general strategies to achieve N-linked glycoproteins across the domains of life.

Larkin, Angelyn; Chang, Michelle M.; Whitworth, Garrett E.; Imperiali, Barbara

2013-01-01

44

IndividualizedPath: identifying genetic alterations contributing to the dysfunctional pathways in glioblastoma individuals.  

PubMed

Due to the extensive complexity and high genetic heterogeneity of genetic alterations in cancer, comprehensively depicting the molecular mechanisms of cancer remains difficult. Characterizing personalized pathogenesis in cancer individuals can help to reveal new details of the complex mechanisms. In this study, we proposed an integrative method called IndividualizedPath to identify genetic alterations and their downstream risk pathways from the perspective of individuals through combining the DNA copy number, gene expression data and topological structures of biological pathways. By applying the method to TCGA glioblastoma multiforme (GBM) samples, we identified 394 gene-pathway pairs in 252 GBM individuals. We found that genes with copy number alterations showed high heterogeneity across GBM individuals, whereas they affected relatively consistent biological pathways. A global landscape of gene-pathway pairs showed that EGFR linked with multiple cancer-related biological pathways confers the highest risk of GBM. GBM individuals with MET-pathway pairs showed significantly shorter survival times than those with only MET amplification. Importantly, we found that the same risk pathways were affected by different genes in distinct groups of GBM individuals with a significant pattern of mutual exclusivity. Similarly, GBM subtype analysis revealed some subtype-specific gene-pathway pairs. In addition, we found that some rare copy number alterations had a large effect on contribution to numerous cancer-related pathways. In summary, our method offers the possibility to identify personalized cancer mechanisms, which can be applied to other types of cancer through the web server (). PMID:24911613

Ping, Yanyan; Zhang, Hongyi; Deng, Yulan; Wang, Li; Zhao, Hongying; Pang, Lin; Fan, Huihui; Xu, Chaohan; Li, Feng; Zhang, Yong; Gong, Yonghui; Xiao, Yun; Li, Xia

2014-07-01

45

Receptor-Drug Interaction: Europium Employment for Studying the Biochemical Pathway of G-Protein-Coupled Receptor Activation  

PubMed Central

In medicinal chemistry field, the biochemical pathways, involved in 7-transmembrane domains G-protein coupled receptors (GPCRs) activation, are commonly studied to establish the activity of ligands towards GPCRs. The most studied steps are the measurement of activated GTP-? subunit and stimulated intracellular cAMP. At the present, many researchers defined agonist or antagonist activity of potential GPCRs drugs employing [35S]GTP?S or [3H]cAMP as probes. Recently, the corresponding lanthanide labels Eu-GTP and Eu-cAMP as alternative to radiochemicals have been developed because they are highly sensitive, easy to automate, easily synthesized, they display a much longer shelf-life and they can be used in multilabel experiments. In the present review, the receptor-drug interaction by europium employment for studying the biochemical pathway of GPCR activation has been focused. Moreover, comparative studies between lanthanide label probes and the corresponding radiolabeled compounds have been carried out.

Antonio, Colabufo Nicola; Grazia, Perrone Maria; Marialessandra, Contino; Francesco, Berardi; Roberto, Perrone

2007-01-01

46

Training Signaling Pathway Maps to Biochemical Data with Constrained Fuzzy Logic: Quantitative Analysis of Liver Cell Responses to Inflammatory Stimuli  

PubMed Central

Predictive understanding of cell signaling network operation based on general prior knowledge but consistent with empirical data in a specific environmental context is a current challenge in computational biology. Recent work has demonstrated that Boolean logic can be used to create context-specific network models by training proteomic pathway maps to dedicated biochemical data; however, the Boolean formalism is restricted to characterizing protein species as either fully active or inactive. To advance beyond this limitation, we propose a novel form of fuzzy logic sufficiently flexible to model quantitative data but also sufficiently simple to efficiently construct models by training pathway maps on dedicated experimental measurements. Our new approach, termed constrained fuzzy logic (cFL), converts a prior knowledge network (obtained from literature or interactome databases) into a computable model that describes graded values of protein activation across multiple pathways. We train a cFL-converted network to experimental data describing hepatocytic protein activation by inflammatory cytokines and demonstrate the application of the resultant trained models for three important purposes: (a) generating experimentally testable biological hypotheses concerning pathway crosstalk, (b) establishing capability for quantitative prediction of protein activity, and (c) prediction and understanding of the cytokine release phenotypic response. Our methodology systematically and quantitatively trains a protein pathway map summarizing curated literature to context-specific biochemical data. This process generates a computable model yielding successful prediction of new test data and offering biological insight into complex datasets that are difficult to fully analyze by intuition alone.

Morris, Melody K.; Saez-Rodriguez, Julio; Clarke, David C.; Sorger, Peter K.; Lauffenburger, Douglas A.

2011-01-01

47

Biochemical and genomic analysis of the denitrification pathway within the genus Neisseria.  

PubMed

Since Neisseria gonorrhoeae and Neisseria meningitidis are obligate human pathogens, a comparison with commensal species of the same genus could reveal differences important in pathogenesis. The recent completion of commensal Neisseria genome draft assemblies allowed us to perform a comparison of the genes involved in the catalysis, assembly and regulation of the denitrification pathway, which has been implicated in the virulence of several bacteria. All species contained a highly conserved nitric oxide reductase (NorB) and a nitrite reductase (AniA or NirK) that was highly conserved in the catalytic but divergent in the N-terminal lipid modification and C-terminal glycosylation domains. Only Neisseria mucosa contained a nitrate reductase (Nar), and only Neisseria lactamica, Neisseria cinerea, Neisseria subflava, Neisseria flavescens and Neisseria sicca contained a nitrous oxide reductase (Nos) complex. The regulators of the denitrification genes, FNR, NarQP and NsrR, were highly conserved, except for the GAF domain of NarQ. Biochemical examination of laboratory strains revealed that all of the neisserial species tested except N. mucosa had a two- to fourfold lower nitrite reductase activity than N. gonorrhoeae, while N. meningitidis and most of the commensal Neisseria species had a two- to fourfold higher nitric oxide (NO) reductase activity. For N. meningitidis and most of the commensal Neisseria, there was a greater than fourfold reduction in the NO steady-state level in the presence of nitrite as compared with N. gonorrhoeae. All of the species tested generated an NO steady-state level in the presence of an NO donor that was similar to that of N. gonorrhoeae. The greatest difference between the Neisseria species was the lack of a functional Nos system in the pathogenic species N. gonorrhoeae and N. meningitidis. PMID:19762442

Barth, Kenneth R; Isabella, Vincent M; Clark, Virginia L

2009-12-01

48

Vascular and Perivascular NO Release and Transport: Biochemical Pathways of NOS1 and NOS3  

PubMed Central

Nitric oxide (NO) derived from nitric oxide synthase (NOS) is an important paracrine effector that maintains vascular tone. The release of NO mediated by NOS isozymes under various O2 conditions critically determines the NO bioavailability in tissues. Because of experimental difficulties, there has been no direct information on how enzymatic NO production and distribution change around arterioles under various oxygen conditions. In this study, we used computational models based on the analysis of biochemical pathways of enzymatic NO synthesis and the availability of NOS isozymes to quantify the NO production by neuronal NOS (NOS1) and endothelial NOS (NOS3). We compared the catalytic activities of NOS1 and NOS3 and their sensitivities to the concentration of substrate O2. Based on the NO release rates predicted from kinetic models, the geometric distribution of NO sources and mass balance analysis, we predicted the NO concentration profiles around an arteriole under various O2 conditions. The results indicated that NOS1-catalyzed NO production was significantly more sensitive to ambient O2 concentration than that catalyzed by NOS3. Also, the high sensitivity of NOS1 catalytic activity to O2 was associated with significantly reduced NO production and therefore NO concentrations, upon hypoxia. Moreover, the major source determining the distribution of NO was NOS1, which was abundantly expressed in the nerve fibers and mast cells close to arterioles, rather than NOS3, which was expressed in the endothelium. Finally, the perivascular NO concentration predicted by the models under conditions of normoxia was paradoxically at least an order of magnitude lower than a number of experimental measurements, suggesting a higher abundance of NOS1 or NOS3 and/or the existence of other enzymatic or non-enzymatic sources of NO in the microvasculature.

Chen, Kejing; Popel, Aleksander S.

2007-01-01

49

CyanoPhyChe: a database for physico-chemical properties, structure and biochemical pathway information of cyanobacterial proteins.  

PubMed

CyanoPhyChe is a user friendly database that one can browse through for physico-chemical properties, structure and biochemical pathway information of cyanobacterial proteins. We downloaded all the protein sequences from the cyanobacterial genome database for calculating the physico-chemical properties, such as molecular weight, net charge of protein, isoelectric point, molar extinction coefficient, canonical variable for solubility, grand average hydropathy, aliphatic index, and number of charged residues. Based on the physico-chemical properties, we provide the polarity, structural stability and probability of a protein entering in to an inclusion body (PEPIB). We used the data generated on physico-chemical properties, structure and biochemical pathway information of all cyanobacterial proteins to construct CyanoPhyChe. The data can be used for optimizing methods of expression and characterization of cyanobacterial proteins. Moreover, the 'Search' and data export options provided will be useful for proteome analysis. Secondary structure was predicted for all the cyanobacterial proteins using PSIPRED tool and the data generated is made accessible to researchers working on cyanobacteria. In addition, external links are provided to biological databases such as PDB and KEGG for molecular structure and biochemical pathway information, respectively. External links are also provided to different cyanobacterial databases. CyanoPhyChe can be accessed from the following URL: http://bif.uohyd.ac.in/cpc. PMID:23185330

Arun, P V Parvati Sai; Bakku, Ranjith Kumar; Subhashini, Mranu; Singh, Pankaj; Prabhu, N Prakash; Suzuki, Iwane; Prakash, Jogadhenu S S

2012-01-01

50

Metabolomic profiling reveals biochemical pathways and biomarkers associated with pathogenesis in cystic fibrosis cells.  

PubMed

Cystic fibrosis (CF) is a life-shortening disease caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. To gain an understanding of the epithelial dysfunction associated with CF mutations and discover biomarkers for therapeutics development, untargeted metabolomic analysis was performed on primary human airway epithelial cell cultures from three separate cohorts of CF patients and non-CF subjects. Statistical analysis revealed a set of reproducible and significant metabolic differences between the CF and non-CF cells. Aside from changes that were consistent with known CF effects, such as diminished cellular regulation against oxidative stress and osmotic stress, new observations on the cellular metabolism in the disease were generated. In the CF cells, the levels of various purine nucleotides, which may function to regulate cellular responses via purinergic signaling, were significantly decreased. Furthermore, CF cells exhibited reduced glucose metabolism in glycolysis, pentose phosphate pathway, and sorbitol pathway, which may further exacerbate oxidative stress and limit the epithelial cell response to environmental pressure. Taken together, these findings reveal novel metabolic abnormalities associated with the CF pathological process and identify a panel of potential biomarkers for therapeutic development using this model system. PMID:20675369

Wetmore, Diana R; Joseloff, Elizabeth; Pilewski, Joseph; Lee, Douglas P; Lawton, Kay A; Mitchell, Matthew W; Milburn, Michael V; Ryals, John A; Guo, Lining

2010-10-01

51

Studies of the lamin proteinase reveal multiple parallel biochemical pathways during apoptotic execution.  

PubMed Central

Although specific proteinases play a critical role in the active phase of apoptosis, their substrates are largely unknown. We previously identified poly(ADP-ribose) polymerase (PARP) as an apoptosis-associated substrate for proteinase(s) related to interleukin 1 beta-converting enzyme (ICE). Now we have used a cell-free system to characterize proteinase(s) that cleave the nuclear lamins during apoptosis. Lamin cleavage during apoptosis requires the action of a second ICE-like enyzme, which exhibits kinetics of cleavage and a profile of sensitivity to specific inhibitors that is distinct from the PARP proteinase. Thus, multiple ICE-like enzymes are required for apoptotic events in these cell-free extracts. Inhibition of the lamin proteinase with tosyllysine "chloromethyl ketone" blocks nuclear apoptosis prior to the packaging of condensed chromatin into apoptotic bodies. Under these conditions, the nuclear DNA is fully cleaved to a nucleosomal ladder. Our studies reveal that the lamin proteinase and the fragmentation nuclease function in independent parallel pathways during the final stages of apoptotic execution. Neither pathway alone is sufficient for completion of nuclear apoptosis. Instead, the various activities cooperate to drive the disassembly of the nucleus. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4

Lazebnik, Y A; Takahashi, A; Moir, R D; Goldman, R D; Poirier, G G; Kaufmann, S H; Earnshaw, W C

1995-01-01

52

Using soil moisture and spatial yield patterns to identify subsurface flow pathways.  

PubMed

Subsurface soil water dynamics can influence crop growth and the fate of surface-applied fertilizers and pesticides. Recently, a method was proposed using only ground-penetrating radar (GPR) and digital elevation maps (DEMs) to identify locations where subsurface water converged into discrete pathways. For this study, the GPR protocol for identifying horizontal subsurface flow pathways was extended to a 3.2-ha field, uncertainty is discussed, and soil moisture and yield patterns are presented as confirming evidence of the extent of the subsurface flow pathways. Observed soil water contents supported the existence of discrete preferential funnel flow processes occurring near the GPR-identified preferential flow pathways. Soil moisture also played a critical role in the formation of corn (Zea mays L.) grain yield patterns with yield spatial patterns being similar for mild and severe drought conditions. A buffer zone protocol was introduced that allowed the impact of subsurface flow pathways on corn grain yield to be quantified. Results indicate that when a GPR-identified subsurface clay layer was within 2 m of the soil surface, there was a beneficial impact on yield during a drought year. Furthermore, the buffer zone analysis demonstrated that corn grain yields decreased as the horizontal distance from the GPR-identified subsurface flow pathways increased during a drought year. Averaged real-time soil moisture contents at 0.1 m also decreased with increasing distance from the GPR-identified flow pathways. This research suggests that subsurface flow pathways exist and influence soil moisture and corn grain yield patterns. PMID:15647558

Gish, T J; Walthall, C L; Daughtry, C S T; Kung, K-J S

2005-01-01

53

The Use of Functional Chemical-Protein Associations to Identify Multi-Pathway Renoprotectants  

PubMed Central

Typically, most nephropathies can be categorized as complex human diseases in which the cumulative effect of multiple minor genes, combined with environmental and lifestyle factors, determines the disease phenotype. Thus, multi-target drugs would be more likely to facilitate comprehensive renoprotection than single-target agents. In this study, functional chemical-protein association analysis was performed to retrieve multi-target drugs of high pathway wideness from the STITCH 3.1 database. Pathway wideness of a drug evaluated the efficiency of regulation of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in quantity. We identified nine experimentally validated renoprotectants that exerted remarkable impact on KEGG pathways by targeting a limited number of proteins. We selected curcumin as an illustrative compound to display the advantage of multi-pathway drugs on renoprotection. We compared curcumin with hemin, an agonist of heme oxygenase-1 (HO-1), which significantly affects only one KEGG pathway, porphyrin and chlorophyll metabolism (adjusted p?=?1.5×10?5). At the same concentration (10 µM), both curcumin and hemin equivalently mitigated oxidative stress in H2O2-treated glomerular mesangial cells. The benefit of using hemin was derived from its agonistic effect on HO-1, providing relief from oxidative stress. Selective inhibition of HO-1 completely blocked the action of hemin but not that of curcumin, suggesting simultaneous multi-pathway intervention by curcumin. Curcumin also increased cellular autophagy levels, enhancing its protective effect; however, hemin had no effects. Based on the fact that the dysregulation of multiple pathways is implicated in the etiology of complex diseases, we proposed a feasible method for identifying multi-pathway drugs from compounds with validated targets. Our efforts will help identify multi-pathway agents capable of providing comprehensive protection against renal injuries.

Xu, Jia; Meng, Kexin; Zhang, Rui; Yang, He; Liao, Chang; Zhu, Wenliang; Jiao, Jundong

2014-01-01

54

Biochemical Characterization of the CDP-d-Arabinitol Biosynthetic Pathway in Streptococcus pneumoniae 17F  

PubMed Central

Streptococcus pneumoniae is a major human pathogen associated with many diseases worldwide. Capsular polysaccharides (CPSs) are the major virulence factor. The biosynthetic pathway of d-arabinitol, which is present in the CPSs of several S. pneumoniae serotypes, has never been identified. In this study, the genes abpA (previously known as abp1) and abpB (previously known as abp2), which have previously been reported to be responsible for nucleoside diphosphate (NDP)-d-arabinitol (the nucleotide-activated form of d-arabinitol) synthesis, were cloned. The enzyme products were overexpressed, purified, and analyzed for their respective activities. Novel products produced by AbpA- and AbpB-catalyzing reactions were detected by capillary electrophoresis, and the structures of the products were elucidated using electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. As a result, abpA was identified to be a d-xylulose-5-phosphate cytidylyltransferase-encoding gene, responsible for the transfer of CTP to d-xylulose-5-phosphate (d-Xlu-5-P) to form CDP-d-xylulose, and abpB was characterized to be a CDP-d-xylulose reductase-encoding gene, responsible for the conversion of CDP-d-xylulose to CDP-d-arabinitol as the final product. The kinetic parameters of AbpA for the substrates d-Xlu-5-P and CTP and those of AbpB for the substrate CDP-d-xylulose and the cofactors NADH or NADPH were measured, and the effects of temperature, pH, and cations on the two enzymes were analyzed. This study confirmed the involvement of the genes abpA and abpB and their products in the biosynthetic pathway of CDP-d-arabinitol.

Wang, Quan; Xu, Yanli; Perepelov, Andrei V.; Knirel, Yuriy A.; Reeves, Peter R.; Shashkov, Alexander S.; Guo, Xi; Ding, Peng

2012-01-01

55

Biochemical characterization of the CDP-D-arabinitol biosynthetic pathway in Streptococcus pneumoniae 17F.  

PubMed

Streptococcus pneumoniae is a major human pathogen associated with many diseases worldwide. Capsular polysaccharides (CPSs) are the major virulence factor. The biosynthetic pathway of D-arabinitol, which is present in the CPSs of several S. pneumoniae serotypes, has never been identified. In this study, the genes abpA (previously known as abp1) and abpB (previously known as abp2), which have previously been reported to be responsible for nucleoside diphosphate (NDP)-D-arabinitol (the nucleotide-activated form of D-arabinitol) synthesis, were cloned. The enzyme products were overexpressed, purified, and analyzed for their respective activities. Novel products produced by AbpA- and AbpB-catalyzing reactions were detected by capillary electrophoresis, and the structures of the products were elucidated using electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. As a result, abpA was identified to be a D-xylulose-5-phosphate cytidylyltransferase-encoding gene, responsible for the transfer of CTP to D-xylulose-5-phosphate (D-Xlu-5-P) to form CDP-D-xylulose, and abpB was characterized to be a CDP-D-xylulose reductase-encoding gene, responsible for the conversion of CDP-D-xylulose to CDP-D-arabinitol as the final product. The kinetic parameters of AbpA for the substrates D-Xlu-5-P and CTP and those of AbpB for the substrate CDP-D-xylulose and the cofactors NADH or NADPH were measured, and the effects of temperature, pH, and cations on the two enzymes were analyzed. This study confirmed the involvement of the genes abpA and abpB and their products in the biosynthetic pathway of CDP-D-arabinitol. PMID:22328666

Wang, Quan; Xu, Yanli; Perepelov, Andrei V; Knirel, Yuriy A; Reeves, Peter R; Shashkov, Alexander S; Guo, Xi; Ding, Peng; Feng, Lu

2012-04-01

56

Influence of anabolic combinations of an androgen plus an estrogen on biochemical pathways in bovine uterine endometrium and ovary.  

PubMed

The application of anabolic steroids in food producing animals is forbidden in the EU since 1988, but the abuse of such drugs is a potential problem. The existing test systems are based on known compounds and can be eluded by newly emerging substances. The examination of physiological effects of anabolic hormones on different tissues to indirectly detect misuse might overcome this problem. Two studies were conducted with post-pubertal 24-months old Nguni heifers and pre-pubertal female 2-4 weeks old Holstein Friesian calves, respectively. The animals of the accordant treatment groups were administered combinations of estrogenic and androgenic compounds. The measurement of the gene expression pattern was undertaken with RT-qPCR. Target genes of different functional groups (receptors, angiogenesis, steroid synthesis, proliferation, apoptosis, nutrient metabolism and others) have been quantified. Several biochemical pathways were shown to be influenced by anabolic treatment. Both studies identified significant regulations in steroid and growth factor receptors (AR, ER?, LHR, FSHR, Flt-1, PR, IGF-1R, Alk-6), angiogenic and tissue remodeling factors (VEGFs, FGFs, BMPs, ANGPT-2, MMPs, TIMP-2, CTSB), steroid synthesis (S5A1, HSD17, CYP19A1), proliferation (TNF?, IGF-1, IGFBPs, p53, c-fos; CEBPD, c-kit), apoptosis (CASP3, FasL, p53) and others (C7, INHA, STAR). Several genes were regulated to opposite directions in post-pubertal compared to pre-pubertal animals. PCA for Nguni heifers demonstrated a distinct separation between the control and the treatment group. In conclusion, anabolics modify hormone sensitivity and steroid synthesis, and they induce proliferative effects in the whole reproductive tract (uterus and ovary) as well as anti-angiogenic effects in the ovary. However, the extent will depend on the developmental stage of the animals. PMID:21272641

Becker, C; Riedmaier, I; Reiter, M; Tichopad, A; Groot, M J; Stolker, A A M; Pfaffl, M W; Nielen, M F W; Meyer, H H D

2011-07-01

57

Targeting the Wnt pathway in zebrafish as a screening method to identify novel therapeutic compounds.  

PubMed

Activating mutations in the Wnt signaling pathway account for the initiation of greater than 90% of all colorectal cancers and this pathway has been implicated in numerous other diseases. Therefore, identifying small molecule inhibitors of this pathway is of critical importance towards identifying clinically relevant drugs. Numerous screens have been employed to identify therapeutic reagents, but none have made it to advanced clinical trials, suggesting that traditional screening methods are ineffective at identifying clinically relevant targets. Here, we describe a novel in vivo screen to identify small molecule inhibitors of the Wnt pathway. Specifically, treatment of zebrafish embryos with LiCl inhibits GSK3 kinase function, resulting in hyperactivation of the signaling pathway and an eyeless phenotype at 1 day post fertilization. Using the small molecule XAV939, a known inhibitor of Wnt signaling, we rescued the LiCl induced eyeless phenotype, confirming efficacy of the screen. We next tested our assay with 400 known small molecule kinase inhibitors, none of which should inhibit Wnt signaling below the level of GSK3 based on their known targets. Accordingly, none of these small molecules rescued the eyeless phenotype, which demonstrates the stringency of the assay. However, several of these small molecule kinase inhibitors did generate a non-Wnt phenotype in accordance with the kinase they targeted. Therefore, combining the efficacy, sensitivity, and stringency of this preliminary screen, this model will provide an alternative to the traditional in vitro screen, generating potentially clinical relevant drugs in a rapid and cost-effective way. PMID:24414478

Robertson, Joshua K; Danzmann, Kestral; Charles, Sherise; Blake, Katherine; Olivares, Annia; Bamikole, Solape; Olson, Meghan; Van Raay, Terence J

2014-02-01

58

Systems analysis utilising pathway interactions identifies sonic hedgehog pathway as a primary biomarker and oncogenic target in hepatocellular carcinoma.  

PubMed

The development and progression of cancer is associated with disruption of biological networks. Historically studies have identified sets of signature genes involved in events ultimately leading to the development of cancer. Identification of such sets does not indicate which biologic processes are oncogenic drivers and makes it difficult to identify key networks to target for interventions. Using a comprehensive, integrated computational approach, the authors identify the sonic hedgehog (SHH) pathway as the gene network that most significantly distinguishes tumour and tumour-adjacent samples in human hepatocellular carcinoma (HCC). The analysis reveals that the SHH pathway is commonly activated in the tumour samples and its activity most significantly differentiates tumour from the non-tumour samples. The authors experimentally validate these in silico findings in the same biologic material using Western blot analysis. This analysis reveals that the expression levels of SHH, phosphorylated cyclin B1, and CDK7 levels are much higher in most tumour tissues as compared to normal tissue. It is also shown that siRNA-mediated silencing of SHH gene expression resulted in a significant reduction of cell proliferation in a liver cancer cell line, SNU449 indicating that SHH plays a major role in promoting cell proliferation in liver cancer. The SHH pathway is a key network underpinning HCC aetiology which may guide the development of interventions for this most common form of human liver cancer. PMID:24712101

Efroni, Sol; Meerzaman, Daoud; Schaefer, Carl F; Greenblum, Sharon; Soo-Lyu, Myung; Hu, Ying; Cultraro, Constance; Meshorer, Eran; Buetow, Kenneth H

2013-12-01

59

A genetic screen in Drosophila for identifying novel components of the hedgehog signaling pathway.  

PubMed

The Hedgehog signaling pathway plays an essential role in the pattern formation and development of metazoan animals. Misregulation of Hedgehog signaling has also been associated with the formation of multiple types of cancer. For these reasons, the Hedgehog pathway has attracted considerable interest. Many proteins required in the Hedgehog pathway have been identified, and while much has been learned about their function in signal transduction, it is clear that this complement of proteins does not comprise the full set necessary for Hedgehog signal transduction. Because significant gaps remain in our knowledge of the molecules required for Hedgehog signaling, we performed an enhancer/suppressor screen in Drosophila melanogaster to identify novel components of the pathway. In addition to the isolation of new alleles of the known pathway components patched and smoothened, this screen identified 14 novel complementation groups and a larger number of loci represented by single alleles. These groups include mutations in the genes encoding the translation factors eRF1 and eIF1A and the kinesin-like protein Pavarotti. It also identified mutations in a gene whose product is necessary for the movement of Hedgehog protein through tissues. PMID:15744048

Collins, Russell T; Cohen, Stephen M

2005-05-01

60

Heterogeneous nuclear ribonucleoproteins C1/C2 identified as autoantigens by biochemical and mass spectrometric methods.  

PubMed

The antigenic specificity of an unusual antinuclear antibody pattern in three patient sera was identified after separating HeLa-cell nuclear extracts by two-dimensional (2D) gel electrophoresis and localizing the antigens by immunoblotting with patient serum. Protein spots were excised from the 2D gel and their contents were analyzed by matrix-assisted laser desorption-ionization (MALDI) or nanoelectrospray ionization time-of-flight (TOF) tandem mass spectrometry (MS) after in-gel digestion with trypsin. A database search identified the proteins as the C1 and C2 heterogeneous nuclear ribonucleoproteins. The clinical spectrum of patients with these autoantibodies includes arthritis, psoriasis, myositis, and scleroderma. None of 59 patients with rheumatoid arthritis, 19 with polymyositis, 33 with scleroderma, and 10 with psoriatic arthritis had similar antibodies. High-resolution protein-separation methods and mass-spectrometric peptide mapping in combination with database searches are powerful tools in the identification of novel autoantigen specificities. PMID:11056675

Heegaard, N H; Larsen, M R; Muncrief, T; Wiik, A; Roepstorff, P

2000-01-01

61

Application of an integrated physical and functional screening approach to identify inhibitors of the Wnt pathway  

PubMed Central

Large-scale proteomic approaches have been used to study signaling pathways. However, identification of biologically relevant hits from a single screen remains challenging due to limitations inherent in each individual approach. To overcome these limitations, we implemented an integrated, multi-dimensional approach and used it to identify Wnt pathway modulators. The LUMIER protein–protein interaction mapping method was used in conjunction with two functional screens that examined the effect of overexpression and siRNA-mediated gene knockdown on Wnt signaling. Meta-analysis of the three data sets yielded a combined pathway score (CPS) for each tested component, a value reflecting the likelihood that an individual protein is a Wnt pathway regulator. We characterized the role of two proteins with high CPSs, Ube2m and Nkd1. We show that Ube2m interacts with and modulates ?-catenin stability, and that the antagonistic effect of Nkd1 on Wnt signaling requires interaction with Axin, itself a negative pathway regulator. Thus, integrated physical and functional mapping in mammalian cells can identify signaling components with high confidence and provides unanticipated insights into pathway regulators.

Miller, Bryan W; Lau, Garnet; Grouios, Chris; Mollica, Emanuela; Barrios-Rodiles, Miriam; Liu, Yongmei; Datti, Alessandro; Morris, Quaid; Wrana, Jeffrey L; Attisano, Liliana

2009-01-01

62

Genetic and Biochemical Characterization of a Pathway for the Degradation of 2-Aminoethylphosphonate in Sinorhizobium meliloti 1021*  

PubMed Central

A variety of microorganisms have the ability to use phosphonic acids as sole sources of phosphorus. Here, a novel pathway for degradation of 2-aminoethylphosphonate in the bacterium Sinorhizobium meliloti 1021 is proposed based on the analysis of the genome sequence. Gene deletion experiments confirmed the involvement of the locus containing phnW, phnA, and phnY genes in the conversion of 2-aminoethylphosphonate to inorganic phosphate. Biochemical studies of the recombinant PhnY and PhnA proteins verified their roles as phosphonoacetaldehyde dehydrogenase and phosphonoacetate hydrolase, respectively. This pathway is likely not limited to S. meliloti as suggested by the presence of homologous gene clusters in other bacterial genomes.

Borisova, Svetlana A.; Christman, Harry D.; Metcalf, M. E. Mourey; Zulkepli, Nurul A.; Zhang, Jun Kai; van der Donk, Wilfred A.; Metcalf, William W.

2011-01-01

63

Bacterial cytological profiling rapidly identifies the cellular pathways targeted by antibacterial molecules  

PubMed Central

Identifying the mechanism of action for antibacterial compounds is essential for understanding how bacteria interact with one another and with other cell types and for antibiotic discovery efforts, but determining a compound's mechanism of action remains a serious challenge that limits both basic research and antibacterial discovery programs. Here, we show that bacterial cytological profiling (BCP) is a rapid and powerful approach for identifying the cellular pathway affected by antibacterial molecules. BCP can distinguish between inhibitors that affect different cellular pathways as well as different targets within the same pathway. We use BCP to demonstrate that spirohexenolide A, a spirotetronate that is active against methicillin-resistant Staphylococcus aureus, rapidly collapses the proton motive force. BCP offers a simple, one-step assay that can be broadly applied, solving the longstanding problem of how to rapidly determine the cellular target of thousands of compounds.

Nonejuie, Poochit; Burkart, Michael; Pogliano, Kit; Pogliano, Joe

2013-01-01

64

Method for identifying biochemical and chemical reactions and micromechanical processes using nanomechanical and electronic signal identification  

DOEpatents

A scanning probe microscope, such as an atomic force microscope (AFM) or a scanning tunneling microscope (STM), is operated in a stationary mode on a site where an activity of interest occurs to measure and identify characteristic time-varying micromotions caused by biological, chemical, mechanical, electrical, optical, or physical processes. The tip and cantilever assembly of an AFM is used as a micromechanical detector of characteristic micromotions transmitted either directly by a site of interest or indirectly through the surrounding medium. Alternatively, the exponential dependence of the tunneling current on the size of the gap in the STM is used to detect micromechanical movement. The stationary mode of operation can be used to observe dynamic biological processes in real time and in a natural environment, such as polymerase processing of DNA for determining the sequence of a DNA molecule. 6 figs.

Holzrichter, J.F.; Siekhaus, W.J.

1997-04-15

65

Combinatorial drug screening identifies compensatory pathway interactions and adaptive resistance mechanisms  

PubMed Central

Constitutively activated signaling molecules are often the primary drivers of malignancy, and are favored targets for therapeutic intervention. However, the effectiveness of targeted inhibition of cell signaling can be blunted by compensatory signaling which generates adaptive resistance mechanisms and reduces therapeutic responses. Therefore, it is important to identify and target these compensatory pathways with combinations of targeted agents to achieve durable clinical benefit. In this report, we demonstrate the use of high-throughput combinatorial drug screening as a discovery tool to identify compensatory pathways that generate resistance to the cytotoxic effects of targeted therapy. We screened 420 drug combinations in 14 different cell lines representing three cancer lineages, and assessed the ability of each combination to cause synergistic cytotoxicity. Drug substitution studies were used to validate the functionally important drug targets. Of the 84 combinations that caused robust synergy in multiple cell lines, none were synergistic in more than half of the lines tested, and we observed no pattern of lineage specificity in the observed synergies. This reflects the plasticity of cell signaling networks, even among cell lines of the same tissue of origin. Mechanistic analysis of one novel synergistic combination identified in the screen, the multi-kinase inhibitor Ro31-8220 and lapatinib, demonstrated compensatory crosstalk between the p70S6 kinase and EGF receptor pathways. In addition, we identified BAD as a node of convergence between these two pathways that may be playing a role in the enhanced apoptosis observed upon combination treatment.

Axelrod, Mark; Gordon, Vicki L.; Conaway, Mark; Tarcsafalvi, Adel; Neitzke, Daniel J.; Gioeli, Daniel; Weber, Michael J.

2013-01-01

66

Integrative analysis of transcript and metabolite profiling data sets to evaluate the regulation of biochemical pathways during photomorphogenesis.  

PubMed

One of the key developmental processes during photomorphogenesis is the differentiation of prolamellar bodies of proplastids into thylakoid membranes containing the photosynthetic pigment-protein complexes of chloroplasts. To study the regulatory events controlling pigment-protein complex assembly, including the biosynthesis of metabolic precursors and pigment end products, etiolated Arabidopsis thaliana seedlings were irradiated with continuous red light (Rc), which led to rapid greening, or continuous far-red light (FRc), which did not result in visible greening, and subjected to analysis by oligonucleotide microarrays and targeted metabolite profiling. An analysis using BioPathAt, a bioinformatic tool that allows the visualization of post-genomic data sets directly on biochemical pathway maps, indicated that in Rc-treated seedlings mRNA expression and metabolite patterns were tightly correlated (e.g., Calvin cycle, biosynthesis of chlorophylls, carotenoids, isoprenoid quinones, thylakoid lipids, sterols, and amino acids). K-means clustering revealed that gene expression patterns across various biochemical pathways were very similar in Rc- and FRc-treated seedlings (despite the visible phenotypic differences), whereas a principal component analysis of metabolite pools allowed a clear distinction between both treatments (in accordance with the visible phenotype). Our results illustrate the general importance of integrative approaches to correlate post-genomic data sets with phenotypic outcomes. PMID:16460663

Ghassemian, Majid; Lutes, Jason; Tepperman, James M; Chang, Hur-Song; Zhu, Tong; Wang, Xun; Quail, Peter H; Lange, B Markus

2006-04-15

67

Rapid biochemical test to identify verocytotoxin-positive strains of Escherichia coli serotype O157.  

PubMed Central

Fluorogenic procedures were used with the substrate 4-methylumbelliferyl-beta-D-glucuronide (MUG) to identify Escherichia coli. Most strains produced beta-glucuronidase and, thus, were MUG positive. A 20-min procedure was developed to detect glucuronidase activity in 1,295 bacterial cultures, representing 23 genera, of strains that were isolated from clinical specimens. Very few organisms other than E. coli were MUG positive. Of 682 E. coli strains that were isolated, 630 (92.4%) were MUG positive. When an additional 188 E. coli serotype O157 isolates were examined, 155 E. coli O157:H7, 10 E. coli O157:H-, and 1 E. coli O157:H (rough) isolate were MUG negative. All 166 cultures were verocytotoxin positive. Of the remaining 22 E. coli O157 isolates, 2 isolates were O157:H-, 1 isolate was O157:H (rough), and 19 isolates were other H types (H6, H16, H19, H25, H42, and H45); these 22 isolates were MUG positive. All 22 cultures were verocytotoxin negative. The rapid MUG procedure can be used to predict verocytotoxin-positive isolates of E. coli O157; that is, there is a very good likelihood that MUG-negative E. coli O157 isolates are verocytotoxin positive.

Thompson, J S; Hodge, D S; Borczyk, A A

1990-01-01

68

Presenilin-Based Genetic Screens in Drosophila melanogaster Identify Novel Notch Pathway Modifiers  

PubMed Central

Presenilin is the enzymatic component of ?-secretase, a multisubunit intramembrane protease that processes several transmembrane receptors, such as the amyloid precursor protein (APP). Mutations in human Presenilins lead to altered APP cleavage and early-onset Alzheimer's disease. Presenilins also play an essential role in Notch receptor cleavage and signaling. The Notch pathway is a highly conserved signaling pathway that functions during the development of multicellular organisms, including vertebrates, Drosophila, and C. elegans. Recent studies have shown that Notch signaling is sensitive to perturbations in subcellular trafficking, although the specific mechanisms are largely unknown. To identify genes that regulate Notch pathway function, we have performed two genetic screens in Drosophila for modifiers of Presenilin-dependent Notch phenotypes. We describe here the cloning and identification of 19 modifiers, including nicastrin and several genes with previously undescribed involvement in Notch biology. The predicted functions of these newly identified genes are consistent with extracellular matrix and vesicular trafficking mechanisms in Presenilin and Notch pathway regulation and suggest a novel role for ?-tubulin in the pathway.

Mahoney, Matt B.; Parks, Annette L.; Ruddy, David A.; Tiong, Stanley Y. K.; Esengil, Hanife; Phan, Alexander C.; Philandrinos, Panos; Winter, Christopher G.; Chatterjee, Runa; Huppert, Kari; Fisher, William W.; L'Archeveque, Lynn; Mapa, Felipa A.; Woo, Wendy; Ellis, Michael C.; Curtis, Daniel

2006-01-01

69

Transcriptomic and biochemical analyses identify a family of chlorhexidine efflux proteins  

PubMed Central

Chlorhexidine is widely used as an antiseptic or disinfectant in both hospital and community settings. A number of bacterial species display resistance to this membrane-active biocide. We examined the transcriptomic response of a representative nosocomial human pathogen, Acinetobacter baumannii, to chlorhexidine to identify the primary chlorhexidine resistance elements. The most highly up-regulated genes encoded components of a major multidrug efflux system, AdeAB. The next most highly overexpressed gene under chlorhexidine stress was annotated as encoding a hypothetical protein, named here as AceI. Orthologs of the aceI gene are conserved within the genomes of a broad range of proteobacterial species. Expression of aceI or its orthologs from several other ?- or ?-proteobacterial species in Escherichia coli resulted in significant increases in resistance to chlorhexidine. Additionally, disruption of the aceI ortholog in Acinetobacter baylyi rendered it more susceptible to chlorhexidine. The AceI protein was localized to the membrane after overexpression in E. coli. This protein was purified, and binding assays demonstrated direct and specific interactions between AceI and chlorhexidine. Transport assays using [14C]-chlorhexidine determined that AceI was able to mediate the energy-dependent efflux of chlorhexidine. An E15Q AceI mutant with a mutation in a conserved acidic residue, although unable to mediate chlorhexidine resistance and transport, was still able to bind chlorhexidine. Taken together, these data are consistent with AceI being an active chlorhexidine efflux protein and the founding member of a family of bacterial drug efflux transporters.

Hassan, Karl A.; Jackson, Scott M.; Penesyan, Anahit; Patching, Simon G.; Tetu, Sasha G.; Eijkelkamp, Bart A.; Brown, Melissa H.; Henderson, Peter J. F.; Paulsen, Ian. T.

2013-01-01

70

Heterogeneous nuclear ribonucleoproteins C1/C2 identified as autoantigens by biochemical and mass spectrometric methods  

PubMed Central

Introduction: The classification of antinuclear antibodies (ANAs) is important for diagnosis and prognosis and for understanding the molecular pathology of autoimmune disease. Many of the proteins that associate with RNA in the ribonucleoprotein (RNP) complexes of the spliceosome have been found to react with some types of ANA [1], including proteins of the heterogeneous nuclear RNP (hnRNP) complex that associate with newly transcribed pre-mRNA. Autoantibodies to the A2, B1, and B2 proteins of hnRNP found in some patients may be markers of several overlap syndromes [2]. However, ANAs with specificity for these proteins as well as for the D protein also appear to occur in many distinct connective-tissue diseases, although epitope specificities may differ [3]. ANAs with specificity for the C component of hnRNP (consisting of the C1 and C2 proteins) have to our knowledge so far been described in only one case [4]. We here describe the approach taken to unambiguously identify the C1/C2 proteins as ANA targets in the sera of some patients. Aims: To determine the fine specificity of sera containing an unusual speckled ANA-staining pattern using a combination of 2D gel electrophoresis and MS. Methods: Patient sera were screened for ANAs by indirect immunofluorescence microscopy on HEp-2 cells (cultured carcinoma cells). Sera with an unusual, very regular, speckled ANA pattern were tested for reactivity with components of nuclear extracts of HeLa cells that were separated by one-dimensional (1D) or 2D gel electrophoresis or by reversed-phase high-performance liquid chromatography (HPLC). IgG reactivity was assessed by immunoblotting. Reactive protein spots from 2D separations were excised from the gels and subjected to in-gel digestion with trypsin for subsequent peptide mapping, partial peptide sequencing, and protein identification by MS and tandem MS on a hybrid electrospray ionization/quadrupole/time-of-flight (ESI-Q-TOF) mass spectrometer [5,6,7]. Results: We observed a strong nuclear staining pattern (titer >1280) with the characteristic even-sized coarse speckles and no staining of nucleoli in sera from three patients. On immunoblots of nuclear extracts from HeLa cells, these sera stained two distinct bands, at Mr 42 000 and 41 000. There activity strongly resembled that of the patient originally described by Stanek et al [4]. The antigens were enriched by fractionating the extract using reversed-phase HPLC on a C4 column, and the two reactive spots on 2D separations were excised for identification. The two components appeared to be of approximately the same isoelectric points, although their molecular masses differed by approximately 2000. Peptide-mass mapping was performed by matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) MS on the tryptic peptide mixture generated by digestion of the two excised proteins. The database search suggested that the two proteins were C1/C2 hnRNPs (Swissprot accession number P07910). The identity of the proteins was further confirmed by tandem MS using an ESI-Q-TOF instrument. One peptide carrying two positive charges (m/z 580.32 Da), corresponding to a peptide mass of 1158.7 Da, was selected as a precursor ion and partially sequenced by collisional fragmentation. The fragmented peptide was found to represent the tryptic fragment VDSLLENLEK, ie amino acids 207-216 (C2 protein numbering). Four other peptides were partially sequenced and all of them matched the human C1/C2 hnRNP sequence. The theoretical masses of C1 and C2 are 32.0 and 33.3 kDa, respectively. The difference between the two sequences is a 13-amino-acid insert in C2 between positions 107 and 108 of C1. The presence of a specific tryptic fragment in the MALDI-TOF peptide-mass map from the higher-molecular-mass spot containing a 13-amino-acid insert that was not present in the lower-molecular-mass spot, further demonstrated that the two components represented the two isoforms of the C class of hnRNPs. The patient whose case prompted us to investigate the specificities of these antibodies was a 72-year-ol

HH Heegaard, Niels; R Larsen, Martin; Muncrief, Terri; Wiik , Allan; Roepstorff, Peter

2000-01-01

71

Genetic and biochemical investigations on bacterial catabolic pathways for lignin-derived aromatic compounds.  

PubMed

Lignins are the most abundant aromatic compounds in nature, and their decomposition is essential to the terrestrial carbon cycle. White rot fungi secreting phenol oxidases are assumed to be involved in the initial degradation of native lignin, whereas bacteria play a main role in the mineralization of lignin-derived low-molecular-weight compounds in soil. There are a number of reports on the degradation pathways for lignin-derived aromatic compounds, but their catabolism has not been enzymatically or genetically characterized. Sphingomonas paucimobilis SYK-6 is one of the best-characterized lignin-degrading bacteria. It can grow on a wide variety of lignin-related biaryls and monoaryls, including beta-aryl ether, biphenyl, diarylpropane, and phenylpropane. These compounds are degraded via the protocatechuate (PCA) 4,5-cleavage pathway or multiple 3-O-methylgallate (3MGA) catabolic pathways. In this review, the enzyme systems for beta-aryl ether and biphenyl degradation, O demethylation linked with one carbon metabolism, the PCA 4,5-cleavage pathway, and the multiple 3MGA catabolic pathways in SYK-6 are outlined. PMID:17213657

Masai, Eiji; Katayama, Yoshihiro; Fukuda, Masao

2007-01-01

72

Biosynthesis of UDP-N,N?-Diacetylbacillosamine in Acinetobacter baumannii: Biochemical Characterization and Correlation to Existing Pathways  

PubMed Central

The Gram-negative, opportunistic pathogen Acinetobacter baumannii has recently captured headlines due to its ability to circumvent current antibiotic therapies. Herein we show that the multi-drug resistant (MDR) AYE strain of A. baumannii contains a gene locus that encodes three enzymes responsible for the biosynthesis of the highly-modified bacterial nucleotide sugar, UDP-N,N -diacetylbacillosamine (UDP-diNAcBac). Previously, this UDP-sugar has been implicated in the pgl pathway of Campylobacter jejuni. Here we report the overexpression, purification, and biochemical characterization of the A. baumannii enzymes WeeK, WeeJ, and WeeI that are responsible for the production of UDP-diNAcBac. We also demonstrate the function of the phosphoglycosyltransferase (WeeH), which transfers the diNAcBac moiety to undecaprenyl-phosphate. UDP-diNAcBac biosynthesis in A. baumannii is also directly compared to the homologous pathways in the pathogens C. jejuni and Neisseria gonorrhoeae. This work demonstrates for the first time the ability of A. baumannii to generate the highly-modified, UDP-diNAcBac nucleotide sugar found previously in other bacteria adding to the growing list of pathogens that assemble glycoconjugates including bacillosamine. Additionally, characterization of these pathway enzymes highlights the opportunity for investigating the significance of highly-modified sugars in bacterial pathogenesis.

Morrison, Michael J.; Imperiali, Barbara

2013-01-01

73

A genome-wide approach identifies that the aspartate metabolism pathway contributes to asparaginase sensitivity.  

PubMed

Asparaginase is an important component for treatment of childhood acute lymphoblastic leukemia (ALL). The basis for interindividual differences in asparaginase sensitivity remains unclear. To comprehensively identify genetic variants important in the cytotoxicity of asparaginase, we used a genome-wide association approach using the HapMap lymphoblastoid cell lines (87 CEU trio members) and 54 primary ALL leukemic blast samples at diagnosis. Asparaginase sensitivity was assessed as the drug concentration necessary to inhibit 50% of growth (inhibitory concentration (IC)(50)). In CEU lines, we tested 2,390,203 single-nucleotide polymorphism (SNP) genotypes at the individual SNP (P<0.001) and gene level (P<0.05), and identified 329 SNPs representing 94 genes that were associated with asparaginase IC(50). The aspartate metabolism pathway was the most overrepresented among 199 pathways evaluated (P=8.1 × 10(-3)), with primary involvement of adenylosuccinate lyase and aspartyl-tRNA synthetase genes. We validated that SNPs in the aspartate metabolism pathway were also associated with asparaginase sensitivity in primary ALL leukemic blast samples (P=5.5 × 10(-5)). Our genome-wide interrogation of CEU cell lines and primary ALL blasts revealed that inherited genomic interindividual variation in a plausible candidate pathway can contribute to asparaginase sensitivity. PMID:21072045

Chen, S-H; Yang, W; Fan, Y; Stocco, G; Crews, K R; Yang, J J; Paugh, S W; Pui, C-H; Evans, W E; Relling, M V

2011-01-01

74

Identifiability and estimation of multiple transmission pathways in cholera and waterborne disease.  

PubMed

Cholera and many waterborne diseases exhibit multiple characteristic timescales or pathways of infection, which can be modeled as direct and indirect transmission. A major public health issue for waterborne diseases involves understanding the modes of transmission in order to improve control and prevention strategies. An important epidemiological question is: given data for an outbreak, can we determine the role and relative importance of direct vs. environmental/waterborne routes of transmission? We examine whether parameters for a differential equation model of waterborne disease transmission dynamics can be identified, both in the ideal setting of noise-free data (structural identifiability) and in the more realistic setting in the presence of noise (practical identifiability). We used a differential algebra approach together with several numerical approaches, with a particular emphasis on identifiability of the transmission rates. To examine these issues in a practical public health context, we apply the model to a recent cholera outbreak in Angola (2006). Our results show that the model parameters-including both water and person-to-person transmission routes-are globally structurally identifiable, although they become unidentifiable when the environmental transmission timescale is fast. Even for water dynamics within the identifiable range, when noisy data are considered, only a combination of the water transmission parameters can practically be estimated. This makes the waterborne transmission parameters difficult to estimate, leading to inaccurate estimates of important epidemiological parameters such as the basic reproduction number (R0). However, measurements of pathogen persistence time in environmental water sources or measurements of pathogen concentration in the water can improve model identifiability and allow for more accurate estimation of waterborne transmission pathway parameters as well as R0. Parameter estimates for the Angola outbreak suggest that both transmission pathways are needed to explain the observed cholera dynamics. These results highlight the importance of incorporating environmental data when examining waterborne disease. PMID:23333764

Eisenberg, Marisa C; Robertson, Suzanne L; Tien, Joseph H

2013-05-01

75

Simulated Annealing Based Algorithm for Identifying Mutated Driver Pathways in Cancer  

PubMed Central

With the development of next-generation DNA sequencing technologies, large-scale cancer genomics projects can be implemented to help researchers to identify driver genes, driver mutations, and driver pathways, which promote cancer proliferation in large numbers of cancer patients. Hence, one of the remaining challenges is to distinguish functional mutations vital for cancer development, and filter out the unfunctional and random “passenger mutations.” In this study, we introduce a modified method to solve the so-called maximum weight submatrix problem which is used to identify mutated driver pathways in cancer. The problem is based on two combinatorial properties, that is, coverage and exclusivity. Particularly, we enhance an integrative model which combines gene mutation and expression data. The experimental results on simulated data show that, compared with the other methods, our method is more efficient. Finally, we apply the proposed method on two real biological datasets. The results show that our proposed method is also applicable in real practice.

Li, Hai-Tao; Zhang, Yu-Lang; Zheng, Chun-Hou; Wang, Hong-Qiang

2014-01-01

76

Simulated annealing based algorithm for identifying mutated driver pathways in cancer.  

PubMed

With the development of next-generation DNA sequencing technologies, large-scale cancer genomics projects can be implemented to help researchers to identify driver genes, driver mutations, and driver pathways, which promote cancer proliferation in large numbers of cancer patients. Hence, one of the remaining challenges is to distinguish functional mutations vital for cancer development, and filter out the unfunctional and random "passenger mutations." In this study, we introduce a modified method to solve the so-called maximum weight submatrix problem which is used to identify mutated driver pathways in cancer. The problem is based on two combinatorial properties, that is, coverage and exclusivity. Particularly, we enhance an integrative model which combines gene mutation and expression data. The experimental results on simulated data show that, compared with the other methods, our method is more efficient. Finally, we apply the proposed method on two real biological datasets. The results show that our proposed method is also applicable in real practice. PMID:24982873

Li, Hai-Tao; Zhang, Yu-Lang; Zheng, Chun-Hou; Wang, Hong-Qiang

2014-01-01

77

Proteomic Profile Identifies Dysregulated Pathways in Cornelia de Lange Syndrome Cells With Distinct Mutations in SMC1A and SMC3 Genes  

PubMed Central

Mutations in cohesin genes have been identified in Cornelia de Lange syndrome (CdLS), but its etiopathogenetic mechanisms are still poorly understood. To define biochemical pathways that are affected in CdLS we analyzed the proteomic profile of CdLS cell lines carrying mutations in the core cohesin genes, SMC1A and SMC3. Dysregulated protein expression was found in CdLS probands compared to controls. The proteomics analysis was able to discriminate between probands harboring mutations in the different domains of the SMC proteins. In particular, proteins involved in the response to oxidative stress were specifically down-regulated in hinge mutated probands. In addition, the finding that CdLS cell lines show an increase in global oxidative stress argues that it could contribute to some CdLS phenotypic features such as premature physiological aging and genome instability. Finally, the c-MYC gene represents a convergent hub lying at the center of dysregulated pathways, and is down-regulated in CdLS. This study allowed us to highlight, for the first time, specific biochemical pathways that are affected in CdLS, providing plausible causal evidence for some of the phenotypic features seen in CdLS.

Gimigliano, Anna; Mannini, Linda; Bianchi, Laura; Puglia, Michele; Deardorff, Matthew A.; Menga, Stefania; Krantz, Ian D; Musio, Antonio; Bini, Luca

2012-01-01

78

Proteomic profile identifies dysregulated pathways in Cornelia de Lange syndrome cells with distinct mutations in SMC1A and SMC3 genes.  

PubMed

Mutations in cohesin genes have been identified in Cornelia de Lange syndrome (CdLS), but its etiopathogenetic mechanisms are still poorly understood. To define biochemical pathways that are affected in CdLS, we analyzed the proteomic profile of CdLS cell lines carrying mutations in the core cohesin genes, SMC1A and SMC3. Dysregulated protein expression was found in CdLS probands compared to controls. The proteomics analysis was able to discriminate between probands harboring mutations in the different domains of the SMC proteins. In particular, proteins involved in the response to oxidative stress were specifically down-regulated in hinge mutated probands. In addition, the finding that CdLS cell lines show an increase in global oxidative stress argues that it could contribute to some CdLS phenotypic features such as premature physiological aging and genome instability. Finally, the c-MYC gene represents a convergent hub lying at the center of dysregulated pathways, and is down-regulated in CdLS. This study allowed us to highlight, for the first time, specific biochemical pathways that are affected in CdLS, providing plausible causal evidence for some of the phenotypic features seen in CdLS. PMID:23106691

Gimigliano, Anna; Mannini, Linda; Bianchi, Laura; Puglia, Michele; Deardorff, Matthew A; Menga, Stefania; Krantz, Ian D; Musio, Antonio; Bini, Luca

2012-12-01

79

Biochemical pathways generating post-mortem volatile compounds co-detected during forensic ethanol analyses  

Microsoft Academic Search

In this contribution are presented the fermentations of the main substrates present in a decaying corpse, namely carbohydrates, amino acids, glycerol and fatty acids, generating the post-mortem volatile compounds that could be detected along with ethanol during the forensic ethanol analysis. The available literature (preferably reviews) on microbial metabolic pathways (enzymes, substrates, conditions) that are implicated in the formation of

Vassiliki A. Boumba; Kallirroe S. Ziavrou; Theodore Vougiouklakis

2008-01-01

80

Evidence from Biochemical Pathways in Favor of Unfinished Evolution Rather than Intelligent Design  

ERIC Educational Resources Information Center

An argument is made in favor of imperfect or unfinished evolution based on some metabolic pathways in which it seems that intelligent design would have done better. The case studies noted indicate the absence of highly intelligent design and are not intended as comprehensive collection but as a limited sample of inefficient situations in…

Behrman, Edward J.; Marzluf, George A.

2004-01-01

81

A Phosphotyrosine Proteomic Screen Identifies Multiple Tyrosine Kinase Signaling Pathways Aberrantly Activated in Malignant Mesothelioma  

PubMed Central

Malignant mesothelioma (MM) is a highly aggressive cancer that is refractory to all current chemotherapeutic regimens. Therefore, uncovering new rational therapeutic targets is imperative in the field. Tyrosine kinase signaling pathways are aberrantly activated in many human cancers and are currently being targeted for chemotherapeutic intervention. Thus, we sought to identify tyrosine kinases hyperactivated in MM. An unbiased phosphotyrosine proteomic screen was employed to identify tyrosine kinases activated in human MM cell lines. From this screen, we have identified novel signaling molecules, such as JAK1, STAT1, cortactin (CTTN), FER, p130Cas (BCAR1), SRC, and FYN as tyrosine phosphorylated in human MM cell lines. Additionally, STAT1 and SRC family kinases (SFK) were confirmed to be active in primary MM specimens. We also confirmed that known signal transduction pathways previously implicated in MM, such as EGFR and MET signaling axes, are coactivated in the majority of human MM specimens and cell lines tested. EGFR, MET, and SFK appear to be coactivated in a significant proportion of MM cell lines, and dual inhibition of these kinases was demonstrated to be more efficacious for inhibiting MM cell viability and downstream effector signaling than inhibition of a single tyrosine kinase. Consequently, these data suggest that tyrosine kinase inhibitor monotherapy may not represent an efficacious strategy for the treatment of MM due to multiple tyrosine kinases potentially signaling redundantly to cellular pathways involved in tumor cell survival and proliferation.

Menges, Craig W.; Chen, Yibai; Mossman, Brooke T.; Chernoff, Jonathan; Yeung, Anthony T.; Testa, Joseph R.

2010-01-01

82

Purification and biochemical characterization of a hydroxyneurosporene desaturase involved in the biosynthetic pathway of the carotenoid spheroidene in Rhodobacter sphaeroides.  

PubMed Central

Hydroxyneurosporene desaturase is involved in the carotenoid biosynthetic pathway of Rhodobacter species. The gene encoding this enzyme was expressed in Escherichia coli, purified, and biochemically characterized. The resulting protein contained an N-terminal six-histidine extension which derived from the cloning vector; this allowed for a one-step purification of the enzyme to homogeneity after solubilization with Nonidet P-40. The hydrogen acceptor in the C-3,4 desaturation reaction was molecular oxygen. NAD+, NADP+, and flavin adenine dinucleotide had no influence on enzymatic activity. Different acyclic 1-hydroxycarotenoids were tested as substrates. Very good conversion was achieved with 1-hydroxyneurosporene and 1-hydroxylycopene, whereas 1-hydroxy-gamma-carotene and 1,1'-dihydroxylycopene were much less effective. From 1'-hydroxy-3,4-didehydrolycopene only trace amounts of product were obtained, and 1-methoxyneurosporene was not converted by purified hydroxyneurosporene desaturase. A Km of 13.4 microM was determined for 1-hydroxyneurosporene.

Albrecht, M; Ruther, A; Sandmann, G

1997-01-01

83

Identifying low variance pathways for free energy calculations of molecular transformations in solution phase  

NASA Astrophysics Data System (ADS)

Improving the efficiency of free energy calculations is important for many biological and materials design applications, such as protein-ligand binding affinities in drug design, partitioning between immiscible liquids, and determining molecular association in soft materials. We show that for any pair potential, moderately accurate estimation of the radial distribution function for a solute molecule is sufficient to accurately estimate the statistical variance of a sampling along a free energy pathway. This allows inexpensive analytical identification of low statistical error free energy pathways. We employ a variety of methods to estimate the radial distribution function (RDF) and find that the computationally cheap two-body ``dilute gas'' limit performs as well or better than 3D-RISM theory and other approximations for identifying low variance free energy pathways. With a RDF estimate in hand, we can search for pairwise interaction potentials that produce low variance. We give an example of a search minimizing statistical variance of solvation free energy over the entire parameter space of a generalized ``soft core'' potential. The free energy pathway arising from this optimization procedure has lower curvature in the variance and reduces the total variance by at least 50% compared to the traditional soft core solvation pathway. We also demonstrate that this optimized pathway allows free energies to be estimated with fewer intermediate states due to its low curvature. This free energy variance optimization technique is generalizable to solvation in any homogeneous fluid and for any type of pairwise potential and can be performed in minutes to hours, depending on the method used to estimate g(r).

Pham, Tri T.; Shirts, Michael R.

2011-07-01

84

Redirection of biochemical pathways for the enhancement of H2 production by Enterobacter cloacae  

Microsoft Academic Search

Improvement in H2 production was achieved through redirection of metabolic pathways by blocking formation of alcohol and some organic acids in Enterobacter cloacae IIT-BT 08. The wild type strain was more susceptible to allyl alcohol (7 mM) and to the combined effect of NaBr and NaBrO3 (40 mM each at pH 5.5) than were double mutants, with defects in both

Narendra Kumar; Agnidipta Ghosh; Debabrata Das

2001-01-01

85

Steady and transient fluid shear stress stimulate NO release in osteoblasts through distinct biochemical pathways  

NASA Technical Reports Server (NTRS)

Fluid flow has been shown to be a potent stimulus in osteoblasts and osteocytes and may therefore play an important role in load-induced bone remodeling. The objective of this study was to investigate the characteristics of flow-activated pathways. Previously we reported that fluid flow stimulates rapid and continuous release of nitric oxide (NO) in primary rat calvarial osteoblasts. Here we demonstrate that flow-induced NO release is mediated by shear stress and that this response is distinctly biphasic. Transients in shear stress associated with the onset of flow stimulated a burst in NO production (8.2 nmol/mg of protein/h), while steady flow stimulated sustained NO production (2.2 nmol/mg of protein/h). Both G-protein inhibition and calcium chelation abolished the burst phase but had no effect on sustained production. Activation of G-proteins stimulated dose-dependent NO release in static cultures of both calvarial osteoblasts and UMR-106 osteoblast-like cells. Pertussis toxin had no effect on NO release. Calcium ionophore stimulated low levels of NO production within 15 minutes but had no effect on sustained production. Taken together, these data suggest that fluid shear stress stimulates NO release by two distinct pathways: a G-protein and calcium-dependent phase sensitive to flow transients, and a G-protein and calcium-independent pathway stimulated by sustained flow.

McAllister, T. N.; Frangos, J. A.

1999-01-01

86

Gene set enrichment analysis identifies key innate immune pathways in primary graft dysfunction after lung transplantation  

PubMed Central

We hypothesized alterations in gene expression could identify important pathways involved in transplant lung injury. Bronchoalveolar lavage fluid was sampled from donors prior to procurement and in recipients within an hour of reperfusion as part of the NIAID Clinical Trials in Organ Transplantation Study. 23 patients with Grade 3 PGD were frequency matched with controls based on donor age and recipient diagnosis. RNA was analyzed using the Human Gene 1.0 ST array. Normalized mRNA expression was transformed and differences between donor and post-reperfusion values were ranked then tested using Gene Set Enrichment Analysis. 362 gene sets were upregulated with 8 meeting significance (FWER p-value <0.05), including the NOD-like receptor inflammasome (NLR; p<0.001), toll-like receptors (TLR; p<0.001), IL-1 receptor (p=0.001), Myeloid differentiation primary response gene 88 (p=0.001), NF?B activation by nontypeable Haemophilus influenzae (p=0.001), TLR 4 (p=0.008), and TLR 9 (p=0.018). The top 5 ranked individual transcripts from these pathways based on rank metric score are predominantly present in the NLR and TLR pathways, including IL1? (1.162), NLRP3 (1.135), IL1? (0.952), IL6 (0.931) and CCL4 (0.842). Gene set enrichment analyses implicate inflammasome mediated and innate immune signaling pathways as key mediators of the development of PGD in lung transplant patients.

Cantu, E.; Lederer, D.J.; Meyer, K.; Milewski, K.; Suzuki, Y.; Shah, R.J.; Diamond, J.M.; Meyer, N.J.; Tobias, J.W.; Baldwin, D.A.; Van Deerlin, V.M.; Olthoff, K.M.; Shaked, A.; Christie, J.D.

2014-01-01

87

Functional genomics identifies type I interferon pathway as central for host defense against Candida albicans  

PubMed Central

Candida albicans is the most common human fungal pathogen causing mucosal and systemic infections. However, human antifungal immunity remains poorly defined. Here, by integrating transcriptional analysis and functional genomics, we identified Candida-specific host defense mechanisms in humans. Candida induced significant expression of genes from the type I interferon (IFN) pathway in human peripheral blood mononuclear cells. This unexpectedly prominent role of type I IFN pathway in anti-Candida host defense was supported by additional evidence. Polymorphisms in type I IFN genes modulated Candida-induced cytokine production and were correlated with susceptibility to systemic candidiasis. In in-vitro experiments, type I IFNs skewed Candida-induced inflammation from a Th17-response toward a Th1-response. Patients with chronic mucocutaneaous candidiasis displayed defective expression of genes in the type I IFN pathway. These findings indicate that the type I IFN pathway is a main signature of Candida-induced inflammation and plays a crucial role in anti-Candida host defense in humans.

Smeekens, Sanne P.; Ng, Aylwin; Kumar, Vinod; Johnson, Melissa D.; Plantinga, Theo S.; van Diemen, Cleo; Arts, Peer; Verwiel, Eugene T.P.; Gresnigt, Mark S.; Fransen, Karin; van Sommeren, Suzanne; Oosting, Marije; Cheng, Shih-Chin; Joosten, Leo A.B.; Hoischen, Alexander; Kullberg, Bart-Jan; Scott, William K.; Perfect, John R.; van der Meer, Jos W.M.; Wijmenga, Cisca; Netea, Mihai G.; Xavier, Ramnik J.

2013-01-01

88

A new screening pathway for identifying asymptomatic patients using dental panoramic radiographs  

NASA Astrophysics Data System (ADS)

To identify asymptomatic patients is the challenging task and the essential first step in diagnosis. Findings of dental panoramic radiographs include not only dental conditions but also radiographic signs that are suggestive of possible systemic diseases such as osteoporosis, arteriosclerosis, and maxillary sinusitis. Detection of such signs on panoramic radiographs has a potential to provide supplemental benefits for patients. However, it is not easy for general dental practitioners to pay careful attention to such signs. We addressed the development of a computer-aided detection (CAD) system that detects radiographic signs of pathology on panoramic images, and the design of the framework of new screening pathway by cooperation of dentists and our CAD system. The performance evaluation of our CAD system showed the sensitivity and specificity in the identification of osteoporotic patients were 92.6 % and 100 %, respectively, and those of the maxillary sinus abnormality were 89.6 % and 73.6 %, respectively. The detection rate of carotid artery calcifications that suggests the need for further medical evaluation was approximately 93.6 % with 4.4 false-positives per image. To validate the utility of the new screening pathway, preliminary clinical trials by using our CAD system were conducted. To date, 223 panoramic images were processed and 4 asymptomatic patients with suspected osteoporosis, 7 asymptomatic patients with suspected calcifications, and 40 asymptomatic patients with suspected maxillary sinusitis were detected in our initial trial. It was suggested that our new screening pathway could be useful to identify asymptomatic patients with systemic diseases.

Hayashi, Tatsuro; Matsumoto, Takuya; Sawagashira, Tsuyoshi; Tagami, Motoki; Katsumata, Akitoshi; Hayashi, Yoshinori; Muramatsu, Chisako; Zhou, Xiangrong; Iida, Yukihiro; Matsuoka, Masato; Katagi, Kiyoji; Fujita, Hiroshi

2012-02-01

89

AUTOANTIBODY PROFILING TO IDENTIFY BIOMARKERS OF KEY PATHOGENIC PATHWAYS IN MUCINOUS OVARIAN CANCER  

PubMed Central

Mucinous epithelial ovarian cancers are clinically and morphologically distinct from the other histopathologic subtypes of ovarian cancer. Unlike other ovarian subtypes, epidemiologic studies have indicated that tobacco exposure is a significant risk factor for developing mucinous ovarian cancer. Detection of autoantibody reactivity is useful in biomarker discovery and for explaining the role of important pathophysiologic pathways in disease. In order to study if there are specific antibody biomarkers in the plasma samples of mucinous ovarian cancer patients, we have initiated a screen by employing a “reverse capture antibody microarray” platform that uses native host antigens derived from mucinous ovarian tissues as “baits” for the capture of differentially labeled patient and control autoantibodies. 35 autoantibodies that were significantly elevated in the cancer plasma samples compared with healthy controls, and six autoantibodies that segregated smoking and nonsmoking patients were identified. Functional annotation of the antibody targets has identified nine target antigens involved in integrin and Wnt signaling pathways. Immunohistochemistry of archived ovarian specimens showed significant overexpression of eight of the nine target antigens in mucinous ovarian tumor tissues, suggesting that plasma autoantibodies from mucinous ovarian cancer patients might have heightened reactivities with epitopes presented by these overexpressed antigens. Autoantibody profiling may have an unexpected utility in uncovering key signaling pathways that are dysregulated in the system of interest.

Tang, Liangdan; Yang, Junzheng; Ng, Shu-Kay; Rodriguez, Noah; Choi, Pui-Wah; Vitonis, Allison; Wang, Kui; McLachlan, Geoffrey J.; Caiazzo, Robert J.; Liu, Brian C.-S.; Welch, William R.; Cramer, Daniel W.; Berkowitz, Ross S.; Ng, Shu-Wing

2009-01-01

90

Genome-wide approaches to systematically identify substrates of the ubiquitin/proteasome pathway  

PubMed Central

The ubiquitin/proteasome system handles the majority of controlled proteolysis in eukaryotes. Defects in the ubiquitin/proteasome system have been implicated in diseases ranging from cancers to neurodegenerative disorders. However, the precise role of ubiquitin/proteasome-mediated degradation in health and disease is far from clear. A major challenge is to link specific substrates directly to a particular degradation pathway. Here, we review genome-wide approaches that have been developed in recent years to comprehensively identify ubiquitylated substrates of a particular pathway. The components of the ubiquitin/proteasome system are attractive drug targets, as illustrated by the efficacy of some proteasome inhibitors in the treatment of multiple myeloma. Information that has emerged from these studies could reveal novel drug targets and strategies for treating human diseases.

Liu, Chang; Choe, Vitnary; Rao, Hai

2010-01-01

91

Genome-wide screen identifies signaling pathways that regulate autophagy during Caenorhabditis elegans development.  

PubMed

The mechanisms that coordinate the regulation of autophagy with developmental signaling during multicellular organism development remain largely unknown. Here, we show that impaired function of ribosomal protein RPL-43 causes an accumulation of SQST-1 aggregates in the larval intestine, which are removed upon autophagy induction. Using this model to screen for autophagy regulators, we identify 139 genes that promote autophagy activity upon inactivation. Various signaling pathways, including Sma/Mab TGF-? signaling, lin-35/Rb signaling, the XBP-1-mediated ER stress response, and the ATFS-1-mediated mitochondrial stress response, regulate the expression of autophagy genes independently of the TFEB homolog HLH-30. Our study thus provides a framework for understanding the role of signaling pathways in regulating autophagy under physiological conditions. PMID:24764321

Guo, Bin; Huang, Xinxin; Zhang, Peipei; Qi, Linxiang; Liang, Qianqian; Zhang, Xuebo; Huang, Jie; Fang, Bin; Hou, Wenru; Han, Jinghua; Zhang, Hong

2014-06-01

92

Chemical screen identifies FDA-approved drugs and target pathways that induce precocious pancreatic endocrine differentiation  

PubMed Central

Pancreatic ?-cells are an essential source of insulin and their destruction because of autoimmunity causes type I diabetes. We conducted a chemical screen to identify compounds that would induce the differentiation of insulin-producing ?-cells in vivo. To do this screen, we brought together the use of transgenic zebrafish as a model of ?-cell differentiation, a unique multiwell plate that allows easy visualization of lateral views of swimming larval fish and a library of clinical drugs. We identified six hits that can induce precocious differentiation of secondary islets in larval zebrafish. Three of these six hits were known drugs with a considerable background of published data on mechanism of action. Using pharmacological approaches, we have identified and characterized two unique pathways in ?-cell differentiation in the zebrafish, including down-regulation of GTP production and retinoic acid biosynthesis.

Rovira, Meritxell; Huang, Wei; Yusuff, Shamila; Shim, Joong Sup; Ferrante, Anthony A.; Liu, Jun O.; Parsons, Michael J.

2011-01-01

93

Biochemical characterization of the tetrahydrobiopterin synthesis pathway in the oleaginous fungus Mortierella alpina.  

PubMed

We characterized the de novo biosynthetic pathway of tetrahydrobiopterin (BH?) in the lipid-producing fungus Mortierella alpina. The BH? cofactor is essential for various cell processes, and is probably present in every cell or tissue of higher organisms. Genes encoding two copies of GTP cyclohydrolase I (GTPCH-1 and GTPCH-2) for the conversion of GTP to dihydroneopterin triphosphate (H?-NTP), 6-pyruvoyltetrahydropterin synthase (PTPS) for the conversion of H?-NTP to 6-pyruvoyltetrahydropterin (PPH?), and sepiapterin reductase (SR) for the conversion of PPH? to BH?, were expressed heterologously in Escherichia coli. The recombinant enzymes were produced as His-tagged fusion proteins and were purified to homogeneity to investigate their enzymic activities. Enzyme products were analysed by HPLC and electrospray ionization-MS. Kinetic parameters and other properties of GTPCH, PTPS and SR were investigated. Physiological roles of BH? in M. alpina are discussed, and comparative analyses between GTPCH, PTPS and SR proteins and other homologous proteins were performed. The presence of two functional GTPCH enzymes has, as far as we are aware, not been reported previously, reflecting the unique ability of this fungus to synthesize both BH? and folate, using the GTPCH product as a common substrate. To our knowledge, this study is the first to report the comprehensive characterization of a BH? biosynthesis pathway in a fungus. PMID:21852350

Wang, Hongchao; Yang, Bo; Hao, Guangfei; Feng, Yun; Chen, Haiqin; Feng, Lu; Zhao, Jianxin; Zhang, Hao; Chen, Yong Q; Wang, Lei; Chen, Wei

2011-11-01

94

A genetic screen identifies an LKB1-MARK signalling axis controlling the Hippo-YAP pathway.  

PubMed

The Hippo-YAP pathway is an emerging signalling cascade involved in the regulation of stem cell activity and organ size. To identify components of this pathway, we performed an RNAi-based kinome screen in human cells. Our screen identified several kinases not previously associated with Hippo signalling that control multiple cellular processes. One of the hits, LKB1, is a common tumour suppressor whose mechanism of action is only partially understood. We demonstrate that LKB1 acts through its substrates of the microtubule affinity-regulating kinase family to regulate the localization of the polarity determinant Scribble and the activity of the core Hippo kinases. Our data also indicate that YAP is functionally important for the tumour suppressive effects of LKB1. Our results identify a signalling axis that links YAP activation with LKB1 mutations, and have implications for the treatment of LKB1-mutant human malignancies. In addition, our findings provide insight into upstream signals of the Hippo-YAP signalling cascade. PMID:24362629

Mohseni, Morvarid; Sun, Jianlong; Lau, Allison; Curtis, Stephen; Goldsmith, Jeffrey; Fox, Victor L; Wei, Chongjuan; Frazier, Marsha; Samson, Owen; Wong, Kwok-Kin; Wong, Kwok-Kim; Kim, Carla; Camargo, Fernando D

2014-01-01

95

An intracellular trafficking pathway in the seminiferous epithelium regulating spermatogenesis: a biochemical and molecular perspective.  

PubMed

During spermatogenesis in adult rat testes, fully developed spermatids (i.e. spermatozoa) at the luminal edge of the seminiferous epithelium undergo "spermiation" at stage VIII of the seminiferous epithelial cycle. This is manifested by the disruption of the apical ectoplasmic specialization (apical ES) so that spermatozoa can enter the tubule lumen and to complete their maturation in the epididymis. At the same time, the blood-testis barrier (BTB) located near the basement membrane undergoes extensive restructuring to allow transit of preleptotene spermatocytes so that post-meiotic germ cells complete their development behind the BTB. While spermiation and BTB restructuring take place concurrently at opposite ends of the Sertoli cell epithelium, the biochemical mechanism(s) by which they are coordinated were not known until recently. Studies have shown that fragments of laminin chains are generated from the laminin/integrin protein complex at the apical ES via the action of MMP-2 (matrix metalloprotease-2) at spermiation. These peptides serve as the local autocrine factors to destabilize the BTB. These laminin peptides also exert their effects on hemidesmosome which, in turn, further potentiates BTB restructuring. Thus, a novel apical ES-BTB-hemidesmosome regulatory loop is operating in the seminiferous epithelium to coordinate these two crucial cellular events of spermatogenesis. This functional loop is further assisted by the Par3/Par6-based polarity protein complex in coordination with cytokines and testosterone at the BTB. Herein, we provide a critical review based on the latest findings in the field regarding the regulation of these cellular events. These recent findings also open up a new window for investigators studying blood-tissue barriers. PMID:19622063

Cheng, C Yan; Mruk, Dolores D

2009-01-01

96

Gene expression signatures identify novel regulatory pathways during murine lung development: implications for lung tumorigenesis  

PubMed Central

Oligonucleotide array based analysis was conducted to examine the temporal pattern of gene expression across the various stages of lung development to identify regulatory pathways at key developmental time points. Whole embryo total RNA or embryonic lung total RNA was harvested from A/J mice at seven developmental stages. To investigate changes in gene expression during lung development, four samples from each stage were examined using Affymetrix U74Av2 murine oligonucleotide microarrays. From the over 12 000 genes and ESTs represented on the array, 1346 genes and ESTs were identified as having a significant change in expression between at least one time point and the others (p<0.001, Kruskal-Wallis test). Within this group of ?1300 genes, four patterns of expression were seen: (1) upregulation during the embryonic period of development (up-down); (2) upregulation during the postnatal period of lung development (down-up) and (3) fluctuating expression, up initially, down for one or more time points, and then up again (up-down-up); and (4) vice versa (down-up-down). Expression patterns of genes previously reported to be involved in pulmonary development were also examined. Using the pathway visualisation tool, GenMapp, at least three regulatory pathways were found to contain clusters of differentially expressed genes: Wnt signalling, cell cycle, and apoptosis. Furthermore, we have shown that many of the genes involved in lung development are either known oncogenes or tumour suppressor genes altered in lung cancer, such as Cyr61, Rassf1a, and Dutt1/Robo1, or putative lung cancer genes. In addition, the genes identified pertinent to early development may also serve as candidate susceptibility genes for various inherited lung cancer disorders as well as for various heritable disorders of lung development. These results will contribute to our understanding of novel aspects of the regulatory machinery for embryonic lung development and of the genes involved in lung tumorigenesis.

Bonner, A; Lemon, W; You, M

2003-01-01

97

Physiological and biochemical characteristics of adrenergic receptors and pathways in brown adipocytes  

NASA Technical Reports Server (NTRS)

Mechanisms involved in the thermogenic response of brown adipose tissue (BAT) to sympathetic nervous stimulation (e.g., by cold exposure) and to norepinephrine (NE) release are investigated. Three effects appear to play a role in the increased oxygen consumption (and heat production) of the adipocytes: increased membrane permeability, activation of the beta-adrenergic pathway, and enhancement of Na(+)/K(+) membrane pump activity. Increased passive influx of Na(+) and efflux of K(+) due to greater permeability raise the energy demands of the Na/K pump; the pump is also stimulated by increased cyclic AMP synthesis resulting from activation by NE of membrane-bound adenyl cyclase. Studies with inhibitors such as propanolol, phentolamine, and ouabain support this hypothesis.

Horwitz, B. A.

1975-01-01

98

Biochemical analysis of the N-glycosylation pathway in baculovirus-infected lepidopteran insect cells.  

PubMed

The baculovirus-insect cell system is used routinely for foreign glycoprotein production, but the precise nature of the N-glycosylation pathway in this system remains unclear. Some studies indicate that these cells cannot process N-linked oligosaccharides to complex forms containing outer-chain galactose and sialic acid, while others indicate that they can. In this study, we used the major virion envelope glycoprotein of the baculovirus Autographa california multicapsid nuclear polyhedrosis virus (AcMNPV) to probe the N-glycosylation pathway in baculovirus-infected lepidopteran insect cells. The results showed that gp64 contained mannose, fucose, and probably N-acetylglucosamine, but no detectable galactose or sialic acid. These same results were observed with gp64 produced in any one of three different lepidopteran insect cell lines derived from Spodoptera frugiperda, Trichoplusia ni, or Estigmene acrea, whether it was produced at relatively earlier or later times after infection. These results indicated that the gp64 produced in AcMNPV-infected lepidopteran insect cells lacks complex N-linked oligosaccharides containing outer-chain galactose and sialic acid. By contrast, gp64 produced in mammalian cells contained both galactose and sialic acid, and endoglycosidase digestions revealed that these sugars were constituents of N-linked, not O-linked, oligosaccharides. This showed that at least one N-linked side chain on gp64 has the potential to be processed to a complex form. Together, these results suggest either that AcMNPV-infected lepidopteran insect cells are unable to convert any of the N-linked side chains on gp64 to complex structures or that outer-chain galactose and sialic acid residues are added to gp64 and then removed by cellular or viral exoglycosidases. PMID:7571420

Jarvis, D L; Finn, E E

1995-10-01

99

Biochemical characterization of GDP-L-fucose de novo synthesis pathway in fungus Mortierella alpina  

SciTech Connect

Mortierella alpina is a filamentous fungus commonly found in soil, which is able to produce large amount of polyunsaturated fatty acids. L-Fucose is an important sugar found in a diverse range of organisms, playing a variety of biological roles. In this study, we characterized the de novo biosynthetic pathway of GDP-L-fucose (the nucleotide-activated form of L-fucose) in M. alpina. Genes encoding GDP-D-mannose 4,6-dehydratase (GMD) and GDP-keto-6-deoxymannose 3,5-epimerase/4-reductase (GMER) were expressed heterologously in Escherichia coli. The recombinant enzymes were produced as His-tagged fusion proteins. Conversion of GDP-mannose to GDP-4-keto-6-deoxy mannose by GMD and GDP-4-keto-6-deoxy mannose to GDP-L-fucose by GMER were analyzed by capillary electrophoresis, electro-spray ionization-mass spectrometry, and nuclear magnetic resonance spectroscopy. The k{sub m} values of GMD for GDP-mannose and GMER for GDP-4-keto-6-deoxy mannose were determined to be 0.77 mM and 1.047 mM, respectively. Both NADH and NADPH may be used by GMER as the coenzyme. The optimum temperature and pH were determined to be 37 {sup o}C and pH 9.0 (GMD) or pH 7.0 (GMER). Divalent cations are not required for GMD and GMER activity, and the activities of both enzymes may be enhanced by DTT. To our knowledge this is the first report on the characterization of GDP-L-fucose biosynthetic pathway in fungi.

Ren, Yan [TEDA School of Biological Sciences and Biotechnology, Nankai University, Tianjin Economic-Technological Development Area, Tianjin 300457 (China)] [TEDA School of Biological Sciences and Biotechnology, Nankai University, Tianjin Economic-Technological Development Area, Tianjin 300457 (China); Perepelov, Andrei V. [N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospekt 47, 119991 Moscow (Russian Federation)] [N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospekt 47, 119991 Moscow (Russian Federation); Wang, Haiyan [TEDA School of Biological Sciences and Biotechnology, Nankai University, Tianjin Economic-Technological Development Area, Tianjin 300457 (China)] [TEDA School of Biological Sciences and Biotechnology, Nankai University, Tianjin Economic-Technological Development Area, Tianjin 300457 (China); Zhang, Hao [State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122 (China)] [State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122 (China); Knirel, Yuriy A. [N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospekt 47, 119991 Moscow (Russian Federation)] [N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospekt 47, 119991 Moscow (Russian Federation); Wang, Lei [TEDA School of Biological Sciences and Biotechnology, Nankai University, Tianjin Economic-Technological Development Area, Tianjin 300457 (China)] [TEDA School of Biological Sciences and Biotechnology, Nankai University, Tianjin Economic-Technological Development Area, Tianjin 300457 (China); Chen, Wei, E-mail: weichen@jiangnan.edu.cn [State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122 (China)] [State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122 (China)

2010-01-22

100

Assessment Method for a Power Analysis to Identify Differentially Expressed Pathways  

PubMed Central

Gene expression data can provide a very rich source of information for elucidating the biological function on the pathway level if the experimental design considers the needs of the statistical analysis methods. The purpose of this paper is to provide a comparative analysis of statistical methods for detecting the differentially expression of pathways (DEP). In contrast to many other studies conducted so far, we use three novel simulation types, producing a more realistic correlation structure than previous simulation methods. This includes also the generation of surrogate data from two large-scale microarray experiments from prostate cancer and ALL. As a result from our comprehensive analysis of parameter configurations, we find that each method should only be applied if certain conditions of the data from a pathway are met. Further, we provide method-specific estimates for the optimal sample size for microarray experiments aiming to identify DEP in order to avoid an underpowered design. Our study highlights the sensitivity of the studied methods on the parameters of the system.

Tripathi, Shailesh; Emmert-Streib, Frank

2012-01-01

101

Gene Expression Profiling Identifies Molecular Pathways Associated with Collagen VI Deficiency and Provides Novel Therapeutic Targets  

PubMed Central

Ullrich congenital muscular dystrophy (UCMD), caused by collagen VI deficiency, is a common congenital muscular dystrophy. At present, the role of collagen VI in muscle and the mechanism of disease are not fully understood. To address this we have applied microarrays to analyse the transcriptome of UCMD muscle and compare it to healthy muscle and other muscular dystrophies. We identified 389 genes which are differentially regulated in UCMD relative to controls. In addition, there were 718 genes differentially expressed between UCMD and dystrophin deficient muscle. In contrast, only 29 genes were altered relative to other congenital muscular dystrophies. Changes in gene expression were confirmed by real-time PCR. The set of regulated genes was analysed by Gene Ontology, KEGG pathways and Ingenuity Pathway analysis to reveal the molecular functions and gene networks associated with collagen VI defects. The most significantly regulated pathways were those involved in muscle regeneration, extracellular matrix remodelling and inflammation. We characterised the immune response in UCMD biopsies as being mainly mediated via M2 macrophages and the complement pathway indicating that anti-inflammatory treatment may be beneficial to UCMD as for other dystrophies. We studied the immunolocalisation of ECM components and found that biglycan, a collagen VI interacting proteoglycan, was reduced in the basal lamina of UCMD patients. We propose that biglycan reduction is secondary to collagen VI loss and that it may be contributing towards UCMD pathophysiology. Consequently, strategies aimed at over-expressing biglycan and restore the link between the muscle cell surface and the extracellular matrix should be considered.

Paco, Sonia; Kalko, Susana G.; Jou, Cristina; Rodriguez, Maria A.; Corbera, Joan; Muntoni, Francesco; Feng, Lucy; Rivas, Eloy; Torner, Ferran; Gualandi, Francesca; Gomez-Foix, Anna M.; Ferrer, Anna; Ortez, Carlos; Nascimento, Andres; Colomer, Jaume; Jimenez-Mallebrera, Cecilia

2013-01-01

102

[Analysis of disease-pathway by identifying susceptible genes to primary biliary cirrhosis].  

PubMed

High concordance rate in monozygotic twins and familial clustering of patients with primary biliary cirrhosis (PBC) indicate the involvement of strong genetic factors in the development of PBC. Recent genome-wide association studies (GWASs) and subsequent meta-analyses in European descent have identified HLA and 21 non-HLA susceptibility loci which are involved in IL12/IL12R signaling, TNF/TLR-NFKB signaling and B cell differentiation in the development of PBC. To identify susceptibility loci for PBC in Japanese population, a GWAS and subsequent replication study was performed in a total of 1327 PBC cases and 1120 healthy controls. In addition to the most significant susceptibility region at HLA, two significant (p<5×10(-8)) susceptibility loci (TNFSF15 and POU2AF1) were identified. Although these susceptibility loci are different from those identified in European descent (IL12A, IL12RB2, SPIB), these loci are involved in the same signaling pathways, differentiation of T lymphocyte to Th1 cells and differentiation of B lymphocyte to plasma cells. Among 21 non-HLA susceptibility loci for PBC identified in GWASs of European descent, 10 loci (CD80, IKZF3, IL7R, NFKB1, STAT4, TNFAIP2, CXCR5, MAP3K7IP1, rs6974491, DENND1B) showed significant associations in the Japanese population. The comparative analysis of disease-susceptibility genes in multiple ethnicities may provide an important clue for the dissection of disease-pathogenesis. PMID:23291485

Nakamura, Minoru; Makamura, Minoru

2012-01-01

103

Retroviral insertions in the VISION database identify molecular pathways in mouse lymphoid leukemia and lymphoma  

PubMed Central

AKXD recombinant inbred (RI) strains develop a variety of leukemias and lymphomas due to somatically acquired insertions of retroviral DNA into the genome of hematopoetic cells that can mutate cellular proto-oncogenes and tumor suppressor genes. We generated a new set of tumors from nine AKXD RI strains selected for their propensity to develop B-cell tumors, the most common type of human hematopoietic cancers. We employed a PCR technique called viral insertion site amplification (VISA) to rapidly isolate genomic sequence at the site of provirus insertion. Here we describe 550 VISA sequence tags (VSTs) that identify 74 common insertion sites (CISs), of which 21 have not been identified previously. Several suspected proto-oncogenes and tumor suppressor genes lie near CISs, providing supportive evidence for their roles in cancer. Furthermore, numerous previously uncharacterized genes lie near CISs, providing a pool of candidate disease genes for future research. Pathway analysis of candidate genes identified several signaling pathways as common and powerful routes to blood cancer, including Notch, E-protein, NF?B, and Ras signaling. Misregulation of several Notch signaling genes was confirmed by quantitative RT-PCR. Our data suggest that analyses of insertional mutagenesis on a single genetic background are biased toward the identification of cooperating mutations. This tumor collection represents the most comprehensive study of the genetics of B-cell leukemia and lymphoma development in mice. We have deposited the VST sequences, CISs in a genome viewer, histopathology, and molecular tumor typing data in a public web database called VISION (Viral Insertion Sites Identifying Oncogenes), which is located at http://www.mouse-genome.bcm.tmc.edu/vision.

Weiser, Keith C.; Liu, Bin; Hansen, Gwenn M.; Skapura, Darlene; Hentges, Kathryn E.; Yarlagadda, Sujatha; Morse III, Herbert C.

2007-01-01

104

A whole-cell phenotypic screening platform for identifying methylerythritol phosphate pathway-selective inhibitors as novel antibacterial agents.  

PubMed

Isoprenoid biosynthesis is essential for survival of all living organisms. More than 50,000 unique isoprenoids occur naturally, with each constructed from two simple five-carbon precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Two pathways for the biosynthesis of IPP and DMAPP are found in nature. Humans exclusively use the mevalonate (MVA) pathway, while most bacteria, including all Gram-negative and many Gram-positive species, use the unrelated methylerythritol phosphate (MEP) pathway. Here we report the development of a novel, whole-cell phenotypic screening platform to identify compounds that selectively inhibit the MEP pathway. Strains of Salmonella enterica serovar Typhimurium were engineered to have separately inducible MEP (native) and MVA (nonnative) pathways. These strains, RMC26 and CT31-7d, were then used to differentiate MVA pathway- and MEP pathway-specific perturbation. Compounds that inhibit MEP pathway-dependent bacterial growth but leave MVA-dependent growth unaffected represent MEP pathway-selective antibacterials. This screening platform offers three significant results. First, the compound is antibacterial and is therefore cell permeant, enabling access to the intracellular target. Second, the compound inhibits one or more MEP pathway enzymes. Third, the MVA pathway is unaffected, suggesting selectivity for targeting the bacterial versus host pathway. The cell lines also display increased sensitivity to two reported MEP pathway-specific inhibitors, further biasing the platform toward inhibitors selective for the MEP pathway. We demonstrate development of a robust, high-throughput screening platform that combines phenotypic and target-based screening that can identify MEP pathway-selective antibacterials simply by monitoring optical density as the readout for cell growth/inhibition. PMID:22777049

Testa, Charles A; Johnson, L Jeffrey

2012-09-01

105

A Whole-Cell Phenotypic Screening Platform for Identifying Methylerythritol Phosphate Pathway-Selective Inhibitors as Novel Antibacterial Agents  

PubMed Central

Isoprenoid biosynthesis is essential for survival of all living organisms. More than 50,000 unique isoprenoids occur naturally, with each constructed from two simple five-carbon precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Two pathways for the biosynthesis of IPP and DMAPP are found in nature. Humans exclusively use the mevalonate (MVA) pathway, while most bacteria, including all Gram-negative and many Gram-positive species, use the unrelated methylerythritol phosphate (MEP) pathway. Here we report the development of a novel, whole-cell phenotypic screening platform to identify compounds that selectively inhibit the MEP pathway. Strains of Salmonella enterica serovar Typhimurium were engineered to have separately inducible MEP (native) and MVA (nonnative) pathways. These strains, RMC26 and CT31-7d, were then used to differentiate MVA pathway- and MEP pathway-specific perturbation. Compounds that inhibit MEP pathway-dependent bacterial growth but leave MVA-dependent growth unaffected represent MEP pathway-selective antibacterials. This screening platform offers three significant results. First, the compound is antibacterial and is therefore cell permeant, enabling access to the intracellular target. Second, the compound inhibits one or more MEP pathway enzymes. Third, the MVA pathway is unaffected, suggesting selectivity for targeting the bacterial versus host pathway. The cell lines also display increased sensitivity to two reported MEP pathway-specific inhibitors, further biasing the platform toward inhibitors selective for the MEP pathway. We demonstrate development of a robust, high-throughput screening platform that combines phenotypic and target-based screening that can identify MEP pathway-selective antibacterials simply by monitoring optical density as the readout for cell growth/inhibition.

Johnson, L. Jeffrey

2012-01-01

106

Large-scale integrative network-based analysis identifies common pathways disrupted by copy number alterations across cancers  

PubMed Central

Background Many large-scale studies analyzed high-throughput genomic data to identify altered pathways essential to the development and progression of specific types of cancer. However, no previous study has been extended to provide a comprehensive analysis of pathways disrupted by copy number alterations across different human cancers. Towards this goal, we propose a network-based method to integrate copy number alteration data with human protein-protein interaction networks and pathway databases to identify pathways that are commonly disrupted in many different types of cancer. Results We applied our approach to a data set of 2,172 cancer patients across 16 different types of cancers, and discovered a set of commonly disrupted pathways, which are likely essential for tumor formation in majority of the cancers. We also identified pathways that are only disrupted in specific cancer types, providing molecular markers for different human cancers. Analysis with independent microarray gene expression datasets confirms that the commonly disrupted pathways can be used to identify patient subgroups with significantly different survival outcomes. We also provide a network view of disrupted pathways to explain how copy number alterations affect pathways that regulate cell growth, cycle, and differentiation for tumorigenesis. Conclusions In this work, we demonstrated that the network-based integrative analysis can help to identify pathways disrupted by copy number alterations across 16 types of human cancers, which are not readily identifiable by conventional overrepresentation-based and other pathway-based methods. All the results and source code are available at http://compbio.cs.umn.edu/NetPathID/.

2013-01-01

107

Cerebral Biochemical Pathways in Experimental Autoimmune Encephalomyelitis and Adjuvant Arthritis: A Comparative Metabolomic Study  

PubMed Central

Many diseases, including brain disorders, are associated with perturbations of tissue metabolism. However, an often overlooked issue is the impact that inflammations outside the brain may have on brain metabolism. Our main goal was to study similarities and differences between brain metabolite profiles of animals suffering from experimental autoimmune encephalomyelitis (EAE) and adjuvant arthritis (AA) in Lewis rat models. Our principal objective was the determination of molecular protagonists involved in the metabolism underlying these diseases. EAE was induced by intraplantar injection of complete Freund’s adjuvant (CFA) and spinal-cord homogenate (SC-H), whereas AA was induced by CFA only. Naive rats served as controls (n?=?9 for each group). Two weeks after inoculation, animals were sacrificed, and brains were removed and processed for metabolomic analysis by NMR spectroscopy or for immunohistochemistry. Interestingly, both inflammatory diseases caused similar, though not identical, changes in metabolites involved in regulation of brain cell size and membrane production: among the osmolytes, taurine and the neuronal marker, N-acetylaspartate, were decreased, and the astrocyte marker, myo-inositol, slightly increased in both inoculated groups compared with controls. Also ethanolamine-containing phospholipids, sources of inflammatory agents, and several glycolytic metabolites were increased in both inoculated groups. By contrast, the amino acids, aspartate and isoleucine, were less concentrated in CFA/SC-H and control vs. CFA rats. Our results suggest that inflammatory brain metabolite profiles may indicate the existence of either cerebral (EAE) or extra-cerebral (AA) inflammation. These inflammatory processes may act through distinct pathways that converge toward similar brain metabolic profiles. Our findings open new avenues for future studies aimed at demonstrating whether brain metabolic effects provoked by AA are pain/stress-mediated and/or due to the presence of systemic proinflammatory molecules. Regardless of the nature of these mechanisms, our findings may be of interest for future clinical studies, e.g. by in-vivo magnetic resonance spectroscopy.

Lutz, Norbert W.; Fernandez, Carla; Pellissier, Jean-Francois; Cozzone, Patrick J.; Beraud, Evelyne

2013-01-01

108

In vivo detection by 31P NMR of pentose phosphate pathway block secondary to biochemical modulation.  

PubMed

The chemotherapeutic regimen of N-(phosphonacetyl)-L-aspartate (PALA) followed 17 h later by 6-methylmercaptopurine riboside (MMPR) and 6-aminonicotanamide (6AN) has been shown to be a potent sensitizer of anti-neoplastic therapy. We undertook this study to compare the therapeutic and metabolic effects of this triple drug combination vs one of its components, 6AN, in a murine mammary carcinoma. After treatment with PALA, MMPR and 6AN, a new peak was detected which was assigned to 6-phosphogluconate (6PG), which is a marker of inhibition of the pentose phosphate pathway at the 6-phosphogluconate dehydrogenase step. Treatment with PALA, MMPR and 6AN also induced a decrease in the ratios of nucleoside triphosphate/inorganic phosphate (NTP/Pi) and phosphocreatine/inorganic phosphate (PCr/Pi) similar to previous results with a different tumor model. These effects were most pronounced at 6 and 10 h. In addition, an increase in PME'/phosphocholine (PME' = downfield peak in the phosphomonoester region) was detected, which was expected because of the cytotoxic effect of this regimen. Treatment with 6AN alone also resulted in the detection of 6PG with a maximum intensity at 6 h post-6AN. Treatment with 6AN alone induced a smaller change in PME'/PC and failed to cause a decrease in PCr/Pi or NTP/Pi at 6 and 10 h. The enhanced response to the combination of PALA, MMPR and 6AN vs 6AN alone, both with regard to cytotoxicity and radiosensitization, may be due to energy depletion. PMID:8892397

Mahmood, U; Street, J C; Matei, C; Ballon, D; Martin, D S; Koutcher, J A

1996-05-01

109

A genome-wide deletion mutant screen identifies pathways affected by nickel sulfate in Saccharomyces cerevisiae  

PubMed Central

Background The understanding of the biological function, regulation, and cellular interactions of the yeast genome and proteome, along with the high conservation in gene function found between yeast genes and their human homologues, has allowed for Saccharomyces cerevisiae to be used as a model organism to deduce biological processes in human cells. Here, we have completed a systematic screen of the entire set of 4,733 haploid S. cerevisiae gene deletion strains (the entire set of nonessential genes for this organism) to identify gene products that modulate cellular toxicity to nickel sulfate (NiSO4). Results We have identified 149 genes whose gene deletion causes sensitivity to NiSO4 and 119 genes whose gene deletion confers resistance. Pathways analysis with proteins whose absence renders cells sensitive and resistant to nickel identified a wide range of cellular processes engaged in the toxicity of S. cerevisiae to NiSO4. Functional categories overrepresented with proteins whose absence renders cells sensitive to NiSO4 include homeostasis of protons, cation transport, transport ATPases, endocytosis, siderophore-iron transport, homeostasis of metal ions, and the diphthamide biosynthesis pathway. Functional categories overrepresented with proteins whose absence renders cells resistant to nickel include functioning and transport of the vacuole and lysosome, protein targeting, sorting, and translocation, intra-Golgi transport, regulation of C-compound and carbohydrate metabolism, transcriptional repression, and chromosome segregation/division. Interactome analysis mapped seven nickel toxicity modulating and ten nickel-resistance networks. Additionally, we studied the degree of sensitivity or resistance of the 111 nickel-sensitive and 72 -resistant strains whose gene deletion product has a similar protein in human cells. Conclusion We have undertaken a whole genome approach in order to further understand the mechanism(s) regulating the cell's toxicity to nickel compounds. We have used computational methods to integrate the data and generate global models of the yeast's cellular response to NiSO4. The results of our study shed light on molecular pathways associated with the cellular response of eukaryotic cells to nickel compounds and provide potential implications for further understanding the toxic effects of nickel compounds to human cells.

2009-01-01

110

Identifying transcription factors and microRNAs as key regulators of pathways using Bayesian inference on known pathway structures  

PubMed Central

Background Transcription factors and microRNAs act in concert to regulate gene expression in eukaryotes. Numerous computational methods based on sequence information are available for the prediction of target genes of transcription factors and microRNAs. Although these methods provide a static snapshot of how genes may be regulated, they are not effective for the identification of condition-specific regulators. Results We propose a new method that combines: a) transcription factors and microRNAs that are predicted to target genes in pathways, with b) microarray expression profiles of microRNAs and mRNAs, in conjunction with c) the known structure of molecular pathways. These elements are integrated into a Bayesian network derived from each pathway that, through probability inference, allows for the prediction of the key regulators in the pathway. We demonstrate 1) the steps to discretize the expression data for the computation of conditional probabilities in a Bayesian network, 2) the procedure to construct a Bayesian network using the structure of a known pathway and the transcription factors and microRNAs predicted to target genes in that pathway, and 3) the inference results as potential regulators of three signaling pathways using microarray expression profiles of microRNA and mRNA in estrogen receptor positive and estrogen receptor negative tumors. Conclusions We displayed the ability of our framework to integrate multiple sets of microRNA and mRNA expression data, from two phenotypes, with curated molecular pathway structures by creating Bayesian networks. Moreover, by performing inference on the network using known evidence, e.g., status of differentially expressed genes, or by entering hypotheses to be tested, we obtain a list of potential regulators of the pathways. This, in turn, will help increase our understanding about the regulatory mechanisms relevant to the two phenotypes.

2012-01-01

111

Biochemical Analysis of the Biosynthetic Pathway of an Anticancer Tetracycline SF2575  

PubMed Central

SF2575 1 is a tetracycline polyketide produced by Streptomyces sp. SF2575 and displays exceptionally potent anticancer activity towards a broad range of cancer cell lines. The structure of SF2575 is characterized by a highly substituted tetracycline aglycon. The modifications include methylation of the C-6 and C-12a hydroxyl groups, acylation of the 4-(S)-hydroxyl with salicylic acid, C-glycosylation of the C-9 of the D-ring with d-olivose and further acylation of the C4?-hydroxyl of d-olivose with the unusual angelic acid. Understanding the biosynthesis of SF2575 can therefore expand the repertoire of enzymes that can modify tetracyclines, and facilitate engineered biosynthesis of SF2575 analogs. In this study, we identified, sequenced and functionally analyzed the ssf biosynthetic gene cluster which contains 40 putative open reading frames. Genes encoding enzymes that can assemble the tetracycline aglycon, as well as installing these unique structural features are found in the gene cluster. Biosynthetic intermediates were isolated from the SF2575 culture extract to suggest the order of pendant groups addition is C-9 glycosylation, C-4 salicylation and O-4? angelycylation. Using in vitro assays, two enzymes that are responsible for C-4 acylation of salicylic acid were identified. These enzymes include an ATP-dependent salicylyl-CoA ligase SsfL1 and a putative GDSL family acyltransferase SsfX3, both of which were shown to have relaxed substrate specificity towards substituted benzoic acids. Since the salicylic acid moiety is critically important for the anticancer properties of SF2575, verification of the activities of SsfL1 and SsfX3 sets the stage for biosynthetic modification of the C-4 group towards structural-activity relationship studies of SF2575. Using heterologous biosynthesis in Streptomyces lividans, we also determined that biosynthesis of the SF2575 tetracycline aglycon 8 parallels that of oxytetracycline 4 and diverges after the assembly of 4-keto-anhydrotetracycline 51. The minimal ssf polyketide synthase together with the amidotransferase SsfD produced the amidated decaketide backbone that is required for the formation of 2-naphthacenecarboxamide skeleton. Additional enzymes, such as cyclases, C-6 methyltransferase and C-4/C-12a dihydroxylase were functionally reconstituted.

Pickens, Lauren B.; Kim, Woncheol; Wang, Peng; Zhou, Hui; Watanabe, Kenji; Gomi, Shuichi; Tang, Yi

2009-01-01

112

Biochemical analysis of the biosynthetic pathway of an anticancer tetracycline SF2575.  

PubMed

SF2575 1 is a tetracycline polyketide produced by Streptomyces sp. SF2575 and displays exceptionally potent anticancer activity toward a broad range of cancer cell lines. The structure of SF2575 is characterized by a highly substituted tetracycline aglycon. The modifications include methylation of the C-6 and C-12a hydroxyl groups, acylation of the 4-(S)-hydroxyl with salicylic acid, C-glycosylation of the C-9 of the D-ring with D-olivose and further acylation of the C4'-hydroxyl of D-olivose with the unusual angelic acid. Understanding the biosynthesis of SF2575 can therefore expand the repertoire of enzymes that can modify tetracyclines, and facilitate engineered biosynthesis of SF2575 analogues. In this study, we identified, sequenced, and functionally analyzed the ssf biosynthetic gene cluster which contains 40 putative open reading frames. Genes encoding enzymes that can assemble the tetracycline aglycon, as well as installing these unique structural features, are found in the gene cluster. Biosynthetic intermediates were isolated from the SF2575 culture extract to suggest the order of pendant-group addition is C-9 glycosylation, C-4 salicylation, and O-4' angelylcylation. Using in vitro assays, two enzymes that are responsible for C-4 acylation of salicylic acid were identified. These enzymes include an ATP-dependent salicylyl-CoA ligase SsfL1 and a putative GDSL family acyltransferase SsfX3, both of which were shown to have relaxed substrate specificity toward substituted benzoic acids. Since the salicylic acid moiety is critically important for the anticancer properties of SF2575, verification of the activities of SsfL1 and SsfX3 sets the stage for biosynthetic modification of the C-4 group toward structure-activity relationship studies of SF2575. Using heterologous biosynthesis in Streptomyces lividans, we also determined that biosynthesis of the SF2575 tetracycline aglycon 8 parallels that of oxytetracycline 4 and diverges after the assembly of 4-keto-anhydrotetracycline 51. The minimal ssf polyketide synthase together with the amidotransferase SsfD produced the amidated decaketide backbone that is required for the formation of 2-naphthacenecarboxamide skeleton. Additional enzymes, such as cyclases C-6 methyltransferase and C-4/C-12a dihydroxylase, were functionally reconstituted. PMID:19908837

Pickens, Lauren B; Kim, Woncheol; Wang, Peng; Zhou, Hui; Watanabe, Kenji; Gomi, Shuichi; Tang, Yi

2009-12-01

113

Global gene expression analysis identifies molecular pathways distinguishing blastocyst dormancy and activation.  

PubMed

Delayed implantation (embryonic diapause) occurs when the embryo at the blastocyst stage achieves a state of suspended animation. During this period, blastocyst growth is very slow, with minimal or no cell division. Nearly 100 mammals in seven different orders undergo delayed implantation, but the underlying molecular mechanisms that direct this process remain largely unknown. In mice, ovariectomy before preimplantation ovarian estrogen secretion on day 4 of pregnancy initiates blastocyst dormancy, which normally lasts for 1-2 weeks by continued progesterone treatment, although blastocyst survival decreases with time. An estrogen injection rapidly activates blastocysts and initiates their implantation in the progesterone-primed uterus. Using this model, here we show that among approximately 20,000 genes examined, only 229 are differentially expressed between dormant and activated blastocysts. The major functional categories of altered genes include the cell cycle, cell signaling, and energy metabolic pathways, particularly highlighting the importance of heparin-binding epidermal growth factor-like signaling in blastocyst-uterine crosstalk in implantation. The results provide evidence that the two different physiological states of the blastocyst, dormancy and activation, are molecularly distinguishable in a global perspective and underscore the importance of specific molecular pathways in these processes. This study has identified candidate genes that provide a scope for in-depth analysis of their functions and an opportunity for examining their relevance to blastocyst dormancy and activation in numerous other species for which microarray analysis is not available or possible due to very limited availability of blastocysts. PMID:15232000

Hamatani, Toshio; Daikoku, Takiko; Wang, Haibin; Matsumoto, Hiromichi; Carter, Mark G; Ko, Minoru S H; Dey, Sudhansu K

2004-07-13

114

Parallel genetic and proteomic screens identify Msps as a CLASP-Abl pathway interactor in Drosophila.  

PubMed

Regulation of cytoskeletal structure and dynamics is essential for multiple aspects of cellular behavior, yet there is much to learn about the molecular machinery underlying the coordination between the cytoskeleton and its effector systems. One group of proteins that regulate microtubule behavior and its interaction with other cellular components, such as actin-regulatory proteins and transport machinery, is the plus-end tracking proteins (MT+TIPs). In particular, evidence suggests that the MT+TIP, CLASP, may play a pivotal role in the coordination of microtubules with other cellular structures in multiple contexts, although the molecular mechanism by which it functions is still largely unknown. To gain deeper insight into the functional partners of CLASP, we conducted parallel genetic and proteome-wide screens for CLASP interactors in Drosophila melanogaster. We identified 36 genetic modifiers and 179 candidate physical interactors, including 13 that were identified in both data sets. Grouping interactors according to functional classifications revealed several categories, including cytoskeletal components, signaling proteins, and translation/RNA regulators. We focused our initial investigation on the MT+TIP Minispindles (Msps), identified among the cytoskeletal effectors in both genetic and proteomic screens. Here, we report that Msps is a strong modifier of CLASP and Abl in the retina. Moreover, we show that Msps functions during axon guidance and antagonizes both CLASP and Abl activity. Our data suggest a model in which CLASP and Msps converge in an antagonistic balance in the Abl signaling pathway. PMID:20498300

Lowery, L A; Lee, H; Lu, C; Murphy, R; Obar, R A; Zhai, B; Schedl, M; Van Vactor, D; Zhan, Y

2010-08-01

115

Metabolomics identifies novel Hnf1alpha-dependent physiological pathways in vivo.  

PubMed

Mutations in the HNF1A gene cause maturity-onset diabetes of the young type 3, one of the most common genetic causes of non-insulin-dependent (type 2) diabetes mellitus. Although the whole-body Hnf1a-null mouse recapitulates the low insulin levels and high blood glucose observed in human maturity-onset diabetes of the young type 3 patients, these mice also suffer from Laron dwarfism and aminoaciduria, suggesting a role for hepatocyte nuclear factor 1? (Hnf1?) in pathophysiologies distinct from non-insulin-dependent (type 2) diabetes mellitus. In an effort to identify pathways associated with inactivation of Hnf1?, an ultraperformance liquid chromatography coupled to mass spectrometry-based metabolomics study was conducted on urine samples from wild-type and Hnf1a-null mice. An increase in phenylalanine metabolites is in agreement with the known regulation of the phenylalanine hydroxylase gene by Hnf1?. This metabolomic approach also identified urinary biomarkers for three tissue-specific dysfunctions previously unassociated with Hnf1? function. 1) Elevated indolelactate coupled to decreased xanthurenic acid also indicated defects in the indole and kynurenine pathways of tryptophan metabolism, respectively. 2) An increase in the neutral amino acid proline in the urine of Hnf1a-null mice correlated with loss of renal apical membrane transporters of the Slc6a family. 3) Further investigation into the mechanism of aldosterone increase revealed an overactive adrenal gland in Hnf1a-null mice possibly due to inhibition of negative feedback regulation. Although the phenotype of the Hnf1a-null mouse is complex, metabolomics has opened the door to investigation of several physiological systems in which Hnf1? may be a critical regulatory component. PMID:20943816

Bonzo, Jessica A; Patterson, Andrew D; Krausz, Kristopher W; Gonzalez, Frank J

2010-12-01

116

Integrated Analysis of Mutation Data from Various Sources Identifies Key Genes and Signaling Pathways in Hepatocellular Carcinoma  

PubMed Central

Background Recently, a number of studies have performed genome or exome sequencing of hepatocellular carcinoma (HCC) and identified hundreds or even thousands of mutations in protein-coding genes. However, these studies have only focused on a limited number of candidate genes, and many important mutation resources remain to be explored. Principal Findings In this study, we integrated mutation data obtained from various sources and performed pathway and network analysis. We identified 113 pathways that were significantly mutated in HCC samples and found that the mutated genes included in these pathways contained high percentages of known cancer genes, and damaging genes and also demonstrated high conservation scores, indicating their important roles in liver tumorigenesis. Five classes of pathways that were mutated most frequently included (a) proliferation and apoptosis related pathways, (b) tumor microenvironment related pathways, (c) neural signaling related pathways, (d) metabolic related pathways, and (e) circadian related pathways. Network analysis further revealed that the mutated genes with the highest betweenness coefficients, such as the well-known cancer genes TP53, CTNNB1 and recently identified novel mutated genes GNAL and the ADCY family, may play key roles in these significantly mutated pathways. Finally, we highlight several key genes (e.g., RPS6KA3 and PCLO) and pathways (e.g., axon guidance) in which the mutations were associated with clinical features. Conclusions Our workflow illustrates the increased statistical power of integrating multiple studies of the same subject, which can provide biological insights that would otherwise be masked under individual sample sets. This type of bioinformatics approach is consistent with the necessity of making the best use of the ever increasing data provided in valuable databases, such as TCGA, to enhance the speed of deciphering human cancers.

Wei, Lin; Tang, Ruqi; Lian, Baofeng; Zhao, Yingjun; He, Xianghuo; Xie, Lu

2014-01-01

117

Significant Deregulated Pathways in Diabetes Type II Complications Identified through Expression Based Network Biology  

NASA Astrophysics Data System (ADS)

Type 2 Diabetes is a complex multifactorial disease, which alters several signaling cascades giving rise to serious complications. It is one of the major risk factors for cardiovascular diseases. The present research work describes an integrated functional network biology approach to identify pathways that get transcriptionally altered and lead to complex complications thereby amplifying the phenotypic effect of the impaired disease state. We have identified two sub-network modules, which could be activated under abnormal circumstances in diabetes. Present work describes key proteins such as P85A and SRC serving as important nodes to mediate alternate signaling routes during diseased condition. P85A has been shown to be an important link between stress responsive MAPK and CVD markers involved in fibrosis. MAPK8 has been shown to interact with P85A and further activate CTGF through VEGF signaling. We have traced a novel and unique route correlating inflammation and fibrosis by considering P85A as a key mediator of signals. The next sub-network module shows SRC as a junction for various signaling processes, which results in interaction between NF-kB and beta catenin to cause cell death. The powerful interaction between these important genes in response to transcriptionally altered lipid metabolism and impaired inflammatory response via SRC causes apoptosis of cells. The crosstalk between inflammation, lipid homeostasis and stress, and their serious effects downstream have been explained in the present analyses.

Ukil, Sanchaita; Sinha, Meenakshee; Varshney, Lavneesh; Agrawal, Shipra

118

Serum nitrite sensitively reflects endothelial NO formation in human forearm vasculature: evidence for biochemical assessment of the endothelial L-arginine-NO pathway  

Microsoft Academic Search

Objective: A reduced bioactivity of endothelial nitric oxide (NO) has been implicated in the pathogenesis of atherosclerosis. In humans, the endothelial L-arginine-NO pathway has been indirectly assessed via the flow response to endothelium-dependent vasodilators locally administered into the coronary, pulmonary or forearm circulation. However, biochemical quantification of endothelial NO formation in these organ circulations has been hampered so far because

Malte Kelm; Hagen Preik-Steinhoff; Michael Preik; Bodo E. Strauer

119

Identifying biological pathways that underlie primordial short stature using network analysis.  

PubMed

Mutations in CUL7, OBSL1 and CCDC8, leading to disordered ubiquitination, cause one of the commonest primordial growth disorders, 3-M syndrome. This condition is associated with i) abnormal p53 function, ii) GH and/or IGF1 resistance, which may relate to failure to recycle signalling molecules, and iii) cellular IGF2 deficiency. However the exact molecular mechanisms that may link these abnormalities generating growth restriction remain undefined. In this study, we have used immunoprecipitation/mass spectrometry and transcriptomic studies to generate a 3-M 'interactome', to define key cellular pathways and biological functions associated with growth failure seen in 3-M. We identified 189 proteins which interacted with CUL7, OBSL1 and CCDC8, from which a network including 176 of these proteins was generated. To strengthen the association to 3-M syndrome, these proteins were compared with an inferred network generated from the genes that were differentially expressed in 3-M fibroblasts compared with controls. This resulted in a final 3-M network of 131 proteins, with the most significant biological pathway within the network being mRNA splicing/processing. We have shown using an exogenous insulin receptor (INSR) minigene system that alternative splicing of exon 11 is significantly changed in HEK293 cells with altered expression of CUL7, OBSL1 and CCDC8 and in 3-M fibroblasts. The net result is a reduction in the expression of the mitogenic INSR isoform in 3-M syndrome. From these preliminary data, we hypothesise that disordered ubiquitination could result in aberrant mRNA splicing in 3-M; however, further investigation is required to determine whether this contributes to growth failure. PMID:24711643

Hanson, Dan; Stevens, Adam; Murray, Philip G; Black, Graeme C M; Clayton, Peter E

2014-06-01

120

An integrative framework for Bayesian variable selection with informative priors for identifying genes and pathways.  

PubMed

The discovery of genetic or genomic markers plays a central role in the development of personalized medicine. A notable challenge exists when dealing with the high dimensionality of the data sets, as thousands of genes or millions of genetic variants are collected on a relatively small number of subjects. Traditional gene-wise selection methods using univariate analyses face difficulty to incorporate correlational, structural, or functional structures amongst the molecular measures. For microarray gene expression data, we first summarize solutions in dealing with 'large p, small n' problems, and then propose an integrative Bayesian variable selection (iBVS) framework for simultaneously identifying causal or marker genes and regulatory pathways. A novel partial least squares (PLS) g-prior for iBVS is developed to allow the incorporation of prior knowledge on gene-gene interactions or functional relationships. From the point view of systems biology, iBVS enables user to directly target the joint effects of multiple genes and pathways in a hierarchical modeling diagram to predict disease status or phenotype. The estimated posterior selection probabilities offer probabilitic and biological interpretations. Both simulated data and a set of microarray data in predicting stroke status are used in validating the performance of iBVS in a Probit model with binary outcomes. iBVS offers a general framework for effective discovery of various molecular biomarkers by combining data-based statistics and knowledge-based priors. Guidelines on making posterior inferences, determining Bayesian significance levels, and improving computational efficiencies are also discussed. PMID:23844055

Peng, Bin; Zhu, Dianwen; Ander, Bradley P; Zhang, Xiaoshuai; Xue, Fuzhong; Sharp, Frank R; Yang, Xiaowei

2013-01-01

121

Identifying biological pathways that underlie primordial short stature using network analysis  

PubMed Central

Mutations in CUL7, OBSL1 and CCDC8, leading to disordered ubiquitination, cause one of the commonest primordial growth disorders, 3-M syndrome. This condition is associated with i) abnormal p53 function, ii) GH and/or IGF1 resistance, which may relate to failure to recycle signalling molecules, and iii) cellular IGF2 deficiency. However the exact molecular mechanisms that may link these abnormalities generating growth restriction remain undefined. In this study, we have used immunoprecipitation/mass spectrometry and transcriptomic studies to generate a 3-M ‘interactome’, to define key cellular pathways and biological functions associated with growth failure seen in 3-M. We identified 189 proteins which interacted with CUL7, OBSL1 and CCDC8, from which a network including 176 of these proteins was generated. To strengthen the association to 3-M syndrome, these proteins were compared with an inferred network generated from the genes that were differentially expressed in 3-M fibroblasts compared with controls. This resulted in a final 3-M network of 131 proteins, with the most significant biological pathway within the network being mRNA splicing/processing. We have shown using an exogenous insulin receptor (INSR) minigene system that alternative splicing of exon 11 is significantly changed in HEK293 cells with altered expression of CUL7, OBSL1 and CCDC8 and in 3-M fibroblasts. The net result is a reduction in the expression of the mitogenic INSR isoform in 3-M syndrome. From these preliminary data, we hypothesise that disordered ubiquitination could result in aberrant mRNA splicing in 3-M; however, further investigation is required to determine whether this contributes to growth failure.

Hanson, Dan; Stevens, Adam; Murray, Philip G; Black, Graeme C M; Clayton, Peter E

2014-01-01

122

Jasmonate Biochemical Pathway  

NSDL National Science Digital Library

Plants possess a family of potent fatty acid–derived wound-response and developmental regulators: the jasmonates. These compounds are derived from the tri-unsaturated fatty acids α-linolenic acid (18:3) and, in plants such as Arabidopsis thaliana and tomato, 7(Z)-, 10(Z)-, and 13(Z)-hexadecatrienoic acid (16:3). The lipoxygenase-catalyzed addition of molecular oxygen to α-linolenic acid initiates jasmonate synthesis by providing a 13-hydroperoxide substrate for formation of an unstable allene oxide by allene oxide synthase (AOS). This allene oxide then undergoes enzyme-guided cyclization to produce 12-oxophytodienoic acid (OPDA). These first steps take place in plastids, but further OPDA metabolism occurs in peroxisomes. OPDA has several fates, including esterification into plastid lipids and transformation into the 12-carbon prohormone jasmonic acid (JA). JA is itself a substrate for further diverse modifications, including the production of jasmonoyl-isoleucine (JA-Ile), which is a major biologically active jasmonate among a growing number of jasmonate derivatives. Each new jasmonate family member that is discovered provides another key to understanding the fine control of gene expression in immune responses; in the initiation and maintenance of long-distance signal transfer in response to wounding; in the regulation of fertility; and in the turnover, inactivation, and sequestration of jasmonates, among other processes.

Aurelie Gfeller (Switzerland;University of Lausanne REV); Lucie Dubugnon (Lausanne;Institut de Chimie Clinique REV); Robin Liechti (Lausanne;Swiss Institute of Bioinformatics REV); Edward E. Farmer (Switzerland;University of Lausanne, Biophore REV)

2010-02-16

123

Pathway-based genome-wide association analysis identified the importance of regulation-of-autophagy pathway for ultradistal radius BMD.  

PubMed

Wrist fracture is not only one of the most common osteoporotic fractures but also a predictor of future fractures at other sites. Wrist bone mineral density (BMD) is an important determinant of wrist fracture risk, with high heritability. Specific genes underlying wrist BMD variation are largely unknown. Most published genome-wide association studies (GWASs) have focused only on a few top-ranking single-nucleotide polymorphisms (SNPs)/genes and considered each of the identified SNPs/genes independently. To identify biologic pathways important to wrist BMD variation, we used a novel pathway-based analysis approach in our GWAS of wrist ultradistal radius (UD) BMD, examining approximately 500,000 SNPs genome-wide from 984 unrelated whites. A total of 963 biologic pathways/gene sets were analyzed. We identified the regulation-of-autophagy (ROA) pathway that achieved the most significant result (p = .005, q(fdr) = 0.043, p(fwer) = 0.016) for association with UD BMD. The ROA pathway also showed significant association with arm BMD in the Framingham Heart Study sample containing 2187 subjects, which further confirmed our findings in the discovery cohort. Earlier studies indicated that during endochondral ossification, autophagy occurs prior to apoptosis of hypertrophic chondrocytes, and it also has been shown that some genes in the ROA pathway (e.g., INFG) may play important roles in osteoblastogenesis or osteoclastogenesis. Our study supports the potential role of the ROA pathway in human wrist BMD variation and osteoporosis. Further functional evaluation of this pathway to determine the mechanism by which it regulates wrist BMD should be pursued to provide new insights into the pathogenesis of wrist osteoporosis. PMID:20200951

Zhang, Lishu; Guo, Yan-Fang; Liu, Yao-Zhong; Liu, Yong-Jun; Xiong, Dong-Hai; Liu, Xiao-Gang; Wang, Liang; Yang, Tie-Lin; Lei, Shu-Feng; Guo, Yan; Yan, Han; Pei, Yu-Fang; Zhang, Feng; Papasian, Christopher J; Recker, Robert R; Deng, Hong-Wen

2010-07-01

124

An isogenic cell panel identifies compounds that inhibit proliferation of mTOR-pathway addicted cells by different mechanisms.  

PubMed

The mTOR pathway is a critical integrator of nutrient and growth factor signaling. Once activated, mTOR promotes cell growth and proliferation. Several components of the mTOR pathway are frequently deregulated in tumors, leading to constitutive activation of the pathway and thus contribute to uncontrolled cell growth. We performed a high-throughput screen with an isogenic cell line system to identify compounds specifically inhibiting proliferation of PTEN/mTOR-pathway addicted cells. We show here the characterization and mode of action of two such compound classes. One compound class inhibits components of the PTEN/mTOR signaling pathway, such as S6 ribosomal protein phosphorylation, and leads to cyclin D3 downregulation. These compounds are not adenosine triphosphate competitive inhibitors for kinases in the pathway, nor do they require FKBP12 for activity like rapamycin. The other compound class turned out to be a farnesylation inhibitor, blocking the activity of GTPases, as well as an inducer of oxidative stress. Our results demonstrate that an isogenic cell system with few specific mutations in oncogenes and tumor suppressor genes can identify different classes of compounds selectively inhibiting proliferation of PTEN/mTOR pathway-addicted isogenic clones. The identified mechanisms are in line with the known cellular signaling networks activated by the altered oncogenes and suppressor genes in the isogenic system. PMID:23954931

Wyder Peters, Lorenza; Molle, Klaus D; Thiemeyer, Anke; Knopf, Agnes; Goxe, Marie; Guerry, Philippe; Brodbeck, Daniela; Colombi, Marco; Hall, Michael N; Moroni, Christoph; Regenass, Urs

2014-01-01

125

Bi-directional gene set enrichment and canonical correlation analysis identify key diet-sensitive pathways and biomarkers of metabolic syndrome  

PubMed Central

Background Currently, a number of bioinformatics methods are available to generate appropriate lists of genes from a microarray experiment. While these lists represent an accurate primary analysis of the data, fewer options exist to contextualise those lists. The development and validation of such methods is crucial to the wider application of microarray technology in the clinical setting. Two key challenges in clinical bioinformatics involve appropriate statistical modelling of dynamic transcriptomic changes, and extraction of clinically relevant meaning from very large datasets. Results Here, we apply an approach to gene set enrichment analysis that allows for detection of bi-directional enrichment within a gene set. Furthermore, we apply canonical correlation analysis and Fisher's exact test, using plasma marker data with known clinical relevance to aid identification of the most important gene and pathway changes in our transcriptomic dataset. After a 28-day dietary intervention with high-CLA beef, a range of plasma markers indicated a marked improvement in the metabolic health of genetically obese mice. Tissue transcriptomic profiles indicated that the effects were most dramatic in liver (1270 genes significantly changed; p < 0.05), followed by muscle (601 genes) and adipose (16 genes). Results from modified GSEA showed that the high-CLA beef diet affected diverse biological processes across the three tissues, and that the majority of pathway changes reached significance only with the bi-directional test. Combining the liver tissue microarray results with plasma marker data revealed 110 CLA-sensitive genes showing strong canonical correlation with one or more plasma markers of metabolic health, and 9 significantly overrepresented pathways among this set; each of these pathways was also significantly changed by the high-CLA diet. Closer inspection of two of these pathways - selenoamino acid metabolism and steroid biosynthesis - illustrated clear diet-sensitive changes in constituent genes, as well as strong correlations between gene expression and plasma markers of metabolic syndrome independent of the dietary effect. Conclusion Bi-directional gene set enrichment analysis more accurately reflects dynamic regulatory behaviour in biochemical pathways, and as such highlighted biologically relevant changes that were not detected using a traditional approach. In such cases where transcriptomic response to treatment is exceptionally large, canonical correlation analysis in conjunction with Fisher's exact test highlights the subset of pathways showing strongest correlation with the clinical markers of interest. In this case, we have identified selenoamino acid metabolism and steroid biosynthesis as key pathways mediating the observed relationship between metabolic health and high-CLA beef. These results indicate that this type of analysis has the potential to generate novel transcriptome-based biomarkers of disease.

2010-01-01

126

2D NMR-based metabolomics uncovers interactions between conserved biochemical pathways in the model organism Caenorhabditis elegans.  

PubMed

Ascarosides are small-molecule signals that play a central role in C. elegans biology, including dauer formation, aging, and social behaviors, but many aspects of their biosynthesis remain unknown. Using automated 2D NMR-based comparative metabolomics, we identified ascaroside ethanolamides as shunt metabolites in C. elegans mutants of daf-22, a gene with homology to mammalian 3-ketoacyl-CoA thiolases predicted to function in conserved peroxisomal lipid ?-oxidation. Two groups of ethanolamides feature ?-keto functionalization confirming the predicted role of daf-22 in ascaroside biosynthesis, whereas ?-methyl substitution points to unexpected inclusion of methylmalonate at a late stage in the biosynthesis of long-chain fatty acids in C. elegans. We show that ascaroside ethanolamide formation in response to defects in daf-22 and other peroxisomal genes is associated with severe depletion of endocannabinoid pools. These results indicate unexpected interaction between peroxisomal lipid ?-oxidation and the biosynthesis of endocannabinoids, which are major regulators of lifespan in C. elegans. Our study demonstrates the utility of unbiased comparative metabolomics for investigating biochemical networks in metazoans. PMID:23163760

Izrayelit, Yevgeniy; Robinette, Steven L; Bose, Neelanjan; von Reuss, Stephan H; Schroeder, Frank C

2013-02-15

127

2D NMR-based metabolomics uncovers interactions between conserved biochemical pathways in the model organism Caenorhabditis elegans  

PubMed Central

Ascarosides are small-molecule signals that play a central role in C. elegans biology, including dauer formation, aging, and social behaviors, but many aspects of their biosynthesis remain unknown. Using automated 2D NMR-based comparative metabolomics, we identified ascaroside ethanolamides as shunt metabolites in C. elegans mutants of daf-22, a gene with homology to mammalian 3-ketoacyl-CoA thiolases predicted to function in conserved peroxisomal lipid ?-oxidation. Two groups of ethanolamides feature ?-keto functionalization confirming the predicted role of daf-22 in ascaroside biosynthesis, whereas ?-methyl substitution points to unexpected inclusion of methylmalonte at a late stage in the biosynthesis of long-chain fatty acids in C. elegans. We show that ascaroside ethanolamide formation in response to defects in daf-22 and other peroxisomal genes is associated with severe depletion of endocannabinoid pools. These results indicate unexpected interaction between peroxisomal lipid ?-oxidation and the biosynthesis of endocannabinoids, which are major regulators of lifespan in C. elegans. Our study demonstrates the utility of unbiased comparative metabolomics for investigating biochemical networks in metazoans.

Izrayelit, Yevgeniy; Robinette, Steven L.; Bose, Neelanjan; von Reuss, Stephan H.; Schroeder, Frank C.

2012-01-01

128

Delineation of joint molecule resolution pathways in meiosis identifies a crossover-specific resolvase.  

PubMed

At the final step of homologous recombination, Holliday junction-containing joint molecules (JMs) are resolved to form crossover or noncrossover products. The enzymes responsible for JM resolution in vivo remain uncertain, but three distinct endonucleases capable of resolving JMs in vitro have been identified: Mus81-Mms4(EME1), Slx1-Slx4(BTBD12), and Yen1(GEN1). Using physical monitoring of recombination during budding yeast meiosis, we show that all three endonucleases are capable of promoting JM resolution in vivo. However, in mms4 slx4 yen1 triple mutants, JM resolution and crossing over occur efficiently. Paradoxically, crossing over in this background is strongly dependent on the Blooms helicase ortholog Sgs1, a component of a well-characterized anticrossover activity. Sgs1-dependent crossing over, but not JM resolution per se, also requires XPG family nuclease Exo1 and the MutL? complex Mlh1-Mlh3. Thus, Sgs1, Exo1, and MutL? together define a previously undescribed meiotic JM resolution pathway that produces the majority of crossovers in budding yeast and, by inference, in mammals. PMID:22500800

Zakharyevich, Kseniya; Tang, Shangming; Ma, Yunmei; Hunter, Neil

2012-04-13

129

Delineation of joint molecule resolution pathways in meiosis identifies a crossover-specific resolvase  

PubMed Central

Summary At the final step of homologous recombination, Holliday Junction-containing joint molecules (JMs) are resolved to form crossover or noncrossover products. The enzymes responsible for JM resolution in vivo remain uncertain, but three distinct endonucleases capable of resolving JMs in vitro have been identified: Mus81-Mms4(EME1), Slx1–Slx4(BTBD12) and Yen1(GEN1). Using physical monitoring of recombination during budding yeast meiosis, we show that all three endonucleases are capable of promoting JM resolution in vivo. However, in mms4 slx4 yen1 triple mutants, JM resolution and crossing-over occur efficiently. Paradoxically, crossing-over in this background is strongly dependent on the Blooms helicase ortholog, Sgs1, a component of a well-characterized anti-crossover activity. Sgs1-dependent crossing-over, but not JM resolution per se, also requires XPG-family nuclease, Exo1, and the MutL? complex, Mlh1–Mlh3. Thus, Sgs1, Exo1 and MutL? together define a previously undescribed meiotic JM resolution pathway that produces the majority of crossovers in budding yeast and, by inference, in mammals.

Zakharyevich, Kseniya; Tang, Shangming; Ma, Yunmei; Hunter, Neil

2012-01-01

130

Selective ?v integrin depletion identifies a core, targetable molecular pathway that regulates fibrosis across solid organs  

PubMed Central

Myofibroblasts are the major source of extracellular matrix components that accumulate during tissue fibrosis, and hepatic stellate cells (HSCs) are the major source of myofibroblasts in the liver. To date, robust systems to genetically manipulate these cells have not existed. We report that Pdgfrb-Cre inactivates genes in murine HSCs with high efficiency. We used this system to delete the ?v integrin subunit because of the suggested role of multiple ?v integrins as central mediators of fibrosis in multiple organs. Depletion of the ?v integrin subunit in HSCs protected mice from CCl4-induced hepatic fibrosis, whereas global loss of ?v?3, ?v?5 or ?v?6 or conditional loss of ?v?8 on HSCs did not. Pdgfrb-Cre effectively targeted myofibroblasts in multiple organs, and depletion of ?v integrins using this system was also protective in models of pulmonary and renal fibrosis. Critically, pharmacological blockade of ?v integrins by a novel small molecule (CWHM 12) attenuated both liver and lung fibrosis, even when administered after fibrosis was established. These data identify a core pathway that regulates fibrosis, and suggest that pharmacological targeting of all ?v integrins may have clinical utility in the treatment of patients with a broad range of fibrotic diseases.

Henderson, Neil C; Arnold, Thomas D; Katamura, Yoshio; Giacomini, Marilyn M; Rodriguez, Juan D; McCarty, Joseph H; Pellicoro, Antonella; Raschperger, Elisabeth; Betsholtz, Christer; Ruminski, Peter G; Griggs, David W; Prinsen, Michael J; Maher, Jacquelyn J; Iredale, John P; Lacy-Hulbert, Adam; Adams, Ralf H; Sheppard, Dean

2013-01-01

131

Targeting of ?v integrin identifies a core molecular pathway that regulates fibrosis in several organs.  

PubMed

Myofibroblasts are the major source of extracellular matrix components that accumulate during tissue fibrosis, and hepatic stellate cells (HSCs) are believed to be the major source of myofibroblasts in the liver. To date, robust systems to genetically manipulate these cells have not been developed. We report that Cre under control of the promoter of Pdgfrb (Pdgfrb-Cre) inactivates loxP-flanked genes in mouse HSCs with high efficiency. We used this system to delete the gene encoding ?(v) integrin subunit because various ?(v)-containing integrins have been suggested as central mediators of fibrosis in multiple organs. Such depletion protected mice from carbon tetrachloride-induced hepatic fibrosis, whereas global loss of ??, ?? or ?? integrins or conditional loss of ?? integrins in HSCs did not. We also found that Pdgfrb-Cre effectively targeted myofibroblasts in multiple organs, and depletion of the ?(v) integrin subunit using this system was protective in other models of organ fibrosis, including pulmonary and renal fibrosis. Pharmacological blockade of ?(v)-containing integrins by a small molecule (CWHM 12) attenuated both liver and lung fibrosis, including in a therapeutic manner. These data identify a core pathway that regulates fibrosis and suggest that pharmacological targeting of all ?(v) integrins may have clinical utility in the treatment of patients with a broad range of fibrotic diseases. PMID:24216753

Henderson, Neil C; Arnold, Thomas D; Katamura, Yoshio; Giacomini, Marilyn M; Rodriguez, Juan D; McCarty, Joseph H; Pellicoro, Antonella; Raschperger, Elisabeth; Betsholtz, Christer; Ruminski, Peter G; Griggs, David W; Prinsen, Michael J; Maher, Jacquelyn J; Iredale, John P; Lacy-Hulbert, Adam; Adams, Ralf H; Sheppard, Dean

2013-12-01

132

Biochemical identification of the OsMKK6-OsMPK3 signalling pathway for chilling stress tolerance in rice.  

PubMed

MAPK (mitogen-activated protein kinase) pathways have been implicated in stress signalling in plants. In the present study, we performed yeast two-hybrid screening to identify partner MAPKs for OsMKK (Oryza sativa MAPK kinase) 6, a rice MAPK kinase, and revealed specific interactions of OsMKK6 with OsMPK3 and OsMPK6. OsMPK3 and OsMPK6 each co-immunoprecipitated OsMKK6, and both were directly phosphorylated by OsMKK6 in vitro. An MBP (myelin basic protein) kinase assay of the immunoprecipitation complex indicated that OsMPK3 and OsMPK6 were activated in response to a moderately low temperature (12°C), but not a severely low temperature (4°C) in rice seedlings. A constitutively active form of OsMKK6, OsMKK6DD, showed elevated phosphorylation activity against OsMPK3 and OsMPK6 in vitro. OsMPK3, but not OsMPK6, was constitutively activated in transgenic plants overexpressing OsMKK6DD, indicating that OsMPK3 is an in vivo target of OsMKK6. Enhanced chilling tolerance was observed in the transgenic plants overexpressing OsMKK6DD. Taken together, our data suggest that OsMKK6 and OsMPK3 constitute a moderately low-temperature signalling pathway and regulate cold stress tolerance in rice. PMID:22248149

Xie, Guosheng; Kato, Hideki; Imai, Ryozo

2012-04-01

133

Transcriptional profiles of drought-responsive genes in modulating transcription signal transduction, and biochemical pathways in tomato  

PubMed Central

To unravel the molecular mechanisms of drought responses in tomato, gene expression profiles of two drought-tolerant lines identified from a population of Solanum pennellii introgression lines, and the recurrent parent S. lycopersicum cv. M82, a drought-sensitive cultivar, were investigated under drought stress using tomato microarrays. Around 400 genes identified were responsive to drought stress only in the drought-tolerant lines. These changes in genes expression are most likely caused by the two inserted chromosome segments of S. pennellii, which possibly contain drought-tolerance quantitative trait loci (QTLs). Among these genes are a number of transcription factors and signalling proteins which could be global regulators involved in the tomato responses to drought stress. Genes involved in organism growth and development processes were also specifically regulated by drought stress, including those controlling cell wall structure, wax biosynthesis, and plant height. Moreover, key enzymes in the pathways of gluconeogenesis (fructose-bisphosphate aldolase), purine and pyrimidine nucleotide biosynthesis (adenylate kinase), tryptophan degradation (aldehyde oxidase), starch degradation (?-amylase), methionine biosynthesis (cystathionine ?-lyase), and the removal of superoxide radicals (catalase) were also specifically affected by drought stress. These results indicated that tomato plants could adapt to water-deficit conditions through decreasing energy dissipation, increasing ATP energy provision, and reducing oxidative damage. The drought-responsive genes identified in this study could provide further information for understanding the mechanisms of drought tolerance in tomato.

Gong, Pengjuan; Zhang, Junhong; Li, Hanxia; Yang, Changxian; Zhang, Chanjuan; Zhang, Xiaohui; Khurram, Ziaf; Zhang, Yuyang; Wang, Taotao; Fei, Zhangjun; Ye, Zhibiao

2010-01-01

134

Biochemical and pharmacological assessment of MAP-kinase signaling along pain pathways in experimental rodent models: a potential tool for the discovery of novel antinociceptive therapeutics.  

PubMed

Injury to the peripheral or central nervous system can induce changes within the nervous tissues that promote a state of sensitization that may underlie conditions of pathological chronic pain. A key biochemical event in the initiation and maintenance of peripheral and central neuronal sensitization associated with chronic pain is the phosphorylation and subsequent activation of mitogen-activated protein kinases (MAPKs) and immediate early gene transcription factors, in particular cAMP-response element binding protein (CREB). In this commentary we review the preclinical data that describe anatomical and mechanistic aspects of nociceptive-induced signaling along nociceptive pathways including peripheral cutaneous axons, the dorsal root ganglia, spinal cord dorsal horn and cerebral cortex. In addition to the regional manifestation of nociceptive signaling, investigations have attempted to elucidate the cellular origin of biochemical nociceptive processing in which communication, i.e. cross-talk between neurons and glia is viewed as an essential component of pathogenic pain development. Here, we outline a research strategy by which nociceptive-induced cellular signaling in experimental pain models, specifically MAPK and CREB phosphorylation can be utilized to provide mechanistic insight into drug-target interaction along the nociceptive pathways. We describe a series of studies using nociceptive inflammatory and neuropathic pain models to investigate the effects of known pain therapeutics on nociceptive-induced biochemical signaling and present this as a complementary research strategy for assessing antinociceptive activity useful in the preclinical development of novel pain therapeutics. PMID:24300134

Edelmayer, Rebecca M; Brederson, Jill-Desiree; Jarvis, Michael F; Bitner, Robert S

2014-02-01

135

Combining affinity proteomics and network context to identify new phosphatase substrates and adapters in growth pathways  

PubMed Central

Protein phosphorylation homoeostasis is tightly controlled and pathological conditions are caused by subtle alterations of the cell phosphorylation profile. Altered levels of kinase activities have already been associated to specific diseases. Less is known about the impact of phosphatases, the enzymes that down-regulate phosphorylation by removing the phosphate groups. This is partly due to our poor understanding of the phosphatase-substrate network. Much of phosphatase substrate specificity is not based on intrinsic enzyme specificity with the catalytic pocket recognizing the sequence/structure context of the phosphorylated residue. In addition many phosphatase catalytic subunits do not form a stable complex with their substrates. This makes the inference and validation of phosphatase substrates a non-trivial task. Here, we present a novel approach that builds on the observation that much of phosphatase substrate selection is based on the network of physical interactions linking the phosphatase to the substrate. We first used affinity proteomics coupled to quantitative mass spectrometry to saturate the interactome of eight phosphatases whose down regulations was shown to affect the activation of the RAS-PI3K pathway. By integrating information from functional siRNA with protein interaction information, we develop a strategy that aims at inferring phosphatase physiological substrates. Graph analysis is used to identify protein scaffolds that may link the catalytic subunits to their substrates. By this approach we rediscover several previously described phosphatase substrate interactions and characterize two new protein scaffolds that promote the dephosphorylation of PTPN11 and ERK by DUSP18 and DUSP26, respectively.

Sacco, Francesca; Boldt, Karsten; Calderone, Alberto; Panni, Simona; Paoluzi, Serena; Castagnoli, Luisa; Ueffing, Marius; Cesareni, Gianni

2014-01-01

136

Genome-Wide Expression Profiling Identifies Type 1 Interferon Response Pathways in Active Tuberculosis  

PubMed Central

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), remains the leading cause of mortality from a single infectious agent. Each year around 9 million individuals newly develop active TB disease, and over 2 billion individuals are latently infected with M.tb worldwide, thus being at risk of developing TB reactivation disease later in life. The underlying mechanisms and pathways of protection against TB in humans, as well as the dynamics of the host response to M.tb infection, are incompletely understood. We carried out whole-genome expression profiling on a cohort of TB patients longitudinally sampled along 3 time-points: during active infection, during treatment, and after completion of curative treatment. We identified molecular signatures involving the upregulation of type-1 interferon (?/?) mediated signaling and chronic inflammation during active TB disease in an Indonesian population, in line with results from two recent studies in ethnically and epidemiologically different populations in Europe and South Africa. Expression profiles were captured in neutrophil-depleted blood samples, indicating a major contribution of lymphocytes and myeloid cells. Expression of type-1 interferon (?/?) genes mediated was also upregulated in the lungs of M.tb infected mice and in infected human macrophages. In patients, the regulated gene expression-signature normalized during treatment, including the type-1 interferon mediated signaling and a concurrent opposite regulation of interferon-gamma. Further analysis revealed IL15RA, UBE2L6 and GBP4 as molecules involved in the type-I interferon response in all three experimental models. Our data is highly suggestive that the innate immune type-I interferon signaling cascade could be used as a quantitative tool for monitoring active TB disease, and provide evidence that components of the patient’s blood gene expression signature bear similarities to the pulmonary and macrophage response to mycobacterial infection.

Ottenhoff, Tom H. M.; Zhang, Mingzi M.; Wong, Hazel E. E.; Sahiratmadja, Edhyana; Khor, Chiea Chuen; Alisjahbana, Bachti; van Crevel, Reinout; Marzuki, Sangkot; Seielstad, Mark; van de Vosse, Esther; Hibberd, Martin L.

2012-01-01

137

Genetic and biochemical dissection of a HisKA domain identifies residues required exclusively for kinase and phosphatase activities.  

PubMed

Two-component signal transduction systems, composed of histidine kinases (HK) and response regulators (RR), allow bacteria to respond to diverse environmental stimuli. The HK can control both phosphorylation and subsequent dephosphorylation of its cognate RR. The majority of HKs utilize the HisKA subfamily of dimerization and histidine phosphotransfer (DHp) domains, which contain the phospho-accepting histidine and directly contact the RR. Extensive genetics, biochemistry, and structural biology on several prototypical TCS systems including NtrB-NtrC and EnvZ-OmpR have provided a solid basis for understanding the function of HK-RR signaling. Recently, work on NarX, a HisKA_3 subfamily protein, indicated that two residues in the highly conserved region of the DHp domain are responsible for phosphatase activity. In this study we have carried out both genetic and biochemical analyses on Myxococcus xanthus CrdS, a member of the HisKA subfamily of bacterial HKs. CrdS is required for the regulation of spore formation in response to environmental stress. Following alanine-scanning mutagenesis of the ?1 helix of the DHp domain of CrdS, we determined the role for each mutant protein for both kinase and phosphatase activity. Our results indicate that the conserved acidic residue (E372) immediately adjacent to the site of autophosphorylation (H371) is specifically required for kinase activity but not for phosphatase activity. Conversely, we found that the conserved Thr/Asn residue (N375) was required for phosphatase activity but not for kinase activity. We extended our biochemical analyses to two CrdS homologs from M. xanthus, HK1190 and HK4262, as well as Thermotoga maritima HK853. The results were similar for each HisKA family protein where the conserved acidic residue is required for kinase activity while the conserved Thr/Asn residue is required for phosphatase activity. These data are consistent with conserved mechanisms for kinase and phosphatase activities in the broadly occurring HisKA family of sensor kinases in bacteria. PMID:23226719

Willett, Jonathan W; Kirby, John R

2012-01-01

138

Integrated Pathway-Based Approach Identifies Association between Genomic Regions at CTCF and CACNB2 and Schizophrenia  

PubMed Central

In the present study, an integrated hierarchical approach was applied to: (1) identify pathways associated with susceptibility to schizophrenia; (2) detect genes that may be potentially affected in these pathways since they contain an associated polymorphism; and (3) annotate the functional consequences of such single-nucleotide polymorphisms (SNPs) in the affected genes or their regulatory regions. The Global Test was applied to detect schizophrenia-associated pathways using discovery and replication datasets comprising 5,040 and 5,082 individuals of European ancestry, respectively. Information concerning functional gene-sets was retrieved from the Kyoto Encyclopedia of Genes and Genomes, Gene Ontology, and the Molecular Signatures Database. Fourteen of the gene-sets or pathways identified in the discovery dataset were confirmed in the replication dataset. These include functional processes involved in transcriptional regulation and gene expression, synapse organization, cell adhesion, and apoptosis. For two genes, i.e. CTCF and CACNB2, evidence for association with schizophrenia was available (at the gene-level) in both the discovery study and published data from the Psychiatric Genomics Consortium schizophrenia study. Furthermore, these genes mapped to four of the 14 presently identified pathways. Several of the SNPs assigned to CTCF and CACNB2 have potential functional consequences, and a gene in close proximity to CACNB2, i.e. ARL5B, was identified as a potential gene of interest. Application of the present hierarchical approach thus allowed: (1) identification of novel biological gene-sets or pathways with potential involvement in the etiology of schizophrenia, as well as replication of these findings in an independent cohort; (2) detection of genes of interest for future follow-up studies; and (3) the highlighting of novel genes in previously reported candidate regions for schizophrenia.

Zapatka, Marc; Frank, Josef; Witt, Stephanie H.; Muhleisen, Thomas W.; Treutlein, Jens; Strohmaier, Jana; Meier, Sandra; Degenhardt, Franziska; Giegling, Ina; Ripke, Stephan; Leber, Markus; Lange, Christoph; Schulze, Thomas G.; Mossner, Rainald; Nenadic, Igor; Sauer, Heinrich; Rujescu, Dan; Maier, Wolfgang; B?rglum, Anders; Ophoff, Roel; Cichon, Sven; Nothen, Markus M.; Rietschel, Marcella; Mattheisen, Manuel; Brors, Benedikt

2014-01-01

139

Integrated pathway-based approach identifies association between genomic regions at CTCF and CACNB2 and schizophrenia.  

PubMed

In the present study, an integrated hierarchical approach was applied to: (1) identify pathways associated with susceptibility to schizophrenia; (2) detect genes that may be potentially affected in these pathways since they contain an associated polymorphism; and (3) annotate the functional consequences of such single-nucleotide polymorphisms (SNPs) in the affected genes or their regulatory regions. The Global Test was applied to detect schizophrenia-associated pathways using discovery and replication datasets comprising 5,040 and 5,082 individuals of European ancestry, respectively. Information concerning functional gene-sets was retrieved from the Kyoto Encyclopedia of Genes and Genomes, Gene Ontology, and the Molecular Signatures Database. Fourteen of the gene-sets or pathways identified in the discovery dataset were confirmed in the replication dataset. These include functional processes involved in transcriptional regulation and gene expression, synapse organization, cell adhesion, and apoptosis. For two genes, i.e. CTCF and CACNB2, evidence for association with schizophrenia was available (at the gene-level) in both the discovery study and published data from the Psychiatric Genomics Consortium schizophrenia study. Furthermore, these genes mapped to four of the 14 presently identified pathways. Several of the SNPs assigned to CTCF and CACNB2 have potential functional consequences, and a gene in close proximity to CACNB2, i.e. ARL5B, was identified as a potential gene of interest. Application of the present hierarchical approach thus allowed: (1) identification of novel biological gene-sets or pathways with potential involvement in the etiology of schizophrenia, as well as replication of these findings in an independent cohort; (2) detection of genes of interest for future follow-up studies; and (3) the highlighting of novel genes in previously reported candidate regions for schizophrenia. PMID:24901509

Juraeva, Dilafruz; Haenisch, Britta; Zapatka, Marc; Frank, Josef; Witt, Stephanie H; Mühleisen, Thomas W; Treutlein, Jens; Strohmaier, Jana; Meier, Sandra; Degenhardt, Franziska; Giegling, Ina; Ripke, Stephan; Leber, Markus; Lange, Christoph; Schulze, Thomas G; Mössner, Rainald; Nenadic, Igor; Sauer, Heinrich; Rujescu, Dan; Maier, Wolfgang; Børglum, Anders; Ophoff, Roel; Cichon, Sven; Nöthen, Markus M; Rietschel, Marcella; Mattheisen, Manuel; Brors, Benedikt

2014-06-01

140

Ectopic Pregnancy as a Model to Identify Endometrial Genes and Signaling Pathways Important in Decidualization and Regulated by Local Trophoblast  

Microsoft Academic Search

The endometrium in early pregnancy undergoes decidualization and functional changes induced by local trophoblast, which are not fully understood. We hypothesized that endometrium from tubal ectopic pregnancy (EP) could be interrogated to identify novel genes and pathways involved in these processes. Gestation-matched endometrium was collected from women with EP (n = 11) and intrauterine pregnancies (IUP) (n = 13). RNA

W. Colin Duncan; Julie L. V. Shaw; Stewart Burgess; Sarah E. McDonald; Hilary O. D. Critchley; Andrew W. Horne

2011-01-01

141

Major carcinogenic pathways identified by gene expression analysis of peritoneal mesotheliomas following chemical treatment in F344 rats  

EPA Science Inventory

This study was performed to characterize the gene expression profile and to identify the major carcinogenic pathways involved in rat peritoneal mesothelioma (RPM) formation following treatment of Fischer 344 rats with o-nitrotoluene (o-NT) or bromochloracetic acid (BCA). Oligo a...

142

Pathways Disrupted in Human ALS Motor Neurons Identified through Genetic Correction of Mutant SOD1.  

PubMed

Although many distinct mutations in a variety of genes are known to cause Amyotrophic Lateral Sclerosis (ALS), it remains poorly understood how they selectively impact motor neuron biology and whether they converge on common pathways to cause neuronal degeneration. Here, we have combined reprogramming and stem cell differentiation approaches with genome engineering and RNA sequencing to define the transcriptional and functional changes that are induced in human motor neurons by mutant SOD1. Mutant SOD1 protein induced a transcriptional signature indicative of increased oxidative stress, reduced mitochondrial function, altered subcellular transport, and activation of the ER stress and unfolded protein response pathways. Functional studies demonstrated that these pathways were perturbed in a manner dependent on the SOD1 mutation. Finally, interrogation of stem-cell-derived motor neurons produced from ALS patients harboring a repeat expansion in C9orf72 indicates that at least a subset of these changes are more broadly conserved in ALS. PMID:24704492

Kiskinis, Evangelos; Sandoe, Jackson; Williams, Luis A; Boulting, Gabriella L; Moccia, Rob; Wainger, Brian J; Han, Steve; Peng, Theodore; Thams, Sebastian; Mikkilineni, Shravani; Mellin, Cassidy; Merkle, Florian T; Davis-Dusenbery, Brandi N; Ziller, Michael; Oakley, Derek; Ichida, Justin; Di Costanzo, Stefania; Atwater, Nick; Maeder, Morgan L; Goodwin, Mathew J; Nemesh, James; Handsaker, Robert E; Paull, Daniel; Noggle, Scott; McCarroll, Steven A; Joung, J Keith; Woolf, Clifford J; Brown, Robert H; Eggan, Kevin

2014-06-01

143

Direct evidence of the cyclooxygenase pathway of prostaglandin synthesis in arthropods: genetic and biochemical characterization of two crustacean cyclooxygenases.  

PubMed

Prostaglandins, well-known lipid mediators in vertebrate animals, have also shown to play certain regulatory roles in insects and other arthropods acting on reproduction, immune system and ion transport. However, knowledge of their biosynthetic pathways in arthropods is lacking. In the present study, we report the cloning and expression of cyclooxygenase (COX) from amphipod crustaceans Gammarus spp and Caprella spp. The amphipod COX proteins contain key residues shown to be important for cyclooxygenase and peroxidase activities. Differently from all other known cyclooxygenases the N-terminal signal sequence of amphipod enzymes is not cleaved during protein expression in mammalian cells. The C-terminus of amphipod COX is shorter than that of mammalian isoforms and lacks the KDEL(STEL)-type endoplasmic reticulum retention/retrieval signal. Despite that, amphipod COX proteins are N-glycosylated and locate similarly to the vertebrate COX on the endoplasmic reticulum and nuclear envelope. Both amphipod COX mRNAs encode functional cyclooxygenases that catalyze the transformation of arachidonic acid into prostaglandins. Using bioinformatic analysis we identified a COX-like gene from the human body louse Pediculus humanus corporis genome that encodes a protein with about 30% sequence identity with human COX-1 and COX-2. Although the COX gene is known to be absent from genomes of Drosophila sp., Aedes aegypti, Bombyx mori, and other insects, our studies establish the existence of the COX gene in certain lineages within the insect world. PMID:19854273

Varvas, Külliki; Kurg, Reet; Hansen, Kristella; Järving, Reet; Järving, Ivar; Valmsen, Karin; Lõhelaid, Helike; Samel, Nigulas

2009-12-01

144

Microbial degradation of fomesafen by a newly isolated strain Pseudomonas zeshuii BY-1 and the biochemical degradation pathway.  

PubMed

Fomesafen is a diphenyl ether herbicide used to control the growth of broadleaf weeds in bean fields. Although the degradation of fomesafen in soils was thought to occur primarily by microbial activity, little was known about the kinetic and metabolic behaviors of this herbicide. This paper reported the capability of the newly isolated strain Pseudomonas zeshuii BY-1 to use fomesafen as the sole source of carbon in pure culture for its growth. Up to 88.7% of 50 mg of L(-1) fomesafen was degraded by this bacterium in mineral medium within 3 days. Strain BY-1 could also degrade other diphenyl ethers, including lactofen, acifluorfen, and fluoroglycofen. During the fomesafen degradation, five metabolites were detected and identified by liquid chromatography-mass spectrometry and tandem mass spectrometry. The primary degradation pathway of fomesafen might be the reduction of the nitro group to an amino group, followed by the acetylation of the amino derivative, dechlorination, and cleavage of the S-N bond. The addition of the BY-1 stain into soils treated with fomesafen resulted in a higher degradation rate than that observed in uninoculated soils, and the bacteria community in contaminated soil recovered after inoculation of the BY-1 stain. On the basis of these results, strain P. zeshuii BY-1 has the potential to be used in the bioremediation of fomesafen-contaminated soils. PMID:22757645

Feng, Zhao-zhong; Li, Qin-fen; Zhang, Jun; Zhang, Jing; Huang, Xing; Lu, Peng; Li, Shun-peng

2012-07-25

145

Identifying reaction modules in metabolic pathways: bioinformatic deduction and experimental validation of a new putative route in purine catabolism  

PubMed Central

Background Enzymes belonging to mechanistically diverse superfamilies often display similar catalytic mechanisms. We previously observed such an association in the case of the cyclic amidohydrolase superfamily whose members play a role in related steps of purine and pyrimidine metabolic pathways. To establish a possible link between enzyme homology and chemical similarity, we investigated further the neighbouring steps in the respective pathways. Results We identified that successive reactions of the purine and pyrimidine pathways display similar chemistry. These mechanistically-related reactions are often catalyzed by homologous enzymes. Detection of series of similar catalysis made by succeeding enzyme families suggested some modularity in the architecture of the central metabolism. Accordingly, we introduce the concept of a reaction module to define at least two successive steps catalyzed by homologous enzymes in pathways alignable by similar chemical reactions. Applying such a concept allowed us to propose new function for misannotated paralogues. In particular, we discovered a putative ureidoglycine carbamoyltransferase (UGTCase) activity. Finally, we present experimental data supporting the conclusion that this UGTCase is likely to be involved in a new route in purine catabolism. Conclusions Using the reaction module concept should be of great value. It will help us to trace how the primordial promiscuous enzymes were assembled progressively in functional modules, as the present pathways diverged from ancestral pathways to give birth to the present-day mechanistically diversified superfamilies. In addition, the concept allows the determination of the actual function of misannotated proteins.

2013-01-01

146

A tumor suppressor is identified as an inhibitor of inflammatory pathways  

Cancer.gov

Scientists at NCI have found that a protein, FBXW7, which acts as a tumor suppressor, is also important for the reduction in strength of inflammatory pathways. It has long been recognized that a complex interaction exists between cancer causing mechanisms and inflammation.

147

Pubertal Effects on Adjustment in Girls: Moving from Demonstrating Effects to Identifying Pathways  

ERIC Educational Resources Information Center

The present investigation examines mediated pathways from pubertal development to changes in depressive affect and aggression. Participants were 100 white girls who were between the ages of 10 and 14 (M=12.13, SD=0.80); girls were from well-educated, middle-to upper-middle class families, and attended private schools in a major northeastern urban…

Graber, Julia A.; Brooks-Gunn, Jeanne; Warren, Michelle P.

2006-01-01

148

Identifying pathways and processes affecting nitrate and orthophosphate inputs to streams in agricultural watersheds  

USGS Publications Warehouse

Understanding nutrient pathways to streams will improve nutrient management strategies and estimates of the time lag between when changes in land use practices occur and when water quality effects that result from these changes are observed. Nitrate and orthophosphate (OP) concentrations in several environmental compartments were examined in watersheds having a range of base flow index (BFI) values across the continental United States to determine the dominant pathways for water and nutrient inputs to streams. Estimates of the proportion of stream nitrate that was derived from groundwater increased as BFI increased. Nitrate concentration gradients between groundwater and surface water further supported the groundwater source of nitrate in these high BFI streams. However, nitrate concentrations in stream-bed pore water in all settings were typically lower than stream or upland groundwater concentrations, suggesting that nitrate discharge to streams was not uniform through the bed. Rather, preferential pathways (e.g., springs, seeps) may allow high nitrate groundwater to bypass sites of high biogeochemical transformation. Rapid pathway compartments (e.g., overland flow, tile drains) had OP concentrations that were typically higher than in streams and were important OP conveyers in most of these watersheds. In contrast to nitrate, the proportion of stream OP that is derived from ground water did not systematically increase as BFI increased. While typically not the dominant source of OP, groundwater discharge was an important pathway of OP transport to streams when BFI values were very high and when geochemical conditions favored OP mobility in groundwater. Copyright ?? 2009 by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America. All rights reserved.

Tesoriero, A. J.; Duff, J. H.; Wolock, D. M.; Spahr, N. E.; Almendinger, J. E.

2009-01-01

149

Diverging Alternative Splicing Fingerprints in the Transforming Growth Factor-? Signaling Pathway Identified in Thoracic Aortic Aneurysms  

PubMed Central

Impaired regulation of the transforming growth factor-? (TGF?) signaling pathway has been linked to thoracic aortic aneurysm (TAA). Previous work has indicated that differential splicing is a common phenomenon, potentially influencing the function of proteins. In the present study we investigated the occurrence of differential splicing in the TGF? pathway associated with TAA in patients with bicuspid aortic valve (BAV) and tricuspid aortic valve (TAV). Affymetrix human exon arrays were applied to 81 intima/media tissue samples from dilated (n = 51) and nondilated (n = 30) aortas of TAV and BAV patients. To analyze the occurrence of alternative splicing in the TGF? pathway, multivariate techniques, including principal component analysis and OPLS-DA (orthogonal partial least squares to latent structures discriminant analysis), were applied on all exons (n = 614) of the TGF? pathway. The scores plot, based on the splice index of individual exons, showed separate clusters of patients with both dilated and nondilated aorta, thereby illustrating the potential importance of alternative splicing in TAA. In total, differential splicing was detected in 187 exons. Furthermore, the pattern of alternative splicing is clearly differs between TAV and BAV patients. Differential splicing was specific for BAV and TAV patients in 40 and 86 exons, respectively, and splicings of 61 exons were shared between the two phenotypes. The occurrence of differential splicing was demonstrated in selected genes by reverse transcription–polymerase chain reaction. In summary, alternative splicing is a common feature of TAA formation. Our results suggest that dilatation in TAV and BAV patients has different alternative splicing fingerprints in the TGF? pathway.

Kurtovic, Sanela; Paloschi, Valentina; Folkersen, Lasse; Gottfries, Johan; Franco-Cereceda, Anders; Eriksson, Per

2011-01-01

150

Biochemical studies and molecular dynamics simulations of Smad3-Erbin interaction identify a non-classical Erbin PDZ binding.  

PubMed

In this work, we describe how the Erbin PDZ domain interacts with Smad3, a transductor of the Transforming Growth Factor-beta (TGFbeta) pathway, via its MH2 domain. This interaction was described as important for TGFbeta signaling as it could potentially repress the transcriptional activity of the growth factor. In order to clarify our preliminary experimental observations pointing this interaction, we built a 3D model of the Erbin PDZ/Smad3 MH2 complex and checked its stability using molecular dynamics simulations. This model pointed out charged residues in Smad3 and Erbin which could be important for the interaction. By introducing point mutations of these residues within the proposed binding domains, we experimentally confirmed that arginine 279, glutamic acid 246 in Smad3 and glutamic acid 1321 in Erbin are important for the binding. These data suggest a possible novel interface of binding in the Erbin PDZ domain and reveal an unconventional mode of interaction for a PDZ domain and its ligand. PMID:19013433

Déliot, Nadine; Chavent, Matthieu; Nourry, Claire; Lécine, Patrick; Arnaud, Camille; Hermant, Aurélie; Maigret, Bernard; Borg, Jean-Paul

2009-01-16

151

Pathway-Specific Analysis of Gene Expression Data Identifies the PI3K/Akt Pathway as a Novel Therapeutic Target in Cervical Cancer  

PubMed Central

Purpose Cervical tumor response on posttherapy 2[18F]fluoro-2-deoxy-D-glucose-positron emission tomography (FDG-PET) is predictive of survival outcome. The purpose of this study was to use gene expression profiling to identify pathways associated with tumor metabolic response. Experimental Design This was a prospective tissue collection study for gene expression profiling of 62 pretreatment biopsies from patients with advanced cervical cancer. Patients were treated with definitive radiation. Fifty-three patients received concurrent chemotherapy. All patients underwent a pretreatment and a 3-month posttherapy FDG-PET/computed tomography (CT). Tumor RNA was harvested from fresh frozen tissue and hybridized to Affymetrix U133Plus2 GeneChips. Gene set enrichment analysis (GSEA) was used to identify signaling pathways associated with tumor metabolic response. Immunohistochemistry and in vitro FDG uptake assays were used to confirm our results. Results There were 40 biopsies from patients with a complete metabolic response (PET-negative group) and 22 biopsies from patients with incomplete metabolic response (PET-positive group). The 3-year cause-specific survival estimates were 98% for the PET-negative group and 39% for the PET-positive group (P < 0.0001). GSEA identified alterations in expression of genes associated with the PI3K/Akt signaling pathway in patients with a positive follow-up PET. Immunohistochemistry using a tissue microarray of 174 pretreatment biopsies confirmed p-Akt as a biomarker for poor prognosis in cervical cancer. The phosphoinositide 3-kinase (PI3K) inhibitor LY294002 inhibited FDG uptake in vitro in cervical cancer cell lines. Conclusions Activation of the PI3K/Akt pathway is associated with incomplete metabolic response in cervical cancer. Targeted inhibition of PI3K/Akt may improve response to chemoradiation.

Schwarz, Julie K.; Payton, Jacqueline E.; Rashmi, Ramachandran; Xiang, Tao; Jia, Yunhe; Huettner, Phyllis; Rogers, Buck E.; Yang, Qin; Watson, Mark; Rader, Janet S.; Grigsby, Perry W.

2013-01-01

152

A Comparative Genome Analysis Identifies Distinct Sorting Pathways in Gram-Positive Bacteria  

Microsoft Academic Search

Surface proteins in gram-positive bacteria are frequently required for virulence, and many are attached to the cell wall by sortase enzymes. Bacteria frequently encode more than one sortase enzyme and an even larger number of potential sortase substrates that possess an LPXTG-type cell wall sorting signal. In order to elucidate the sorting pathways present in gram-positive bacteria, we performed a

David Comfort; Robert T. Clubb

2004-01-01

153

Genetic analysis of the two zebrafish patched homologues identifies novel roles for the hedgehog signaling pathway  

PubMed Central

Background Aberrant activation of the Hedgehog (Hh) signaling pathway in different organisms has shown the importance of this family of morphogens during development. Genetic screens in zebrafish have assigned specific roles for Hh in proliferation, differentiation and patterning, but mainly as a result of a loss of its activity. We attempted to fully activate the Hh pathway by removing both receptors for the Hh proteins, called Patched1 and 2, which are functioning as negative regulators in this pathway. Results Here we describe a splice-donor mutation in Ptc1, called ptc1hu1602, which in a homozygous state results in a subtle eye and somite phenotype. Since we recently positionally cloned a ptc2 mutant, a ptc1;ptc2 double mutant was generated, showing severely increased levels of ptc1, gli1 and nkx2.2a, confirming an aberrant activation of Hh signaling. As a consequence, a number of phenotypes were observed that have not been reported previously using Shh mRNA overexpression. Somites of ptc1;ptc2 double mutants do not express anteroposterior polarity markers, however initial segmentation of the somites itself is not affected. This is the first evidence that segmentation and anterior/posterior (A/P) patterning of the somites are genetically uncoupled processes. Furthermore, a novel negative function of Hh signaling is observed in the induction of the fin field, acting well before any of the previously reported function of Shh in fin formation and in a way that is different from the proposed early role of Gli3 in limb/fin bud patterning. Conclusion The generation and characterization of the ptc1;ptc2 double mutant assigned novel and unexpected functions to the Hh signaling pathway. Additionally, these mutants will provide a useful system to further investigate the consequences of constitutively activated Hh signaling during vertebrate development.

Koudijs, Marco J; den Broeder, Marjo J; Groot, Evelyn; van Eeden, Fredericus JM

2008-01-01

154

Three-Dimensional Pharmacophore Design and Biochemical Screening Identifies Substituted 1,2,4-Triazoles as Inhibitors of the Annexin A2-S100A10 Protein Interaction  

PubMed Central

Abstract Protein interactions are increasingly appreciated as targets in small-molecule drug discovery. The interaction between the adapter protein S100A10 and its binding partner annexin?A2 is a potentially important drug target. To obtain small-molecule starting points for inhibitors of this interaction, a three-dimensional pharmacophore model was constructed from the X-ray crystal structure of the complex between S100A10 and annexin A2. The pharmacophore model represents the favourable hydrophobic and hydrogen bond interactions between the two partners, as well as spatial and receptor site constraints (excluded volume spheres). Using this pharmacophore model, UNITY flex searches were carried out on a 3D library of 0.7 million commercially available compounds. This resulted in 568 hit compounds. Subsequently, GOLD docking studies were performed on these hits, and a set of 190 compounds were purchased and tested biochemically for inhibition of the protein interaction. Three compounds of similar chemical structure were identified as genuine inhibitors of the binding of annexin A2 to S100A10. The binding modes predicted by GOLD were in good agreement with their UNITY-generated conformations. We synthesised a series of analogues revealing areas critical for binding. Thus computational predictions and biochemical screening can be used successfully to derive novel chemical classes of protein–protein interaction blockers.

Reddy, Tummala R K; Li, Chan; Fischer, Peter M; Dekker, Lodewijk V

2012-01-01

155

Consensus Pathways Implicated in Prognosis of Colorectal Cancer Identified Through Systematic Enrichment Analysis of Gene Expression Profiling Studies  

PubMed Central

Background A large number of gene expression profiling (GEP) studies on prognosis of colorectal cancer (CRC) has been performed, but no reliable gene signature for prediction of CRC prognosis has been found. Bioinformatic enrichment tools are a powerful approach to identify biological processes in high-throughput data analysis. Principal Findings We have for the first time collected the results from the 23 so far published independent GEP studies on CRC prognosis. In these 23 studies, 1475 unique, mapped genes were identified, from which 124 (8.4%) were reported in at least two studies, with 54 of them showing consisting direction in expression change between the single studies. Using these data, we attempted to overcome the lack of reproducibility observed in the genes reported in individual GEP studies by carrying out a pathway-based enrichment analysis. We used up to ten tools for overrepresentation analysis of Gene Ontology (GO) categories or Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in each of the three gene lists (1475, 124 and 54 genes). This strategy, based on testing multiple tools, allowed us to identify the oxidative phosphorylation chain and the extracellular matrix receptor interaction categories, as well as a general category related to cell proliferation and apoptosis, as the only significantly and consistently overrepresented pathways in the three gene lists, which were reported by several enrichment tools. Conclusions Our pathway-based enrichment analysis of 23 independent gene expression profiling studies on prognosis of CRC identified significantly and consistently overrepresented prognostic categories for CRC. These overrepresented categories have been functionally clearly related with cancer progression, and deserve further investigation.

Lascorz, Jesus; Chen, Bowang; Hemminki, Kari; Forsti, Asta

2011-01-01

156

Quantitatively and Kinetically Identifying Binding Motifs of Amelogenin Proteins to Mineral Crystals Through Biochemical and Spectroscopic Assays  

PubMed Central

Tooth enamel is the hardest tissue in vertebrate animals. Consisting of millions of carbonated hydroxyapatite crystals, this highly mineralized tissue develops from a protein matrix in which amelogenin is the predominant component. The enamel matrix proteins are eventually and completely degraded and removed by proteinases to form mineral-enriched tooth enamel. Identification of the apatite-binding motifs in amelogenin is critical for understanding the amelogenin–crystal interactions and amelogenin–proteinases interactions during tooth enamel biomineralization. A stepwise strategy is introduced to kinetically and quantitatively identify the crystal-binding motifs in amelogenin, including a peptide screening assay, a competitive adsorption assay, and a kinetic-binding assay using amelogenin and gene-engineered amelogenin mutants. A modified enzyme-linked immunosorbent assay on crystal surfaces is also applied to compare binding amounts of amelogenin and its mutants on different planes of apatite crystals. We describe the detailed protocols for these assays and provide the considerations for these experiments in this chapter.

Zhu, Li; Hwang, Peter; Witkowska, H. Ewa; Liu, Haichuan; Li, Wu

2014-01-01

157

An EGFR and AKT Signaling Pathway was Identified with Mediation Model in Osteosarcomas Clinical Study.  

PubMed

Identification of correlation pattern and signal pathway among biomarkers in patients has become increasingly interesting for its potential values in diagnosis, treatment and prognosis. EGFR and p-AKT signaling in osteosarcoma (OS) patients were analyzed for its relationship with cancer cell proliferation maker, Ki-67, using causal procedures and statistical tests. A total of 69 patients were collected who present to Vanderbilt University Medical Center with newly diagnosed, previously untreated osteosarcomas during the clinical study period 1994 through 2003. Tissue microarrays were constructed for EGFR, p-AKT and Ki-67. The mediation model was constructed with structural equation model (SEM) for the causal analysis of the three biomarkers in osteosarcoma patients. The results suggested a mediating effect of p-AKT for the causal relationship between EGFR and Ki-67. The study also found significant associations between EGFR and Ki-67 (p = 0.002), EGFR and p-AKT (p = 0.027), and p-AKT and Ki-67 controlling EGFR (p = 0.004). After the impact of EGFR on Ki-67 was accounted for by p-AKT, the relation between EGFR and Ki-67 was no longer significant (p = 0.381). The mediating effect was confirmed with Sobel test (p < 0.001) and Goodman (I) test (p < 0.001). The study indicated that a mediation model could be an approach to exploring the correlation pattern of EGFR and AKT signal pathway for cancer cell proliferation in OS patients in clinical study. PMID:19662227

Wu, Huiyun; Muscato, Nicole E; Gonzalez, Adriana; Shyr, Yu

2007-01-01

158

An EGFR and AKT Signaling Pathway was Identified with Mediation Model in Osteosarcomas Clinical Study  

PubMed Central

Identification of correlation pattern and signal pathway among biomarkers in patients has become increasingly interesting for its potential values in diagnosis, treatment and prognosis. EGFR and p-AKT signaling in osteosarcoma (OS) patients were analyzed for its relationship with cancer cell proliferation maker, Ki-67, using causal procedures and statistical tests. A total of 69 patients were collected who present to Vanderbilt University Medical Center with newly diagnosed, previously untreated osteosarcomas during the clinical study period 1994 through 2003. Tissue microarrays were constructed for EGFR, p-AKT and Ki-67. The mediation model was constructed with structural equation model (SEM) for the causal analysis of the three biomarkers in osteosarcoma patients. The results suggested a mediating effect of p-AKT for the causal relationship between EGFR and Ki-67. The study also found significant associations between EGFR and Ki-67 (p = 0.002), EGFR and p-AKT (p = 0.027), and p-AKT and Ki-67 controlling EGFR (p = 0.004). After the impact of EGFR on Ki-67 was accounted for by p-AKT, the relation between EGFR and Ki-67 was no longer significant (p = 0.381). The mediating effect was confirmed with Sobel test (p < 0.001) and Goodman (I) test (p < 0.001). The study indicated that a mediation model could be an approach to exploring the correlation pattern of EGFR and AKT signal pathway for cancer cell proliferation in OS patients in clinical study.

Wu, Huiyun; Muscato, Nicole E.; Gonzalez, Adriana; Shyr, Yu

2007-01-01

159

Metabolic, Genomic, and Biochemical Analyses of Glandular Trichomes from the Wild Tomato Species Lycopersicon hirsutum Identify a Key Enzyme in the Biosynthesis of MethylketonesW?  

PubMed Central

Medium-length methylketones (C7-C15) are highly effective in protecting plants from numerous pests. We used a biochemical genomics approach to elucidate the pathway leading to synthesis of methylketones in the glandular trichomes of the wild tomato Lycopersicon hirsutum f glabratum (accession PI126449). A comparison of gland EST databases from accession PI126449 and a second L. hirsutum accession, LA1777, whose glands do not contain methylketones, showed that the expression of genes for fatty acid biosynthesis is elevated in PI126449 glands, suggesting de novo biosynthesis of methylketones. A cDNA abundant in the PI126449 gland EST database but rare in the LA1777 database was similar in sequence to plant esterases. This cDNA, designated Methylketone Synthase 1 (MKS1), was expressed in Escherichia coli and the purified protein used to catalyze in vitro reactions in which C12, C14, and C16 ?-ketoacyl–acyl-carrier-proteins (intermediates in fatty acid biosynthesis) were hydrolyzed and decarboxylated to give C11, C13, and C15 methylketones, respectively. Although MKS1 does not contain a classical transit peptide, in vitro import assays showed that it was targeted to the stroma of plastids, where fatty acid biosynthesis occurs. Levels of MKS1 transcript, protein, and enzymatic activity were correlated with levels of methylketones and gland density in a variety of tomato accessions and in different plant organs.

Fridman, Eyal; Wang, Jihong; Iijima, Yoko; Froehlich, John E.; Gang, David R.; Ohlrogge, John; Pichersky, Eran

2005-01-01

160

Dominant Mutations in S. cerevisiae PMS1 Identify the Mlh1-Pms1 Endonuclease Active Site and an Exonuclease 1-Independent Mismatch Repair Pathway  

PubMed Central

Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC) is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. To better understand the function of Mlh1-Pms1 in MMR, we used Saccharomyces cerevisiae to identify six pms1 mutations (pms1-G683E, pms1-C817R, pms1-C848S, pms1-H850R, pms1-H703A and pms1-E707A) that were weakly dominant in wild-type cells, which surprisingly caused a strong MMR defect when present on low copy plasmids in an exo1? mutant. Molecular modeling showed these mutations caused amino acid substitutions in the metal coordination pocket of the Pms1 endonuclease active site and biochemical studies showed that they inactivated the endonuclease activity. This model of Mlh1-Pms1 suggested that the Mlh1-FERC motif contributes to the endonuclease active site. Consistent with this, the mlh1-E767stp mutation caused both MMR and endonuclease defects similar to those caused by the dominant pms1 mutations whereas mutations affecting the predicted metal coordinating residue Mlh1-C769 had no effect. These studies establish that the Mlh1-Pms1 endonuclease is required for MMR in a previously uncharacterized Exo1-independent MMR pathway.

Smith, Catherine E.; Mendillo, Marc L.; Bowen, Nikki; Hombauer, Hans; Campbell, Christopher S.; Desai, Arshad; Putnam, Christopher D.; Kolodner, Richard D.

2013-01-01

161

Study identifies pathway in human lymphoma, points way to new blood cancer treatments  

Cancer.gov

A pathway called the "Unfolded Protein Response," or UPR, a cell's way of responding to unfolded and misfolded proteins, helps tumor cells escape programmed cell death during the development of lymphoma. Research, led by scientists in the Department of Radiation Oncology from the Perelman School of Medicine, University of Pennsylvania (home of the Abramson Cancer Center), and the Department of Urology, University of California, San Francisco (home of the UCSF Helen Diller Family Comprehensive Cancer Center), shows for the first time that the UPR is active in patients with human lymphomas and mice genetically bred to develop lymphomas. Importantly, when the UPR is inactivated, lymphoma cells readily undergo cell death. Their findings appear online in the Journal of Clinical Investigation and will appear in the December 2012 issue.

162

Candidate Gene Approach Identifies Multiple Genes and Signaling Pathways Downstream of Tbx4 in the Developing Allantois  

PubMed Central

Loss of Tbx4 results in absence of chorio-allantoic fusion and failure of formation of the primary vascular plexus of the allantois leading to embryonic death at E10.5. We reviewed the literature for genes implicated in chorio-allantoic fusion, cavitation and vascular plexus formation, processes affected in Tbx4 mutant allantoises. Using this candidate gene approach, we identified a number of genes downstream of Tbx4 in the allantois including extracellular matrix molecules Vcan, Has2, and Itg?5, transcription factors Snai1 and Twist, and signaling molecules Bmp2, Bmp7, Notch2, Jag1 and Wnt2. In addition, we show that the canonical Wnt signaling pathway contributes to the vessel-forming potential of the allantois. Ex vivo, the Tbx4 mutant phenotype can be rescued using agonists of the Wnt signaling pathway and, in wildtype allantoises, an inhibitor of the canonical Wnt signaling pathway disrupts vascular plexus formation. In vivo, Tbx4 and Wnt2 double heterozygous placentas show decreased vasculature suggesting interactions between Tbx4 and the canonical Wnt signaling pathway in the process of allantois-derived blood vessel formation.

Arora, Ripla; del Alcazar, Chelsea M.; Morrisey, Edward E.; Naiche, L. A.; Papaioannou, Virginia E.

2012-01-01

163

Major carcinogenic pathways identified by gene expression analysis of peritoneal mesotheliomas following chemical treatment in F344 rats  

SciTech Connect

This study was performed to characterize the gene expression profile and to identify the major carcinogenic pathways involved in rat peritoneal mesothelioma (RPM) formation following treatment of Fischer 344 rats with o-nitrotoluene (o-NT) or bromochloracetic acid (BCA). Oligo arrays, with over 20,000 target genes, were used to evaluate o-NT- and BCA-induced RPMs, when compared to a non-transformed mesothelial cell line (Fred-PE). Analysis using Ingenuity Pathway Analysis software revealed 169 cancer-related genes that were categorized into binding activity, growth and proliferation, cell cycle progression, apoptosis, and invasion and metastasis. The microarray data were validated by positive correlation with quantitative real-time RT-PCR on 16 selected genes including igf1, tgfb3 and nov. Important carcinogenic pathways involved in RPM formation included insulin-like growth factor 1 (IGF-1), p38 MAPkinase, Wnt/{beta}-catenin and integrin signaling pathways. This study demonstrated that mesotheliomas in rats exposed to o-NT- and BCA were similar to mesotheliomas in humans, at least at the cellular and molecular level.

Kim, Yongbaek [Environmental Toxicology Program, National Institute of Environmental Health Sciences, MD B3-08, 111 Alexander Drive, Research Triangle Park, NC 27709 (United States); Thai-Vu Ton [Environmental Toxicology Program, National Institute of Environmental Health Sciences, MD B3-08, 111 Alexander Drive, Research Triangle Park, NC 27709 (United States); De Angelo, Anthony B. [Environmental Protection Agency, Research Triangle Park, NC 27709 (United States); Morgan, Kevin [Aventis, Bridgewater, NJ 08807 (United States); Devereux, Theodora R. [Environmental Carcinogenesis Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 (United States); Anna, Colleen [Environmental Carcinogenesis Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 (United States); Collins, Jennifer B. [Microarray Group, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 (United States); Paules, Richard S. [Microarray Group, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 (United States); Crosby, Lynn M. [Wyeth Research, Chazy, NY 12921 (United States); Sills, Robert C. [Environmental Toxicology Program, National Institute of Environmental Health Sciences, MD B3-08, 111 Alexander Drive, Research Triangle Park, NC 27709 (United States)]. E-mail: sills@niehs.nih.gov

2006-07-15

164

The Prognostic Value of the Apoptosis Pathway in Colorectal Cancer: A Review of the Literature on Biomarkers Identified by Immunohistochemistry  

PubMed Central

Research towards biomarkers that predict patient outcome in colorectal cancer (CRC) is rapidly expanding. However, none of these biomarkers have been recommended by the American Association of Clinical Oncology or the European Group on Tumor Markers. Current staging criteria result in substantial under-and over-treatment of CRC patients. Evasion of apoptosis, a characteristic feature of tumorigenesis, is known to correlate with patient outcome. We reviewed the literature on immunohistochemistry-based studies between 1998 and 2011 describing biomarkers in this pathway in CRC and identified 26 markers. Most frequently described were p53, Bcl-2, survivin, and the Fas and TRAILR1 receptors and their ligands. None of the studies reviewed provided sufficient support for implementing a single marker into current clinical practice. This is likely due to the complex biology of this pathway. We suggest focusing on the combination of key markers within the apoptosis pathway that together represent an ‘apoptotic tumor profile’, which better reflects the status of this pathway in a tumor.

Zeestraten, Eliane C.M.; Benard, Anne; Reimers, Marlies S.; Schouten, Philip C.; Liefers, Gerrit J.; van de Velde, Cornelis J.H.; Kuppen, Peter J.K.

2013-01-01

165

Adolescents Can Know Best: Using concept mapping to identify factors and pathways driving adolescent sexuality in Lima, Peru  

PubMed Central

The primary objective of this study was to identify and describe individual- and environmental-level factors that Peruvian adolescents perceive to be related to adolescent sexuality. A series of concept mapping sessions were carried out from January-March 2006 with 63 15–17 year olds from a low-income community near Lima in order for adolescents to (1) brainstorm items that they thought were related to sexuality (2) sort, group and rate items to score their importance for sexuality-related outcomes, and (3) create pathways from the groups of items to engaging in sex. Brainstorming resulted in 61 items, which participants grouped into 11 clusters. The highest rated clusters were personal values, respect and confidence in relationships, future achievements and parent-child communication. The pathway of decision-making about having sex primarily contained items rated as only moderately important. This study identified important understudied factors, new perspectives on previously-recognized factors, and possible pathways to sexual behavior. These interesting, provocative findings underscore the importance of directly integrating adolescent voices into future sexual and reproductive health research, policies and programs that target this population.

Bayer, Angela M.; Cabrera, Lilia Z.; Gilman, Robert H.; Hindin, Michelle J.; Tsui, Amy O.

2011-01-01

166

Novel Angiogenic Domains: Use in Identifying Unique Transforming and Tumor Promoting Pathways in Human Breast Cancer.  

National Technical Information Service (NTIS)

Our research program focus on novel observations to seek oncogenes in human breast cancer that will cooperate with stable expression of the angiogenic domains of pleiotrophin(PTN) and midkine(MK) to establish an 'angiogenic switch' and, to identify downst...

T. F. Deuel

2001-01-01

167

Ventral encoding of functional affordances: a neural pathway for identifying errors in action.  

PubMed

Functional tool usage is a critical aspect of our daily lives. Not only must we know which tools to use for a specific action goal, we must also know how to manipulate those tools in meaningful way to achieve the goal of the action. The purpose of this study was to identify the regions of the brain critical to supporting the process of understanding errors in tool manipulation. Using fMRI, neural activations were recorded while subjects were presented with images demonstrating typical action scenes (screwdriver used on a screw), but with the tool being manipulated either correctly (screwdriver held by handle) or incorrectly (screwdriver held by bit rather than handle). Activations in fMRI for identifying correct over incorrect tool manipulation were seen along the canonical parietofrontal action network, while activations for identifying incorrect over correct tool manipulation were primarily seen at superior temporal areas and insula. We expand our hypotheses about ventral brain networks identifying contextual error to further suggest mechanisms for understanding functional tool actions, which collectively we regard as functional affordances. This proposes a fundamental role for ventral brain areas in functional action understanding. PMID:23733029

Mizelle, J C; Kelly, Rachel L; Wheaton, Lewis A

2013-08-01

168

Wnt signal transduction pathways  

PubMed Central

The Wnt signaling pathway is an ancient and evolutionarily conserved pathway that regulates crucial aspects of cell fate determination, cell migration, cell polarity, neural patterning and organogenesis during embryonic development. The Wnts are secreted glycoproteins and comprise a large family of nineteen proteins in humans hinting to a daunting complexity of signaling regulation, function and biological output. To date major signaling branches downstream of the Fz receptor have been identified including a canonical or Wnt/?-catenin dependent pathway and the non-canonical or ?-catenin-independent pathway which can be further divided into the Planar Cell Polarity and the Wnt/Ca2+ pathways, and these branches are being actively dissected at the molecular and biochemical levels. In this review, we will summarize the most recent advances in our understanding of these Wnt signaling pathways and the role of these pathways in regulating key events during embryonic patterning and morphogenesis.

Komiya, Yuko

2008-01-01

169

A Comparative Genome Analysis Identifies Distinct Sorting Pathways in Gram-Positive Bacteria  

PubMed Central

Surface proteins in gram-positive bacteria are frequently required for virulence, and many are attached to the cell wall by sortase enzymes. Bacteria frequently encode more than one sortase enzyme and an even larger number of potential sortase substrates that possess an LPXTG-type cell wall sorting signal. In order to elucidate the sorting pathways present in gram-positive bacteria, we performed a comparative analysis of 72 sequenced microbial genomes. We show that sortase enzymes can be partitioned into five distinct subfamilies based upon their primary sequences and that most of their substrates can be predicted by making a few conservative assumptions. Most bacteria encode sortases from two or more subfamilies, which are predicted to function nonredundantly in sorting proteins to the cell surface. Only ?20% of sortase-related proteins are most closely related to the well-characterized Staphylococcus aureus SrtA protein, but nonetheless, these proteins are responsible for anchoring the majority of surface proteins in gram-positive bacteria. In contrast, most sortase-like proteins are predicted to play a more specialized role, with each anchoring far fewer proteins that contain unusual sequence motifs. The functional sortase-substrate linkage predictions are available online (http://www.doe-mbi.ucla.edu/Services/Sortase/) in a searchable database.

Comfort, David; Clubb, Robert T.

2004-01-01

170

A metabolomics approach using juvenile cystic mice to identify urinary biomarkers and altered pathways in polycystic kidney disease  

PubMed Central

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease and affects 1 in 1,000 individuals. Ultrasound is most often used to diagnose ADPKD; such a modality is only useful late in the disease after macroscopic cysts are present. There is accumulating evidence suggesting that there are common cellular and molecular mechanisms responsible for cystogenesis in human and murine PKD regardless of the genes mutated, and, in the case of complex metabolomic analysis, the use of a mouse model has distinct advantages for proof of principle over a human study. Therefore, in this study we utilized a urinary metabolomics-based investigation using gas chromatography-time of flight mass spectrometry to demonstrate that the cystic mouse can be discriminated from its wild-type counterpart by urine analysis alone. At day 26 of life, before there is serological evidence of kidney dysfunction, affected mice are distinguishable by urine metabolomic analysis; this finding persists through 45 days until 64 days, at which time body weight differences confound the results. Using functional score analysis and the KEGG pathway database, we identify several biologically relevant metabolic pathways which are altered very early in this disease, the most highly represented being the purine and galactose metabolism pathways. In addition, we identify several specific candidate biomarkers, including allantoic acid and adenosine, which are augmented in the urine of young cystic mice. These markers and pathway components, once extended to human disease, may prove useful as a noninvasive means of diagnosing cystic kidney diseases and to suggest novel therapeutic approaches. Thus, urine metabolomics has great diagnostic potential for cystic renal disorders and deserves further study.

Taylor, Sandra L.; Ganti, Sheila; Bukanov, Nikolay O.; Chapman, Arlene; Fiehn, Oliver; Osier, Michael; Kim, Kyoungmi

2010-01-01

171

Meta-analyses identify 13 novel loci associated with age at menopause and highlights DNA repair and immune pathways  

PubMed Central

To identify novel loci for age at natural menopause, we performed a meta-analysis of 22 genome-wide association studies in 38,968 women of European descent, with replication in up to 14,435 women. In addition to four known loci, we identified 13 new age at natural menopause loci (P < 5 × 10?8). The new loci included genes implicated in DNA repair (EXO1, HELQ, UIMC1, FAM175A, FANCI, TLK1, POLG, PRIM1) and immune function (IL11, NLRP11, BAT2). Gene-set enrichment pathway analyses using the full GWAS dataset identified exodeoxyribonuclease, NF?B signalling and mitochondrial dysfunction as biological processes related to timing of menopause.

Stolk, Lisette; Perry, John RB; Chasman, Daniel I; He, Chunyan; Mangino, Massimo; Sulem, Patrick; Barbalic, Maja; Broer, Linda; Byrne, Enda M; Ernst, Florian; Esko, Tonu; Franceschini, Nora; Gudbjartsson, Daniel F; Hottenga, Jouke-Jan; Kraft, Peter; McArdle, Patick F; Porcu, Eleonora; Shin, So-Youn; Smith, Albert V; van Wingerden, Sophie; Zhai, Guangju; Zhuang, Wei V; Albrecht, Eva; Alizadeh, Behrooz Z; Aspelund, Thor; Bandinelli, Stefania; Lauc, Lovorka Barac; Beckmann, Jacques S; Boban, Mladen; Boerwinkle, Eric; Broekmans, Frank J; Burri, Andrea; Campbell, Harry; Chanock, Stephen J; Chen, Constance; Cornelis, Marilyn C; Corre, Tanguy; Coviello, Andrea D; d'Adamo, Pio; Davies, Gail; de Faire, Ulf; de Geus, Eco JC; Deary, Ian J; Dedoussis, George VZ; Deloukas, Panagiotis; Ebrahim, Shah; Eiriksdottir, Gudny; Emilsson, Valur; Eriksson, Johan G; Fauser, Bart CJM; Ferreli, Liana; Ferrucci, Luigi; Fischer, Krista; Folsom, Aaron R; Garcia, Melissa E; Gasparini, Paolo; Gieger, Christian; Glazer, Nicole; Grobbee, Diederick E; Hall, Per; Haller, Toomas; Hankinson, Susan E; Hass, Merli; Hayward, Caroline; Heath, Andrew C; Hofman, Albert; Ingelsson, Erik; Janssens, A Cecile JW; Johnson, Andrew D; Karasik, David; Kardia, Sharon LR; Keyzer, Jules; Kiel, Douglas P; Kolcic, Ivana; Kutalik, Zoltan; Lahti, Jari; Lai, Sandra; Laisk, Triin; Laven, Joop SE; Lawlor, Debbie A; Liu, Jianjun; Lopez, Lorna M; Louwers, Yvonne V; Magnusson, Patrik KE; Marongiu, Mara; Martin, Nicholas G; Klaric, Irena Martinovic; Masciullo, Corrado; McKnight, Barbara; Medland, Sarah E; Melzer, David; Mooser, Vincent; Navarro, Pau; Newman, Anne B; Nyholt, Dale R; Onland-Moret, N. Charlotte; Palotie, Aarno; Pare, Guillaume; Parker, Alex N; Pedersen, Nancy L; Peeters, Petra HM; Pistis, Giorgio; Plump, Andrew S; Polasek, Ozren; Pop, Victor JM; Psaty, Bruce M; Raikkonen, Katri; Rehnberg, Emil; Rotter, Jerome I; Rudan, Igor; Sala, Cinzia; Salumets, Andres; Scuteri, Angelo; Singleton, Andrew; Smith, Jennifer A; Snieder, Harold; Soranzo, Nicole; Stacey, Simon N; Starr, John M; Stathopoulou, Maria G; Stirrups, Kathleen; Stolk, Ronald P; Styrkarsdottir, Unnur; Sun, Yan V; Tenesa, Albert; Thorand, Barbara; Toniolo, Daniela; Tryggvadottir, Laufey; Tsui, Kim; Ulivi, Sheila; van Dam, Rob M; van der Schouw, Yvonne T; van Gils, Carla H; van Nierop, Peter; Vink, Jacqueline M; Visscher, Peter M; Voorhuis, Marlies; Waeber, Gerard; Wallaschofski, Henri; Wichmann, H Erich; Widen, Elisabeth; Gent, Colette JM Wijnands-van; Willemsen, Gonneke; Wilson, James F; Wolffenbuttel, Bruce HR; Wright, Alan F; Yerges-Armstrong, Laura M; Zemunik, Tatijana; Zgaga, Lina; Zillikens, M. Carola; Zygmunt, Marek; Arnold, Alice M; Boomsma, Dorret I; Buring, Julie E.; Crisponi, Laura; Demerath, Ellen W; Gudnason, Vilmundur; Harris, Tamara B; Hu, Frank B; Hunter, David J; Launer, Lenore J; Metspalu, Andres; Montgomery, Grant W; Oostra, Ben A; Ridker, Paul M; Sanna, Serena; Schlessinger, David; Spector, Tim D; Stefansson, Kari; Streeten, Elizabeth A; Thorsteinsdottir, Unnur; Uda, Manuela; Uitterlinden, Andre G; van Duijn, Cornelia M; Volzke, Henry; Murray, Anna; Murabito, Joanne M; Visser, Jenny A; Lunetta, Kathryn L

2011-01-01

172

Systems approaches to unraveling plant metabolism: identifying biosynthetic genes of secondary metabolic pathways.  

PubMed

The diversity of useful compounds produced by plant secondary metabolism has stimulated broad systems biology approaches to identify the genes involved in their biosynthesis. Systems biology studies in non-model plants pose interesting but addressable challenges, and have been greatly facilitated by the ability to grow and maintain plants, develop laboratory culture systems, and profile key metabolites in order to identify critical genes involved their biosynthesis. In this chapter we describe a suite of approaches that have been useful in Actaea racemosa (L.; syn. Cimicifuga racemosa, Nutt., black coshosh), a non-model medicinal plant with no genome sequence and little horticultural information available, that have led to the development of initial gene-metabolite relationships for the production of several bioactive metabolites in this multicomponent botanical therapeutic, and that can be readily applied to a wide variety of under-characterized medicinal plants. PMID:24218220

Spiering, Martin J; Kaur, Bhavneet; Parsons, James F; Eisenstein, Edward

2014-01-01

173

Predicting and analyzing DNA-binding domains using a systematic approach to identifying a set of informative physicochemical and biochemical properties  

PubMed Central

Background Existing methods of predicting DNA-binding proteins used valuable features of physicochemical properties to design support vector machine (SVM) based classifiers. Generally, selection of physicochemical properties and determination of their corresponding feature vectors rely mainly on known properties of binding mechanism and experience of designers. However, there exists a troublesome problem for designers that some different physicochemical properties have similar vectors of representing 20 amino acids and some closely related physicochemical properties have dissimilar vectors. Results This study proposes a systematic approach (named Auto-IDPCPs) to automatically identify a set of physicochemical and biochemical properties in the AAindex database to design SVM-based classifiers for predicting and analyzing DNA-binding domains/proteins. Auto-IDPCPs consists of 1) clustering 531 amino acid indices in AAindex into 20 clusters using a fuzzy c-means algorithm, 2) utilizing an efficient genetic algorithm based optimization method IBCGA to select an informative feature set of size m to represent sequences, and 3) analyzing the selected features to identify related physicochemical properties which may affect the binding mechanism of DNA-binding domains/proteins. The proposed Auto-IDPCPs identified m=22 features of properties belonging to five clusters for predicting DNA-binding domains with a five-fold cross-validation accuracy of 87.12%, which is promising compared with the accuracy of 86.62% of the existing method PSSM-400. For predicting DNA-binding sequences, the accuracy of 75.50% was obtained using m=28 features, where PSSM-400 has an accuracy of 74.22%. Auto-IDPCPs and PSSM-400 have accuracies of 80.73% and 82.81%, respectively, applied to an independent test data set of DNA-binding domains. Some typical physicochemical properties discovered are hydrophobicity, secondary structure, charge, solvent accessibility, polarity, flexibility, normalized Van Der Waals volume, pK (pK-C, pK-N, pK-COOH and pK-a(RCOOH)), etc. Conclusions The proposed approach Auto-IDPCPs would help designers to investigate informative physicochemical and biochemical properties by considering both prediction accuracy and analysis of binding mechanism simultaneously. The approach Auto-IDPCPs can be also applicable to predict and analyze other protein functions from sequences.

2011-01-01

174

How metal films de-wet substrates—identifying the kinetic pathways and energetic driving forces  

NASA Astrophysics Data System (ADS)

We study how single-crystal chromium films of uniform thickness on W(110) substrates are converted to arrays of three-dimensional (3D) Cr islands during annealing. We use low-energy electron microscopy (LEEM) to directly observe a kinetic pathway that produces trenches that expose the wetting layer. Adjacent film steps move simultaneously uphill and downhill relative to the staircase of atomic steps on the substrate. This step motion thickens the film regions where steps advance. Where film steps retract, the film thins, eventually exposing the stable wetting layer. Since our analysis shows that thick Cr films have a lattice constant close to bulk Cr, we propose that surface and interface stress provide a possible driving force for the observed morphological instability. Atomistic simulations and analytic elastic models show that surface and interface stress can cause a dependence of film energy on thickness that leads to an instability to simultaneous thinning and thickening. We observe that de-wetting is also initiated at bunches of substrate steps in two other systems, Ag/W(110) and Ag/Ru(0001). We additionally describe how Cr films are converted into patterns of unidirectional stripes as the trenches that expose the wetting layer lengthen along the W[001] direction. Finally, we observe how 3D Cr islands form directly during film growth at elevated temperature. The Cr mesas (wedges) form as Cr film steps advance down the staircase of substrate steps, another example of the critical role that substrate steps play in 3D island formation.

McCarty, Kevin F.; Hamilton, John C.; Sato, Yu; Saá, Angela; Stumpf, Roland; de la Figuera, Juan; Thürmer, Konrad; Jones, Frank; Schmid, Andreas K.; Talin, A. Alec; Bartelt, Norman C.

2009-04-01

175

Biochemical Plant Responses to Ozone (IV. Cross-Induction of Defensive Pathways in Parsley (Petroselinum crispum L.) Plants).  

PubMed Central

Parsley (Petroselinum crispum L.) is known to respond to ultraviolet irradiation by the synthesis of flavone glycosides, whereas fungal or elicitor stress leads to the synthesis of furanocoumarin phytoalexins. We tested how these defensive pathways are affected by a single ozone treatment (200 nL L-1; 10 h). Assays were performed at the levels of transcripts, for enzyme activities, and for secondary products. The most rapid transcript accumulation was maximal at 3 h, whereas flavone glycosides and furanocoumarins were maximally induced at 12 and 24 h, respectively, after the start of ozone treatment. Ozone acted as a cross-inducer because the two distinct pathways were simultaneously induced. These results are consistent with the previously observed ozone induction of fungal and viral defense reactions in tobacco, spruce, and pine.

Eckey-Kaltenbach, H.; Ernst, D.; Heller, W.; Sandermann, H.

1994-01-01

176

Integrative network analysis identifies key genes and pathways in the progression of hepatitis C virus induced hepatocellular carcinoma  

PubMed Central

Background Incidence of hepatitis C virus (HCV) induced hepatocellular carcinoma (HCC) has been increasing in the United States and Europe during recent years. Although HCV-associated HCC shares many pathological characteristics with other types of HCC, its molecular mechanisms of progression remain elusive. Methods To investigate the underlying pathology, we developed a systematic approach to identify deregulated biological networks in HCC by integrating gene expression profiles with high-throughput protein-protein interaction data. We examined five stages including normal (control) liver, cirrhotic liver, dysplasia, early HCC and advanced HCC. Results Among the five consecutive pathological stages, we identified four networks including precancerous networks (Normal-Cirrhosis and Cirrhosis-Dysplasia) and cancerous networks (Dysplasia-Early HCC, Early-Advanced HCC). We found little overlap between precancerous and cancerous networks, opposite to a substantial overlap within precancerous or cancerous networks. We further found that the hub proteins interacted with HCV proteins, suggesting direct interventions of these networks by the virus. The functional annotation of each network demonstrates a high degree of consistency with current knowledge in HCC. By assembling these functions into a module map, we could depict the stepwise biological functions that are deregulated in HCV-induced hepatocarcinogenesis. Additionally, these networks enable us to identify important genes and pathways by developmental stage, such as LCK signalling pathways in cirrhosis, MMP genes and TIMP genes in dysplastic liver, and CDC2-mediated cell cycle signalling in early and advanced HCC. CDC2 (alternative symbol CDK1), a cell cycle regulatory gene, is particularly interesting due to its topological position in temporally deregulated networks. Conclusions Our study uncovers a temporal spectrum of functional deregulation and prioritizes key genes and pathways in the progression of HCV induced HCC. These findings present a wealth of information for further investigation.

2011-01-01

177

Combining Cheminformatics Methods and Pathway Analysis To Identify Molecules With Whole-Cell Activity Against Mycobacterium tuberculosis  

PubMed Central

Purpose New strategies for developing inhibitors of Mycobacterium tuberculosis (Mtb) are required in order to identify the next generation of tuberculosis (TB) drugs. Our approach leverages the integration of intensive data mining and curation and computational approaches, including cheminformatics combined with bioinformatics, to suggest biological targets and their small molecule modulators. Knowledge of which biological targets are essential for Mtb viability, under a given set of in vitro or in vivo assay conditions, and absent in the human host is a crucial input. We draw on the mimicry of the associated “essential metabolites” to suggest small molecule inhibitors of the essential protein target. Empirical studies are then utilized to delineate the effect of the small molecule putative mimic on cultured Mtb growth. Methods We now describe a combined cheminformatics and bioinformatics approach that uses the TBCyc pathway and genome database, the Collaborative Drug Discovery database of molecules with activity against Mtb and their associated targets, a 3D pharmacophore approach and Bayesian models of TB activity in order to select pathways and metabolites and ultimately prioritize molecules that may be acting as metabolite mimics and exhibit activity against TB. Results In this study we combined the TB cheminformatics and pathways databases that enabled us to computationally search >80,000 vendor available molecules and ultimately test 23 compounds in vitro that resulted in two compounds (N-(2-furylmethyl)-N?-[(5-nitro-3-thienyl)carbonyl]thioureaand N-[(5-nitro-3-thienyl)carbonyl]-N?-(2-thienylmethyl)thiourea) proposed as mimics of D-fructose 1,6 bisphosphate, (MIC of 20 and 40?g/ml, respectively). Conclusion This is a simple yet novel approach that has the potential to identify inhibitors of bacterial growth as illustrated by compounds identified in this study that have activity against Mtb.

Sarker, Malabika; Talcott, Carolyn; Madrid, Peter; Chopra, Sidharth; Bunin, Barry A.; Lamichhane, Gyanu; Freundlich, Joel S.; Ekins, Sean

2013-01-01

178

Gain-of-function assays in the axolotl (Ambystoma mexicanum) to identify signaling pathways that induce and regulate limb regeneration.  

PubMed

The adult salamander has been studied as a model for regeneration of complex tissues for many decades. Only recently with the development of gain-of-function assays for regeneration, has it been possible to screen for and assay the function of the multitude of signaling factors that have been identified in studies of embryonic development and tumorigenesis. Given the conservation of function of these regulatory pathways controlling growth and pattern formation, it is now possible to use the functional assays in the salamander to test the ability of endogenous as well as small-molecule signaling factors to induce a regenerative response. PMID:24029949

Lee, Jangwoo; Aguilar, Cristian; Gardiner, David

2013-01-01

179

Quantitative Phosphoproteomic Analysis Identifies Activation of the RET and IGF-1R/IR Signaling Pathways in Neuroblastoma  

PubMed Central

Neuroblastoma is an embryonal tumor of childhood with a heterogenous clinical presentation that reflects differences in activation of complex biological signaling pathways. Protein phosphorylation is a key component of cellular signal transduction and plays a critical role in processes that control cancer cell growth and survival. We used shotgun LC/MS to compare phosphorylation between a human MYCN amplified neuroblastoma cell line (NB10), modeling a resistant tumor, and a human neural precursor cell line (NPC), modeling a normal baseline neural crest cell. 2181 unique phosphorylation sites representing 1171 proteins and 2598 phosphopeptides were found. Protein kinases accounted for 6% of the proteome, with a predominance of tyrosine kinases, supporting their prominent role in oncogenic signaling pathways. Highly abundant receptor tyrosine kinase (RTK) phosphopeptides in the NB10 cell line relative to the NPC cell line included RET, insulin-like growth factor 1 receptor/insulin receptor (IGF-1R/IR), and fibroblast growth factor receptor 1 (FGFR1). Multiple phosphorylated peptides from downstream mediators of the PI3K/AKT/mTOR and RAS pathways were also highly abundant in NB10 relative to NPC. Our analysis highlights the importance of RET, IGF-1R/IR and FGFR1 as RTKs in neuroblastoma and suggests a methodology that can be used to identify potential novel biological therapeutic targets. Furthermore, application of this previously unexploited technology in the clinic opens the possibility of providing a new wide-scale molecular signature to assess disease progression and prognosis.

DeNardo, Bradley D.; Holloway, Michael P.; Ji, Qinqin; Nguyen, Kevin T.; Cheng, Yan; Valentine, Marcus B.; Salomon, Arthur; Altura, Rachel A.

2013-01-01

180

Kinetic Studies and Biochemical Pathway Analysis of Anaerobic Poly-(R)-3-Hydroxybutyric Acid Synthesis in Escherichia coli  

PubMed Central

Poly-(R)-3-hydroxybutyric acid (PHB) was synthesized anaerobically in recombinant Escherichia coli. The host anaerobically accumulated PHB to more than 50% of its cell dry weight during cultivation in either growth or nongrowth medium. The maximum specific PHB production rate during growth-associated synthesis was approximately 2.3 ± 0.2 mmol of PHB/g of residual cell dry weight/h. The by-product secretion profiles differed significantly between the PHB-synthesizing strain and the control strain. PHB production decreased acetate accumulation for both growth and nongrowth-associated PHB synthesis. For instance under nongrowth cultivation, the PHB-synthesizing culture produced approximately 66% less acetate on a glucose yield basis as compared to a control culture. A theoretical biochemical network model was used to provide a rational basis to interpret the experimental results like the fermentation product secretion profiles and to study E. coli network capabilities under anaerobic conditions. For example, the maximum theoretical carbon yield for anaerobic PHB synthesis in E. coli is 0.8. The presented study is expected to be generally useful for analyzing, interpreting, and engineering cellular metabolisms.

Carlson, Ross; Wlaschin, Aaron; Srienc, Friedrich

2005-01-01

181

Proteomic Approaches Identify Members of Cofilin Pathway Involved in Oral Tumorigenesis  

PubMed Central

The prediction of tumor behavior for patients with oral carcinomas remains a challenge for clinicians. The presence of lymph node metastasis is the most important prognostic factor but it is limited in predicting local relapse or survival. This highlights the need for identifying biomarkers that may effectively contribute to prediction of recurrence and tumor spread. In this study, we used one- and two-dimensional gel electrophoresis, mass spectrometry and immunodetection methods to analyze protein expression in oral squamous cell carcinomas. Using a refinement for classifying oral carcinomas in regard to prognosis, we analyzed small but lymph node metastasis-positive versus large, lymph node metastasis-negative tumors in order to contribute to the molecular characterization of subgroups with risk of dissemination. Specific protein patterns favoring metastasis were observed in the “more-aggressive” group defined by the present study. This group displayed upregulation of proteins involved in migration, adhesion, angiogenesis, cell cycle regulation, anti-apoptosis and epithelial to mesenchymal transition, whereas the “less-aggressive” group was engaged in keratinocyte differentiation, epidermis development, inflammation and immune response. Besides the identification of several proteins not yet described as deregulated in oral carcinomas, the present study demonstrated for the first time the role of cofilin-1 in modulating cell invasion in oral carcinomas.

Polachini, Giovana M.; Sobral, Lays M.; Mercante, Ana M. C.; Paes-Leme, Adriana F.; Xavier, Flavia C. A.; Henrique, Tiago; Guimaraes, Douglas M.; Vidotto, Alessandra; Fukuyama, Erica E.; Gois-Filho, Jose F.; Cury, Patricia M.; Curioni, Otavio A.; Michaluart Jr, Pedro; Silva, Adriana M. A.; Wunsch-Filho, Victor; Nunes, Fabio D.; Leopoldino, Andreia M.; Tajara, Eloiza H.

2012-01-01

182

Forward genetic screen for malignant peripheral nerve sheath tumor formation identifies new genes and pathways driving tumorigenesis.  

PubMed

Malignant peripheral nerve sheath tumors (MPNSTs) are sarcomas of Schwann cell lineage origin that occur sporadically or in association with the inherited syndrome neurofibromatosis type 1. To identify genetic drivers of MPNST development, we used the Sleeping Beauty (SB) transposon-based somatic mutagenesis system in mice with somatic loss of transformation-related protein p53 (Trp53) function and/or overexpression of human epidermal growth factor receptor (EGFR). Common insertion site (CIS) analysis of 269 neurofibromas and 106 MPNSTs identified 695 and 87 sites with a statistically significant number of recurrent transposon insertions, respectively. Comparison to human data sets identified new and known driver genes for MPNST formation at these sites. Pairwise co-occurrence analysis of CIS-associated genes identified many cooperating mutations that are enriched in Wnt/?-catenin, PI3K-AKT-mTOR and growth factor receptor signaling pathways. Lastly, we identified several new proto-oncogenes, including Foxr2 (encoding forkhead box R2), which we functionally validated as a proto-oncogene involved in MPNST maintenance. PMID:23685747

Rahrmann, Eric P; Watson, Adrienne L; Keng, Vincent W; Choi, Kwangmin; Moriarity, Branden S; Beckmann, Dominic A; Wolf, Natalie K; Sarver, Aaron; Collins, Margaret H; Moertel, Christopher L; Wallace, Margaret R; Gel, Bernat; Serra, Eduard; Ratner, Nancy; Largaespada, David A

2013-07-01

183

Novel MEK1 Mutation Identified by Mutational Analysis of Epidermal Growth Factor Receptor Signaling Pathway Genes in Lung Adenocarcinoma  

PubMed Central

Genetic lesions affecting a number of kinases and other elements within the epidermal growth factor receptor (EGFR) signaling pathway have been implicated in the pathogenesis of human non–small-cell lung cancer (NSCLC). We performed mutational profiling of a large cohort of lung adenocarcinomas to uncover other potential somatic mutations in genes of this pathway that could contribute to lung tumorigenesis. We have identified in 2 of 207 primary lung tumors a somatic activating mutation in exon 2 of MEK1 (i.e., mitogen-activated protein kinase kinase 1 or MAP2K1) that substitutes asparagine for lysine at amino acid 57 (K57N) in the nonkinase portion of the kinase. Neither of these two tumors harbored known mutations in other genes encoding components of the EGFR signaling pathway (i.e., EGFR, HER2, KRAS, PIK3CA, and BRAF). Expression of mutant, but not wild-type, MEK1 leads to constitutive activity of extracellular signal–regulated kinase (ERK)-1/2 in human 293T cells and to growth factor–independent proliferation of murine Ba/F3 cells. A selective MEK inhibitor, AZD6244, inhibits mutant-induced ERK activity in 293T cells and growth of mutant-bearing Ba/F3 cells. We also screened 85 NSCLC cell lines for MEK1 exon 2 mutations; one line (NCI-H1437) harbors a Q56P substitution, a known transformation-competent allele of MEK1 originally identified in rat fibroblasts, and is sensitive to treatment with AZD6244. MEK1 mutants have not previously been reported in lung cancer and may provide a target for effective therapy in a small subset of patients with lung adenocarcinoma.

Marks, Jenifer L.; Gong, Yixuan; Chitale, Dhananjay; Golas, Ben; McLellan, Michael D.; Kasai, Yumi; Ding, Li; Mardis, Elaine R.; Wilson, Richard K.; Solit, David; Levine, Ross; Michel, Kathrin; Thomas, Roman K.; Rusch, Valerie W.; Ladanyi, Marc; Pao, William

2008-01-01

184

Proteomic profiling identifies dysregulated pathways in small cell lung cancer and novel therapeutic targets including PARP1.  

PubMed

Small cell lung cancer (SCLC) is an aggressive malignancy distinct from non-small cell lung cancer (NSCLC) in its metastatic potential and treatment response. Using an integrative proteomic and transcriptomic analysis, we investigated molecular differences contributing to the distinct clinical behavior of SCLCs and NSCLCs. SCLCs showed lower levels of several receptor tyrosine kinases and decreased activation of phosphoinositide 3-kinase (PI3K) and Ras/mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK) pathways but significantly increased levels of E2F1-regulated factors including enhancer of zeste homolog 2 (EZH2), thymidylate synthase, apoptosis mediators, and DNA repair proteins. In addition, PARP1, a DNA repair protein and E2F1 co-activator, was highly expressed at the mRNA and protein levels in SCLCs. SCLC growth was inhibited by PARP1 and EZH2 knockdown. Furthermore, SCLC was significantly more sensitive to PARP inhibitors than were NSCLCs, and PARP inhibition downregulated key components of the DNA repair machinery and enhanced the efficacy of chemotherapy. Significance: SCLC is a highly lethal cancer with a 5-year survival rate of less than 10%. To date, no molecularly targeted agents have prolonged survival in patients with SCLCs. As a step toward identifying new targets, we systematically profiled SCLCs with a focus on therapeutically relevant signaling pathways. Our data reveal fundamental differences in the patterns of pathway activation in SCLCs and NSCLCs and identify several potential therapeutic targets for SCLCs, including PARP1 and EZH2. On the basis of these results, clinical studies evaluating PARP and EZH2 inhibition, together with chemotherapy or other agents, warrant further investigation. PMID:22961666

Byers, Lauren Averett; Wang, Jing; Nilsson, Monique B; Fujimoto, Junya; Saintigny, Pierre; Yordy, John; Giri, Uma; Peyton, Michael; Fan, You Hong; Diao, Lixia; Masrorpour, Fatemeh; Shen, Li; Liu, Wenbin; Duchemann, Boris; Tumula, Praveen; Bhardwaj, Vikas; Welsh, James; Weber, Stephanie; Glisson, Bonnie S; Kalhor, Neda; Wistuba, Ignacio I; Girard, Luc; Lippman, Scott M; Mills, Gordon B; Coombes, Kevin R; Weinstein, John N; Minna, John D; Heymach, John V

2012-09-01

185

Comparative genomic analyses identify common molecular pathways modulated upon exposure to low doses of arsenic and cadmium  

PubMed Central

Background Exposure to the toxic metals arsenic and cadmium is associated with detrimental health effects including cancers of various organs. While arsenic and cadmium are well known to cause adverse health effects at high doses, the molecular impact resulting from exposure to environmentally relevant doses of these metals remains largely unexplored. Results In this study, we examined the effects of in vitro exposure to either arsenic or cadmium in human TK6 lymphoblastoid cells using genomics and systems level pathway mapping approaches. A total of 167 genes with differential expression were identified following exposure to either metal with surprisingly no overlap between the two. Real-time PCR was used to confirm target gene expression changes. The gene sets were overlaid onto protein-protein interaction maps to identify metal-induced transcriptional networks. Interestingly, both metal-induced networks were significantly enriched for proteins involved in common biological processes such as tumorigenesis, inflammation, and cell signaling. These findings were further supported by gene set enrichment analysis. Conclusions This study is the first to compare the transcriptional responses induced by low dose exposure to cadmium and arsenic in human lymphoblastoid cells. These results highlight that even at low levels of exposure both metals can dramatically influence the expression of important cellular pathways.

2011-01-01

186

Filaggrin-stratified transcriptomic analysis of pediatric skin identifies mechanistic pathways in patients with atopic dermatitis  

PubMed Central

Background Atopic dermatitis (AD; eczema) is characterized by a widespread abnormality in cutaneous barrier function and propensity to inflammation. Filaggrin is a multifunctional protein and plays a key role in skin barrier formation. Loss-of-function mutations in the gene encoding filaggrin (FLG) are a highly significant risk factor for atopic disease, but the molecular mechanisms leading to dermatitis remain unclear. Objective We sought to interrogate tissue-specific variations in the expressed genome in the skin of children with AD and to investigate underlying pathomechanisms in atopic skin. Methods We applied single-molecule direct RNA sequencing to analyze the whole transcriptome using minimal tissue samples. Uninvolved skin biopsy specimens from 26 pediatric patients with AD were compared with site-matched samples from 10 nonatopic teenage control subjects. Cases and control subjects were screened for FLG genotype to stratify the data set. Results Two thousand four hundred thirty differentially expressed genes (false discovery rate, P < .05) were identified, of which 211 were significantly upregulated and 490 downregulated by greater than 2-fold. Gene ontology terms for “extracellular space” and “defense response” were enriched, whereas “lipid metabolic processes” were downregulated. The subset of FLG wild-type cases showed dysregulation of genes involved with lipid metabolism, whereas filaggrin haploinsufficiency affected global gene expression and was characterized by a type 1 interferon–mediated stress response. Conclusion These analyses demonstrate the importance of extracellular space and lipid metabolism in atopic skin pathology independent of FLG genotype, whereas an aberrant defense response is seen in subjects with FLG mutations. Genotype stratification of the large data set has facilitated functional interpretation and might guide future therapy development.

Cole, Christian; Kroboth, Karin; Schurch, Nicholas J.; Sandilands, Aileen; Sherstnev, Alexander; O'Regan, Grainne M.; Watson, Rosemarie M.; Irwin McLean, W.H.; Barton, Geoffrey J.; Irvine, Alan D.; Brown, Sara J.

2014-01-01

187

Gene, pathway and network frameworks to identify epistatic interactions of single nucleotide polymorphisms derived from GWAS data  

PubMed Central

Background Interactions among genomic loci (also known as epistasis) have been suggested as one of the potential sources of missing heritability in single locus analysis of genome-wide association studies (GWAS). The computational burden of searching for interactions is compounded by the extremely low threshold for identifying significant p-values due to multiple hypothesis testing corrections. Utilizing prior biological knowledge to restrict the set of candidate SNP pairs to be tested can alleviate this problem, but systematic studies that investigate the relative merits of integrating different biological frameworks and GWAS data have not been conducted. Results We developed four biologically based frameworks to identify pairwise interactions among candidate SNP pairs as follows: (1) for each human protein-coding gene, a set of SNPs associated with that gene was constructed providing a gene-based interaction model, (2) for each known biological pathway, a set of SNPs associated with the genes in the pathway was constructed providing a pathway-based interaction model, (3) a set of SNPs associated with genes in a disease-related subnetwork provides a network-based interaction model, and (4) a framework is based on the function of SNPs. The last approach uses expression SNPs (eSNPs or eQTLs), which are SNPs or loci that have defined effects on the abundance of transcripts of other genes. We constructed pairs of eSNPs and SNPs located in the target genes whose expression is regulated by eSNPs. For all four frameworks the SNP sets were exhaustively tested for pairwise interactions within the sets using a traditional logistic regression model after excluding genes that were previously identified to associate with the trait. Using previously published GWAS data for type 2 diabetes (T2D) and the biologically based pair-wise interaction modeling, we identify twelve genes not seen in the previous single locus analysis. Conclusion We present four approaches to detect interactions associated with complex diseases. The results show our approaches outperform the traditional single locus approaches in detecting genes that previously did not reach significance; the results also provide novel drug targets and biomarkers relevant to the underlying mechanisms of disease.

2012-01-01

188

Oxygen and Hydrogen Peroxide in the Early Evolution of Life on Earth: In silico Comparative Analysis of Biochemical Pathways  

PubMed Central

Abstract In the Universe, oxygen is the third most widespread element, while on Earth it is the most abundant one. Moreover, oxygen is a major constituent of all biopolymers fundamental to living organisms. Besides O2, reactive oxygen species (ROS), among them hydrogen peroxide (H2O2), are also important reactants in the present aerobic metabolism. According to a widely accepted hypothesis, aerobic metabolism and many other reactions/pathways involving O2 appeared after the evolution of oxygenic photosynthesis. In this study, the hypothesis was formulated that the Last Universal Common Ancestor (LUCA) was at least able to tolerate O2 and detoxify ROS in a primordial environment. A comparative analysis was carried out of a number of the O2-and H2O2-involving metabolic reactions that occur in strict anaerobes, facultative anaerobes, and aerobes. The results indicate that the most likely LUCA possessed O2-and H2O2-involving pathways, mainly reactions to remove ROS, and had, at least in part, the components of aerobic respiration. Based on this, the presence of a low, but significant, quantity of H2O2 and O2 should be taken into account in theoretical models of the early Archean atmosphere and oceans and the evolution of life. It is suggested that the early metabolism involving O2/H2O2 was a key adaptation of LUCA to already existing weakly oxic zones in Earth's primordial environment. Key Words: Hydrogen peroxide—Oxygen—Origin of life—Photosynthesis—Superoxide dismutase—Superoxide reductase. Astrobiology 12, 775–784.

Slesak, Halina; Kruk, Jerzy

2012-01-01

189

Forward genetic screen for malignant peripheral nerve sheath tumor formation identifies new genes and genetic pathways driving tumorigenesis  

PubMed Central

Malignant peripheral nerve sheath tumors (MPNSTs) are sarcomas of Schwann cell-lineage origin that occur sporadically or in association with the inherited syndrome, Neurofibromatosis Type 1. To identify genetic drivers of MPNST development, we utilized the Sleeping Beauty (SB) transposon-based somatic mutagenesis system in mice with somatic loss of tumor protein p53 (Trp53) function and/or overexpression of epidermal growth factor receptor (EGFR). Common insertion site (CIS) analysis of 269 neurofibromas and 106 MPNSTs identified 695 and 87 sites with a statistically significant number of recurrent transposon insertions, respectively. Comparison to human data sets revealed novel and known driver genes for MPNST formation at these sites. Pairwise co-occurrence analysis of CIS-associated genes identified many cooperating mutations that are enriched for in Wnt/CTNNB1, PI3K/Akt/mTor, and growth factor receptor signaling pathways. Lastly, we identified several novel proto-oncogenes including forkhead box R2 (Foxr2), which we functionally validated as a proto-oncogene involved in MPNST maintenance.

Rahrmann, Eric P; Watson, Adrienne L; Keng, Vincent W; Choi, Kwangmin; Moriarity, Branden S; Beckmann, Dominic A; Wolf, Natalie; Sarver, Aaron; Collins, Margaret H; Moertel, Christopher L; Wallace, Margaret R; Gel, Bernat; Serra, Eduard; Ratner, Nancy; Largaespada, David A

2013-01-01

190

P439Comparative metabolomics identifies conserved metabolic pathways that control mitochondrial ROS production during ischaemia reperfusion injury.  

PubMed

Ischaemia-reperfusion (IR) injury, which occurs when blood supply to an organ is disrupted and then restored, underlies many disorders, notably heart attack and stroke. Thus while reperfusion of ischaemic tissue is essential for survival, it also initiates oxidative damage, cell death, and aberrant immune responses, driven in large part by generation of mitochondrial reactive oxygen species (ROS). Although mitochondrial ROS production in IR is established, it has generally been considered a non-specific response to reperfusion, consistent with the many apparently unconnected interventions that decrease IR damage. In contrast to this view, we have identified a critical post-translational modification on mitochondrial complex I that is sufficient to abolish mitochondrial ROS during IR. Using MitoSNO - a novel selective inhibitor of complex I - we establish that S-nitrosation of a single cysteine residue on the ND3 subunit sufficient to impair oxidoreductase activity on in the target protein.This inhibitory modification in turn diminished both ROS and tissue necrosis at reperfusion. These findings led us to pursue the intriguing hypothesis that mitochondrial ROS production during IR could be controlled by a distinct metabolic process that interacts with mitochondrial complex I. We developed a comparative in vivo metabolomic analysis and unexpectedly identified conserved metabolic pathways responsible for controlling mitochondrial ROS production during IR. In doing so, we establish a universal metabolic signature of ischaemic injury in a range of tissues that are predictive for mitochondrial ROS production through mitochondrial complex I during reperfusion of ischaemic tissue. Pharmacological inhibition of these pathways is sufficient to ameliorate in vivo IR injury in murine models of heart attack, stroke and kidney damage. Thus our work identifies a general metabolic response of tissues to ischaemia and reperfusion that provides a unifying explanation for many hitherto unconnected aspects of IR injury. PMID:25020824

Chouchani, E; Pell, V; Gaude, E; Aksentijevic, D; Shattock, M; Davidson, S; Duchen, M; Frezza, C; Krieg, T; Murphy, M

2014-07-15

191

Notch pathway activity identifies cells with cancer stem cell-like properties and correlates with worse survival in lung adenocarcinoma  

PubMed Central

Purpose The cancer stem cell theory postulates that tumors contain a subset of cells with stem cell properties of self-renewal, differentiation and tumor-initiation. The purpose of this study is to determine the role of Notch activity in identifying lung cancer stem cells. Experimental Design We investigated the role of Notch activity in lung adenocarcinoma utilizing a Notch GFP-reporter construct and a gamma-secretase inhibitor (GSI), which inhibits Notch pathway activity. Results Transduction of lung cancer cells with Notch GFP-reporter construct identified a subset of cells with high Notch activity (GFP-bright). GFP-bright cells had the ability to form more tumor spheres in serum-free media, and were able to generate both GFP-bright and GFP-dim (lower Notch activity) cell populations. GFP-bright cells were resistant to chemotherapy and were tumorigenic in serial xenotransplantation assays. Tumor xenografts of mice treated with GSI had decreased expression of downstream effectors of Notch pathway and failed to regenerate tumors upon reimplantation in NOD/SCID mice. Using multivariate analysis, we detected a statistically significant correlation between poor clinical outcome and Notch activity (reflected in increased Notch ligand expression or decreased expression of the negative modulators), in a group of 441 lung adenocarcinoma patients. This correlation was further confirmed in an independent group of 89 adenocarcinoma patients where Hes-1 overexpression correlated with poor overall survival. Conclusions Notch activity can identify lung cancer stem cell-like population and its inhibition may be an appropriate target for treating lung adenocarcinoma.

Hassan, Khaled A.; Wang, Luo; Korkaya, Hasan; Chen, Guoan; Maillard, Ivan; Beer, David G.; Kalemkerian, Gregory P.; Wicha, Max S.

2013-01-01

192

A pathway closely related to the (D)-tagatose pathway of gram-negative enterobacteria identified in the gram-positive bacterium Bacillus licheniformis.  

PubMed

We report the first identification of a gene cluster involved in d-tagatose catabolism in Bacillus licheniformis. The pathway is closely related to the d-tagatose pathway of the Gram-negative bacterium Klebsiella oxytoca, in contrast to the d-tagatose 6-phosphate pathway described in the Gram-positive bacterium Staphylococcus aureus. PMID:23524682

Van der Heiden, Edwige; Delmarcelle, Michaël; Lebrun, Sarah; Freichels, Régine; Brans, Alain; Vastenavond, Christian M; Galleni, Moreno; Joris, Bernard

2013-06-01

193

Transcriptomic Analysis in a Drosophila Model Identifies Previously Implicated and Novel Pathways in the Therapeutic Mechanism in Neuropsychiatric Disorders  

PubMed Central

We have taken advantage of a newly described Drosophila model to gain insights into the potential mechanism of antiepileptic drugs (AEDs), a group of drugs that are widely used in the treatment of several neurological and psychiatric conditions besides epilepsy. In the recently described Drosophila model that is inspired by pentylenetetrazole (PTZ) induced kindling epileptogenesis in rodents, chronic PTZ treatment for 7?days causes a decreased climbing speed and an altered CNS transcriptome, with the latter mimicking gene expression alterations reported in epileptogenesis. In the model, an increased climbing speed is further observed 7?days after withdrawal from chronic PTZ. We used this post-PTZ withdrawal regime to identify potential AED mechanism. In this regime, treatment with each of the five AEDs tested, namely, ethosuximide, gabapentin, vigabatrin, sodium valproate, and levetiracetam, resulted in rescuing of the altered climbing behavior. The AEDs also normalized PTZ withdrawal induced transcriptomic perturbation in fly heads; whereas AED untreated flies showed a large number of up- and down-regulated genes which were enriched in several processes including gene expression and cell communication, the AED treated flies showed differential expression of only a small number of genes that did not enrich gene expression and cell communication processes. Gene expression and cell communication related upregulated genes in AED untreated flies overrepresented several pathways – spliceosome, RNA degradation, and ribosome in the former category, and inositol phosphate metabolism, phosphatidylinositol signaling, endocytosis, and hedgehog signaling in the latter. Transcriptome remodeling effect of AEDs was overall confirmed by microarray clustering that clearly separated the profiles of AED treated and untreated flies. Besides being consistent with previously implicated pathways, our results provide evidence for a role of other pathways in psychiatric drug mechanism. Overall, we provide an amenable model to understand neuropsychiatric mechanism in cellular and molecular terms.

Singh, Priyanka; Mohammad, Farhan; Sharma, Abhay

2011-01-01

194

Computer-assisted morphometry and microdensitometry of transmitter- identified neurons with special reference to the mesostriatal dopamine pathway. Methodological aspects.  

PubMed

New morphometrical and microdensitometrical approaches for evaluation of transmitter-identified neurons in the central nervous system have been developed. These rely at the presynaptic level on the use of immunocytochemistry and at the postsynaptic level on the use of receptor autoradiography. The immunocytochemical analysis involves the indirect immunofluorescence method and the indirect immunoperoxidase method utilizing cryostat and vibratome sections, respectively. In the postsynaptic analysis cryostat sections and tritium-sensitive film were employed. A block diagram representation of the system of the image analyzer used and its connection with its host computer is given. Furthermore, flow charts of the original software developed by our group in presented. The morphometrical analysis has been performed on coronal sections of rat brain resulting in determinations of cell body and cell group parameters. Based on this information, objective criteria have been introduced to assess the existence of a cell group of transmitter-identified neurons in a three-dimensional frame and to give a morphometrical description of this group in the space. Moreover, new quantitative approaches to describe the dendritic and terminal fields have been introduced and for the first time in this type of morphometrical analysis, the Lorenz curves and the Gini index have been utilized in the description of the pattern of dendritic and terminal networks. By means of these morphometrical approaches it became possible to analyze topological and biochemical heterogeneities within cell groups defined in the rostrocaudal frame. In particular, it has been possible to develop a quantitative method for the evaluation of coexistence in nerve cell bodies. This method has been called the overlap method and allows an analysis cell by cell of the possible coexistence of two or more antigens. PMID:6149667

Agnati, L F; Fuxe, K; Benfenati, F; Zini, I; Zoli, M; Fabbri, L; Härfstrand, A

1984-01-01

195

Molecular and Biochemical Characterization of the 5-Nitroanthranilic Acid Degradation Pathway in Bradyrhizobium sp. Strain JS329 ? †  

PubMed Central

Biodegradation pathways of synthetic nitroaromatic compounds and anilines are well documented, but little is known about those of nitroanilines. We previously reported that the initial step in 5-nitroanthranilic acid (5NAA) degradation by Bradyrhizobium sp. strain JS329 is a hydrolytic deamination to form 5-nitrosalicylic acid (5NSA), followed by ring fission catalyzed by 5NSA dioxygenase. The mechanism of release of the nitro group was unknown. In this study, we subcloned, sequenced, and expressed the genes encoding 5NAA deaminase (5NAA aminohydrolase, NaaA), 5NSA dioxygenase (NaaB) and lactonase (NaaC), the key genes responsible for 5NAA degradation. Sequence analysis and enzyme characterization revealed that NaaA is a hydrolytic metalloenzyme with a narrow substrate range. The nitro group is spontaneously eliminated as nitrite concomitant with the formation of a lactone from the ring fission product of 5NSA dioxygenation. The elimination of the nitro group during lactone formation is a previously unreported mechanism for denitration of nitro aliphatic compounds.

Qu, Yi; Spain, Jim C.

2011-01-01

196

A Sulfite Respiration Pathway from Thermus thermophilus and the Key Role of Newly Identified Cytochrome c550 ?  

PubMed Central

Sulfite, produced for instance during amino acid metabolism, is a very reactive and toxic compound. Various detoxification mechanisms exist, but sulfite oxidoreductases (SORs) are one of the major actors in sulfite remediation in bacteria and animals. Here we describe the existence of an operon in the extreme thermophilic bacterium Thermus thermophilus HB8 encoding both a SOR and a diheme c-type cytochrome. The in vitro analysis clearly showed that the newly identified cytochrome c550 acts as an acceptor of the electrons generated by the SOR enzyme during the oxidation of sulfite. The electrons are then rapidly shuttled via cytochrome c552 to the terminal ba3- and caa3-type oxidases, thereby unveiling a novel electron transfer pathway, linking sulfite oxidation to oxygen reduction in T. thermophilus: sulfite ? SORHB8 ? cytochrome c550 ? cytochrome c552 ? ba3 oxidase/caa3 oxidase ? O2. The description of the complete pathway reveals that electrons generated during sulfite oxidation by the SOR are funneled into the respiratory chain, participating in the energy production of T. thermophilus.

Robin, Sylvain; Arese, Marzia; Forte, Elena; Sarti, Paolo; Giuffre, Alessandro; Soulimane, Tewfik

2011-01-01

197

Functional genomics identifies five distinct molecular subtypes with clinical relevance and pathways for growth control in epithelial ovarian cancer  

PubMed Central

Epithelial ovarian cancer (EOC) is hallmarked by a high degree of heterogeneity. To address this heterogeneity, a classification scheme was developed based on gene expression patterns of 1538 tumours. Five, biologically distinct subgroups — Epi-A, Epi-B, Mes, Stem-A and Stem-B — exhibited significantly distinct clinicopathological characteristics, deregulated pathways and patient prognoses, and were validated using independent datasets. To identify subtype-specific molecular targets, ovarian cancer cell lines representing these molecular subtypes were screened against a genome-wide shRNA library. Focusing on the poor-prognosis Stem-A subtype, we found that two genes involved in tubulin processing, TUBGCP4 and NAT10, were essential for cell growth, an observation supported by a pathway analysis that also predicted involvement of microtubule-related processes. Furthermore, we observed that Stem-A cell lines were indeed more sensitive to inhibitors of tubulin polymerization, vincristine and vinorelbine, than the other subtypes. This subtyping offers new insights into the development of novel diagnostic and personalized treatment for EOC patients.

Tan, Tuan Zea; Miow, Qing Hao; Huang, Ruby Yun-Ju; Wong, Meng Kang; Ye, Jieru; Lau, Jieying Amelia; Wu, Meng Chu; Bin Abdul Hadi, Luqman Hakim; Soong, Richie; Choolani, Mahesh; Davidson, Ben; Nesland, Jahn M; Wang, Ling-Zhi; Matsumura, Noriomi; Mandai, Masaki; Konishi, Ikuo; Goh, Boon-Cher; Chang, Jeffrey T; Thiery, Jean Paul; Mori, Seiichi

2013-01-01

198

Pathway-Centric Integrative Analysis Identifies RRM2 as a Prognostic Marker in Breast Cancer Associated with Poor Survival and Tamoxifen Resistance.  

PubMed

Breast cancer (BCa) molecular subtypes include luminal A, luminal B, normal-like, HER-2-enriched, and basal-like tumors, among which luminal B and basal-like cancers are highly aggressive. Biochemical pathways associated with patient survival or treatment response in these more aggressive subtypes are not well understood. With the limited availability of pathologically verified clinical specimens, cell line models are routinely used for pathway-centric studies. We measured the metabolome of luminal and basal-like BCa cell lines using mass spectrometry, linked metabolites to biochemical pathways using Gene Set Analysis, and developed a novel rank-based method to select pathways on the basis of their enrichment in patient-derived omics data sets and prognostic relevance. Key mediators of the pathway were then characterized for their role in disease progression. Pyrimidine metabolism was altered in luminal versus basal BCa, whereas the combined expression of its associated genes or expression of one key gene, ribonucleotide reductase subunit M2 (RRM2) alone, associated significantly with decreased survival across all BCa subtypes, as well as in luminal patients resistant to tamoxifen. Increased RRM2 expression in tamoxifen-resistant patients was verified using tissue microarrays, whereas the metabolic products of RRM2 were higher in tamoxifen-resistant cells and in xenograft tumors. Both genetic and pharmacological inhibition of this key enzyme in tamoxifen-resistant cells significantly decreased proliferation, reduced expression of cell cycle genes, and sensitized the cells to tamoxifen treatment. Our study suggests for evaluating RRM2-associated metabolites as noninvasive markers for tamoxifen resistance and its pharmacological inhibition as a novel approach to overcome tamoxifen resistance in BCa. PMID:25016594

Putluri, Nagireddy; Maity, Suman; Kommangani, Ramakrishna; Creighton, Chad J; Putluri, Vasanta; Chen, Fengju; Nanda, Sarmishta; Bhowmik, Salil Kumar; Terunuma, Atsushi; Dorsey, Tiffany; Nardone, Agostina; Fu, Xiaoyong; Shaw, Chad; Sarkar, Tapasree Roy; Schiff, Rachel; Lydon, John P; O'Malley, Bert W; Ambs, Stefan; Das, Gokul M; Michailidis, George; Sreekumar, Arun

2014-05-01

199

Structural and Biochemical Characterization of the Salicylyl-acyltranferase SsfX3 from a Tetracycline Biosynthetic Pathway*?  

PubMed Central

SsfX3 is a GDSL family acyltransferase that transfers salicylate to the C-4 hydroxyl of a tetracycline intermediate in the penultimate step during biosynthesis of the anticancer natural product SF2575. The C-4 salicylate takes the place of the more common C-4 dimethylamine functionality, making SsfX3 the first acyltransferase identified to act on a tetracycline substrate. The crystal structure of SsfX3 was determined at 2.5 Å, revealing two distinct domains as follows: an N-terminal ?-sandwich domain that resembles a carbohydrate-binding module, and a C-terminal catalytic domain that contains the atypical ?/?-hydrolase fold found in the GDSL hydrolase family of enzymes. The active site lies at one end of a large open binding pocket, which is spatially defined by structural elements from both the N- and C-terminal domains. Mutational analysis in the putative substrate binding pocket identified residues from both domains that are important for binding the acyl donor and acceptor. Furthermore, removal of the N-terminal carbohydrate-binding module-like domain rendered the stand-alone ?/?-hydrolase domain inactive. The additional noncatalytic module is therefore proposed to be required to define the binding pocket and provide sufficient interactions with the spatially extended tetracyclic substrate. SsfX3 was also demonstrated to accept a variety of non-native acyl groups. This relaxed substrate specificity toward the acyl donor allowed the chemoenzymatic biosynthesis of C-4-modified analogs of the immediate precursor to the bioactive SF2575; these were used to assay the structure activity relationships at the C-4 position.

Pickens, Lauren B.; Sawaya, Michael R.; Rasool, Huma; Pashkov, Inna; Yeates, Todd O.; Tang, Yi

2011-01-01

200

Microarray and Biochemical Analysis of Lovastatin-Induced Apoptosis of Squamous Cell Carcinomas  

Microsoft Academic Search

We recently identified 3 - hydroxy - 3 - methylglutaryl coenzyme A (HMG -CoA ) reductase, the rate -limiting enzyme of the mevalonate pathway, as a potential therapeutic target of the head and neck squamous cell carcinomas ( HNSCC) and cervical carcinomas (CC ). The products of this complex biochemical pathway, including de novo cholesterol, are vital for a variety

Jim Dimitroulakos; Wilson H. Marhin; Jason Tokunaga; Jonathan Irishy; Patrick Gullaney; Linda Z. Penn; Suzanne Kamel-Reid

2002-01-01

201

Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma  

PubMed Central

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy. High-resolution copy number analysis of 125 tumors of which 24 were subjected to whole-exome sequencing identified 135 homozygous deletions and 994 somatic gene mutations with predicted functional consequences. We identified new recurrent alterations in 6 genes (ARID1A, RPS6KA3, NFE2L2, IRF2, CDH8 and PROKR2) not previously described in HCC. Functional analyses demonstrated tumor suppressor properties for IRF2 whose inactivation, exclusively found in hepatitis B virus related tumors, leads to impaired TP53 function. Alternatively, inactivation of proteins involved in chromatin remodeling was frequent and predominant in alcohol related tumors. Moreover, activation of the oxidative stress metabolism and inactivation of RPS6KA3 were new pathways associated with WNT/?-catenin activation, thereby suggesting a cooperative effect in tumorigenesis. This study shows the dramatic somatic genetic diversity in HCC, it reveals interactions between oncogene and tumor suppressor gene mutations markedly related to specific risk factors.

Guichard, Cecile; Amaddeo, Giuliana; Imbeaud, Sandrine; Ladeiro, Yannick; Pelletier, Laura; Maad, Ichrafe Ben; Calderaro, Julien; Bioulac-Sage, Paulette; Letexier, Melanie; Degos, Francoise; Clement, Bruno; Balabaud, Charles; Chevet, Eric; Laurent, Alexis; Couchy, Gabrielle; Letouze, Eric; Calvo, Fabien; Zucman-Rossi, Jessica

2012-01-01

202

Mutations in PRDM5 in brittle cornea syndrome identify a pathway regulating extracellular matrix development and maintenance.  

PubMed

Extreme corneal fragility and thinning, which have a high risk of catastrophic spontaneous rupture, are the cardinal features of brittle cornea syndrome (BCS), an autosomal-recessive generalized connective tissue disorder. Enucleation is frequently the only management option for this condition, resulting in blindness and psychosocial distress. Even when the cornea remains grossly intact, visual function could also be impaired by a high degree of myopia and keratoconus. Deafness is another common feature and results in combined sensory deprivation. Using autozygosity mapping, we identified mutations in PRDM5 in families with BCS. We demonstrate that regulation of expression of extracellular matrix components, particularly fibrillar collagens, by PRDM5 is a key molecular mechanism that underlies corneal fragility in BCS and controls normal corneal development and maintenance. ZNF469, encoding a zinc finger protein of hitherto undefined function, has been identified as a quantitative trait locus for central corneal thickness, and mutations in this gene have been demonstrated in Tunisian Jewish and Palestinian kindreds with BCS. We show that ZNF469 and PRDM5, two genes that when mutated cause BCS, participate in the same regulatory pathway. PMID:21664999

Burkitt Wright, Emma M M; Spencer, Helen L; Daly, Sarah B; Manson, Forbes D C; Zeef, Leo A H; Urquhart, Jill; Zoppi, Nicoletta; Bonshek, Richard; Tosounidis, Ioannis; Mohan, Meyyammai; Madden, Colm; Dodds, Annabel; Chandler, Kate E; Banka, Siddharth; Au, Leon; Clayton-Smith, Jill; Khan, Naz; Biesecker, Leslie G; Wilson, Meredith; Rohrbach, Marianne; Colombi, Marina; Giunta, Cecilia; Black, Graeme C M

2011-06-10

203

MelanomaDB: A Web Tool for Integrative Analysis of Melanoma Genomic Information to Identify Disease-Associated Molecular Pathways  

PubMed Central

Despite on-going research, metastatic melanoma survival rates remain low and treatment options are limited. Researchers can now access a rapidly growing amount of molecular and clinical information about melanoma. This information is becoming difficult to assemble and interpret due to its dispersed nature, yet as it grows it becomes increasingly valuable for understanding melanoma. Integration of this information into a comprehensive resource to aid rational experimental design and patient stratification is needed. As an initial step in this direction, we have assembled a web-accessible melanoma database, MelanomaDB, which incorporates clinical and molecular data from publically available sources, which will be regularly updated as new information becomes available. This database allows complex links to be drawn between many different aspects of melanoma biology: genetic changes (e.g., mutations) in individual melanomas revealed by DNA sequencing, associations between gene expression and patient survival, data concerning drug targets, biomarkers, druggability, and clinical trials, as well as our own statistical analysis of relationships between molecular pathways and clinical parameters that have been produced using these data sets. The database is freely available at http://genesetdb.auckland.ac.nz/melanomadb/about.html. A subset of the information in the database can also be accessed through a freely available web application in the Illumina genomic cloud computing platform BaseSpace at http://www.biomatters.com/apps/melanoma-profiler-for-research. The MelanomaDB database illustrates dysregulation of specific signaling pathways across 310 exome-sequenced melanomas and in individual tumors and identifies the distribution of somatic variants in melanoma. We suggest that MelanomaDB can provide a context in which to interpret the tumor molecular profiles of individual melanoma patients relative to biological information and available drug therapies.

Trevarton, Alexander J.; Mann, Michael B.; Knapp, Christoph; Araki, Hiromitsu; Wren, Jonathan D.; Stones-Havas, Steven; Black, Michael A.; Print, Cristin G.

2013-01-01

204

Implementation of a High-Throughput Screen for Identifying Small Molecules to Activate the Keap1-Nrf2-ARE Pathway  

PubMed Central

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that induces a battery of cytoprotective genes involved in antioxidant defense through binding to Antioxidant Response Elements (ARE) located in the promoter regions of these genes. To identify Nrf2 activators for the treatment of oxidative/electrophilic stress-induced diseases, the present study developed a high-throughput assay to evaluate Nrf2 activation using AREc32 cells that contain a luciferase gene under the control of ARE promoters. Of the 47,000 compounds screened, 238 (top 0.5% hits) of the chemicals increased the luminescent signal more than 14.4-fold and were re-tested at eleven concentrations in a range of 0.01–30 µM. Of these 238 compounds, 231 (96%) increased the luminescence signal in a concentration-dependent manner. Chemical structure relationship analysis of these 231 compounds indicated enrichment of four chemical scaffolds (diaryl amides and diaryl ureas, oxazoles and thiazoles, pyranones and thiapyranones, and pyridinones and pyridazinones). In addition, 30 of these 231 compounds were highly effective and/or potent in activating Nrf2, with a greater than 80-fold increase in luminescence, or an EC50 lower than 1.6 µM. These top 30 compounds were also screened in Hepa1c1c7 cells for an increase in Nqo1 mRNA, the prototypical Nrf2-target gene. Of these 30 compounds, 17 increased Nqo1 mRNA in a concentration-dependent manner. In conclusion, the present study documents the development, implementation, and validation of a high-throughput screen to identify activators of the Keap1-Nrf2-ARE pathway. Results from this screening identified Nrf2 activators, and provide novel insights into chemical scaffolds that might prevent oxidative/electrophilic stress-induced toxicity and carcinogenesis.

Liu, Jie Jerry; Chaguturu, Rathnam; Klaassen, Curtis D.

2012-01-01

205

Adaptative biochemical pathways and regulatory networks in Klebsiella oxytoca BAS-10 producing a biotechnologically relevant exopolysaccharide during Fe(III)-citrate fermentation  

PubMed Central

Background A bacterial strain previously isolated from pyrite mine drainage and named BAS-10 was tentatively identified as Klebsiella oxytoca. Unlikely other enterobacteria, BAS-10 is able to grow on Fe(III)-citrate as sole carbon and energy source, yielding acetic acid and CO2 coupled with Fe(III) reduction to Fe(II) and showing unusual physiological characteristics. In fact, under this growth condition, BAS-10 produces an exopolysaccharide (EPS) having a high rhamnose content and metal-binding properties, whose biotechnological applications were proven as very relevant. Results Further phylogenetic analysis, based on 16S rDNA sequence, definitively confirmed that BAS-10 belongs to K. oxytoca species. In order to rationalize the biochemical peculiarities of this unusual enterobacteriun, combined 2D-Differential Gel Electrophoresis (2D-DIGE) analysis and mass spectrometry procedures were used to investigate its proteomic changes: i) under aerobic or anaerobic cultivation with Fe(III)-citrate as sole carbon source; ii) under anaerobic cultivations using Na(I)-citrate or Fe(III)-citrate as sole carbon source. Combining data from these differential studies peculiar levels of outer membrane proteins, key regulatory factors of carbon and nitrogen metabolism and enzymes involved in TCA cycle and sugar biosynthesis or required for citrate fermentation and stress response during anaerobic growth on Fe(III)-citrate were revealed. The protein differential regulation seems to ensure efficient cell growth coupled with EPS production by adapting metabolic and biochemical processes in order to face iron toxicity and to optimize energy production. Conclusion Differential proteomics provided insights on the molecular mechanisms necessary for anaeorobic utilization of Fe(III)-citrate in a biotechnologically promising enterobacteriun, also revealing genes that can be targeted for the rational design of high-yielding EPS producer strains.

2012-01-01

206

Extragenic Suppressors of the Arabidopsis Det1 Mutant Identify Elements of Flowering-Time and Light-Response Regulatory Pathways  

PubMed Central

Light regulation of seedling morphogenesis is mediated by photoreceptors that perceive red, far-red, blue and UV light. Photomorphogenetic mutants of Arabidopsis have identified several of the primary photoreceptors, as well as a set of negative regulators of seedling photomorphogenesis, including DET1, that appear to act downstream of the photoreceptors. To study the regulatory context in which DET1 acts to repress photomorphogenesis, we used a simple morphological screen to isolate extragenic mutations in six loci, designated ted (for reversal of the det phenotype), that partially or fully suppress the seedling morphological phenotype of det1-1. Genetic analyses indicate that mutations in the ted4 and ted5 loci identify new alleles of the previously described photomorphogenetic loci hy1 and hy5, respectively. Molecular analyses indicate that the ted mutations partially suppress the dark-grown gene expression phenotype of det1-1, and that the mechanism of suppression does not involve direct remediation of the splicing defect caused by the det1-1 mutation. The ted mutations also partially suppress the light-grown morphological phenotype of mature det1-1 plants, and ted1 and ted2 suppress a daylength insensitivity phenotype of det1. TED1, TED2 and TED3 are newly described genes, whose function appears closely associated with that of DET1. In addition, alleles of ted1 are associated with a moderate late-flowering phenotype, suggesting that TED1 plays a role in the pathways that regulate both seedling morphogenesis and the initiation of flowering.

Pepper, A. E.; Chory, J.

1997-01-01

207

Mutations in TLR/MYD88 pathway identify a subset of young chronic lymphocytic leukemia patients with favorable outcome.  

PubMed

Mutations in Toll-like receptor (TLR) and myeloid differentiation primary response 88 (MYD88) genes have been found in chronic lymphocytic leukemia (CLL) at low frequency. We analyzed the incidence, clinicobiological characteristics, and outcome of patients with TLR/MYD88 mutations in 587 CLL patients. Twenty-three patients (3.9%) had mutations, 19 in MYD88 (one with concurrent IRAK1 mutation), 2 TLR2 (one with concomitant TLR6 mutation), 1 IRAK1, and 1 TLR5. No mutations were found in IRAK2 and IRAK4. TLR/MYD88-mutated CLL overexpressed genes of the nuclear factor ?B pathway. Patients with TLR/MYD88 mutations were significantly younger (83% age ?50 years) than those with no mutations. TLR/MYD88 mutations were the most frequent in young patients. Patients with mutated TLR/MYD88 CLL had a higher frequency of mutated IGHV and low expression of CD38 and ZAP-70. Overall survival (OS) was better in TLR/MYD88-mutated than unmutated patients in the whole series (10-year OS, 100% vs 62%; P = .002), and in the subset of patients age ?50 years (100% vs 70%; P = .02). In addition, relative OS of TLR/MYD88-mutated patients was similar to that in the age- and gender-matched population. In summary, TLR/MYD88 mutations identify a population of young CLL patients with favorable outcome. PMID:24782504

Martínez-Trillos, Alejandra; Pinyol, Magda; Navarro, Alba; Aymerich, Marta; Jares, Pedro; Juan, Manel; Rozman, María; Colomer, Dolors; Delgado, Julio; Giné, Eva; González-Díaz, Marcos; Hernández-Rivas, Jesús M; Colado, Enrique; Rayón, Consolación; Payer, Angel R; Terol, Maria José; Navarro, Blanca; Quesada, Victor; Puente, Xosé S; Rozman, Ciril; López-Otín, Carlos; Campo, Elías; López-Guillermo, Armando; Villamor, Neus

2014-06-12

208

The combination of transcriptomics and informatics identifies pathways targeted by miR-204 during neurogenesis and axon guidance.  

PubMed

Vertebrate organogenesis is critically sensitive to gene dosage and even subtle variations in the expression levels of key genes may result in a variety of tissue anomalies. MicroRNAs (miRNAs) are fundamental regulators of gene expression and their role in vertebrate tissue patterning is just beginning to be elucidated. To gain further insight into this issue, we analysed the transcriptomic consequences of manipulating the expression of miR-204 in the Medaka fish model system. We used RNA-Seq and an innovative bioinformatics approach, which combines conventional differential expression analysis with the behavior expected by miR-204 targets after its overexpression and knockdown. With this approach combined with a correlative analysis of the putative targets, we identified a wider set of miR-204 target genes belonging to different pathways. Together, these approaches confirmed that miR-204 has a key role in eye development and further highlighted its putative function in neural differentiation processes, including axon guidance as supported by in vivo functional studies. Together, our results demonstrate the advantage of integrating next-generation sequencing and bioinformatics approaches to investigate miRNA biology and provide new important information on the role of miRNAs in the control of axon guidance and more broadly in nervous system development. PMID:24895435

Conte, Ivan; Merella, Stefania; Garcia-Manteiga, Jose Manuel; Migliore, Chiara; Lazarevic, Dejan; Carrella, Sabrina; Marco-Ferreres, Raquel; Avellino, Raffaella; Davidson, Nathan Paul; Emmett, Warren; Sanges, Remo; Bockett, Nicholas; Van Heel, David; Meroni, Germana; Bovolenta, Paola; Stupka, Elia; Banfi, Sandro

2014-08-01

209

Eimeria falciformis infection of the mouse caecum identifies opposing roles of IFN?-regulated host pathways for the parasite development.  

PubMed

Intracellular parasites reprogram host functions for their survival and reproduction. The extent and relevance of parasite-mediated host responses in vivo remains poorly studied, however. We utilized Eimeria falciformis, a parasite infecting the mouse intestinal epithelium, to identify and validate host determinants of parasite infection. Most prominent mouse genes induced during the onset of asexual and sexual growth of parasite comprise interferon ? (IFN?)-regulated factors, e.g., immunity-related GTPases (IRGA6/B6/D/M2/M3), guanylate-binding proteins (GBP2/3/5/6/8), chemokines (CxCL9-11), and several enzymes of the kynurenine pathway including indoleamine 2,3-dioxygenase 1 (IDO1). These results indicated a multifarious innate defense (tryptophan catabolism, IRG, GBP, and chemokine signaling), and a consequential adaptive immune response (chemokine-cytokine signaling and lymphocyte recruitment). The inflammation- and immunity-associated transcripts were increased during the course of infection, following influx of B cells, T cells, and macrophages to the parasitized caecum tissue. Consistently, parasite growth was enhanced in animals inhibited for CxCr3, a major receptor for CxCL9-11 present on immune cells. Interestingly, despite a prominent induction, mouse IRGB6 failed to bind and disrupt the parasitophorous vacuole, implying an immune evasion by E. falciformis. Furthermore, oocyst output was impaired in IFN?-R(-/-) and IDO1(-/-) mice, both of which suggest a subversion of IFN? signaling by the parasite to promote its growth. PMID:24368565

Schmid, Manuela; Heitlinger, Emanuel; Spork, Simone; Mollenkopf, Hans-Joachim; Lucius, Richard; Gupta, Nishith

2014-07-01

210

The combination of transcriptomics and informatics identifies pathways targeted by miR-204 during neurogenesis and axon guidance  

PubMed Central

Vertebrate organogenesis is critically sensitive to gene dosage and even subtle variations in the expression levels of key genes may result in a variety of tissue anomalies. MicroRNAs (miRNAs) are fundamental regulators of gene expression and their role in vertebrate tissue patterning is just beginning to be elucidated. To gain further insight into this issue, we analysed the transcriptomic consequences of manipulating the expression of miR-204 in the Medaka fish model system. We used RNA-Seq and an innovative bioinformatics approach, which combines conventional differential expression analysis with the behavior expected by miR-204 targets after its overexpression and knockdown. With this approach combined with a correlative analysis of the putative targets, we identified a wider set of miR-204 target genes belonging to different pathways. Together, these approaches confirmed that miR-204 has a key role in eye development and further highlighted its putative function in neural differentiation processes, including axon guidance as supported by in vivo functional studies. Together, our results demonstrate the advantage of integrating next-generation sequencing and bioinformatics approaches to investigate miRNA biology and provide new important information on the role of miRNAs in the control of axon guidance and more broadly in nervous system development.

Conte, Ivan; Merella, Stefania; Garcia-Manteiga, Jose Manuel; Migliore, Chiara; Lazarevic, Dejan; Carrella, Sabrina; Marco-Ferreres, Raquel; Avellino, Raffaella; Davidson, Nathan Paul; Emmett, Warren; Sanges, Remo; Bockett, Nicholas; Van Heel, David; Meroni, Germana; Bovolenta, Paola; Stupka, Elia; Banfi, Sandro

2014-01-01

211

Regulatory gene networks and signaling pathways from primary osteoarthritis and Kashin-Beck disease, an endemic osteoarthritis, identified by three analysis software.  

PubMed

Three new software systems, Ingenuity pathway analysis(IPA, TranscriptomeBrowser and MetaCore, were compared by analyzing chondrocyte microarray data of Kashin-Beck disease (KBD) and primary knee osteoarthritis(OA) to understand the pathway or network analysis software which has a superior function to identify target genes with easy operation and effective for differential diagnosis and treatment of KBD and OA. RNA was isolated from cartilage samples taken from KBD patients and OA ones. Agilent 44K human whole genome oligonucleotide microarrays were used to detect differentially expressed genes. From IPA, we identified one significant canonical pathway and two significant networks. From GeneHub analysis, we got three networks. One significant canonical pathway and one significant network were obtained from TranscriptomeBrowser analysis. POSTN and LEF1 which were got from IPA, RAC2 which was identified by both of the IPA and TranscriptomeBrowser may be most closely related to the etiopathogenesis of KBD. According to our data analysis, IPA and TranscriptomeBrowser are suitable for pathway analysis, while, TranscriptomeBrowser is suitable for network analysis. The significant genes obtained from IPA and TranscriptomeBrowser analysis may thus provide a better understanding of the molecular details in the pathogenesis of KBD and also provide useful pathways and network maps for future research in osteochondrosis. PMID:23069848

Wang, Sen; Duan, Chen; Zhang, Feng; Ma, Weijuan; Guo, Xiong

2013-01-01

212

Study of biochemical pathway and enzyme involved in metsulfuron-methyl degradation by Ancylobacter sp. XJ-412-1 isolated from soil.  

PubMed

Ancylobacter sp. XJ-412-1, capable of degrading metsulfuron-methyl, was isolated from sulfonylurea-contaminated soil. When metsulfuron-methyl was provided as the sole carbon source, more than 90.5% of metsulfuron-methyl at concentration of 50 mg l(-1) was degraded by strain XJ-412-1 after incubation at 30°C for 7 days. The initial degradation products of metsulfuron-methyl (MSM), thifensulfuron-methyl (TSM), and bensulfuron-methyl (BSM) by XJ-412-1 were identified as corresponding deesterified derivatives by liquid chromatography-mass spectrometry, which indicated a primary pathway of the deesterification of these three sulfonylurea herbicides. The carboxyesterase activity of the cell-free extracts was assayed and strongly inhibited by 4-chloromercuribenzoic acid (PCMB), diethyl pyrocarbonate (DEPC), phenylmethylsulfonyl fluoride (PMSF), and malathion. PMID:21431836

Lu, Peng; Jin, Lei; Liang, Bin; Zhang, Jing; Li, Shunpeng; Feng, Zhaozhong; Huang, Xing

2011-06-01

213

Construction and analysis of biochemical networks  

NASA Astrophysics Data System (ADS)

Bioprocesses are being implemented for a range of different applications including the production of fuels, chemicals and drugs. Hence, it is becoming increasingly important to understand and model how they function and how they can be modified or designed to give the optimal performance. Here we discuss the construction and analysis of biochemical networks which are the first logical steps towards this goal. The construction of a reaction network is possible through reconstruction: extracting information from literature and from databases. This can be supplemented by reaction prediction methods which can identify steps which are missing from the current knowledge base. Analysis of biochemical systems generally requires some experimental input but can be used to identify important reactions and targets for enhancing the performance of the organism involved. Metabolic flux, pathway and metabolic control analysis can be used to determine the limits, capabilities and potential targets for enhancement respectively.

Binns, Michael; Theodoropoulos, Constantinos

2012-09-01

214

Use of an Activated Beta-Catenin to Identify Wnt Pathway Target Genes in Caenorhabditis elegans, Including a Subset of Collagen Genes Expressed in Late Larval Development.  

PubMed

The Wnt signaling pathway plays a fundamental role during metazoan development, where it regulates diverse processes, including cell fate specification, cell migration, and stem cell renewal. Activation of the beta-catenin-dependent/canonical Wnt pathway up-regulates expression of Wnt target genes to mediate a cellular response. In the nematode Caenorhabditis elegans, a canonical Wnt signaling pathway regulates several processes during larval development; however, few target genes of this pathway have been identified. To address this deficit, we used a novel approach of conditionally activated Wnt signaling during a defined stage of larval life by overexpressing an activated beta-catenin protein, then used microarray analysis to identify genes showing altered expression compared with control animals. We identified 166 differentially expressed genes, of which 104 were up-regulated. A subset of the up-regulated genes was shown to have altered expression in mutants with decreased or increased Wnt signaling; we consider these genes to be bona fide C. elegans Wnt pathway targets. Among these was a group of six genes, including the cuticular collagen genes, bli-1 col-38, col-49, and col-71. These genes show a peak of expression in the mid L4 stage during normal development, suggesting a role in adult cuticle formation. Consistent with this finding, reduction of function for several of the genes causes phenotypes suggestive of defects in cuticle function or integrity. Therefore, this work has identified a large number of putative Wnt pathway target genes during larval life, including a small subset of Wnt-regulated collagen genes that may function in synthesis of the adult cuticle. PMID:24569038

Jackson, Belinda M; Abete-Luzi, Patricia; Krause, Michael W; Eisenmann, David M

2014-01-01

215

Use of an Activated Beta-Catenin to Identify Wnt Pathway Target Genes in Caenorhabditis elegans, Including a Subset of Collagen Genes Expressed in Late Larval Development  

PubMed Central

The Wnt signaling pathway plays a fundamental role during metazoan development, where it regulates diverse processes, including cell fate specification, cell migration, and stem cell renewal. Activation of the beta-catenin?dependent/canonical Wnt pathway up-regulates expression of Wnt target genes to mediate a cellular response. In the nematode Caenorhabditis elegans, a canonical Wnt signaling pathway regulates several processes during larval development; however, few target genes of this pathway have been identified. To address this deficit, we used a novel approach of conditionally activated Wnt signaling during a defined stage of larval life by overexpressing an activated beta-catenin protein, then used microarray analysis to identify genes showing altered expression compared with control animals. We identified 166 differentially expressed genes, of which 104 were up-regulated. A subset of the up-regulated genes was shown to have altered expression in mutants with decreased or increased Wnt signaling; we consider these genes to be bona fide C. elegans Wnt pathway targets. Among these was a group of six genes, including the cuticular collagen genes, bli-1col-38, col-49, and col-71. These genes show a peak of expression in the mid L4 stage during normal development, suggesting a role in adult cuticle formation. Consistent with this finding, reduction of function for several of the genes causes phenotypes suggestive of defects in cuticle function or integrity. Therefore, this work has identified a large number of putative Wnt pathway target genes during larval life, including a small subset of Wnt-regulated collagen genes that may function in synthesis of the adult cuticle.

Jackson, Belinda M.; Abete-Luzi, Patricia; Krause, Michael W.; Eisenmann, David M.

2014-01-01

216

PROFILING GENE EXPRESSION IN HUMAN H295R ADRENOCORTICAL CARCINOMA CELLS AND RAT TESTES TO IDENTIFY PATHWAYS OF TOXICITY FOR CONAZOLE FUNGICIDES  

EPA Science Inventory

Profiling Gene Expression in Human H295R Adrenocortical Carcinoma Cells and Rat Testes to Identify Pathways of Toxicity for Conazole Fungicides Ren1, H., Schmid1, J., Retief2, J., Turpaz2, Y.,Zhang3, X.,Jones3, P., Newsted3, J.,Giesy3, J., Wolf1, D.,Wood1, C., Bao1, W., Dix1, ...

217

New Variants Including ARG1 Polymorphisms Associated with C-Reactive Protein Levels Identified by Genome-Wide Association and Pathway Analysis  

PubMed Central

C-reactive protein (CRP) is a general marker of systemic inflammation and cardiovascular disease (CVD). The genetic contribution to differences in CRP levels remains to be explained, especially in non-European populations. Thus, the aim of this study was to identify genetic loci associated with CRP levels in Korean population. We performed genome-wide association studies (GWAS) using SNPs from 8,529 Korean individuals (7,626 for stage 1 and 903 for stage 2). We also performed pathway analysis. We identified a new genetic locus associated with CRP levels upstream of ARG1 gene (top significant SNP: rs9375813, Pmeta?=?2.85×10?8), which encodes a key enzyme of the urea cycle counteract the effects of nitric oxide, in addition to known CRP (rs7553007, Pmeta?=?1.72×10?16) and HNF1A loci (rs2259816, Pmeta?=?2.90×10?10). When we evaluated the associations between the CRP-related SNPs with cardiovascular disease phenotypes, rs9375813 (ARG1) showed a marginal association with hypertension (P?=?0.0440). To identify more variants and pathways, we performed pathway analysis and identified six candidate pathways comprised of genes related to inflammatory processes and CVDs (CRP, HNF1A, PCSK6, CD36, and ABCA1). In addition to the previously reported loci (CRP, HNF1A, and IL6) in diverse ethnic groups, we identified novel variants in the ARG1 locus associated with CRP levels in Korean population and a number of interesting genes related to inflammatory processes and CVD through pathway analysis.

Yim, Seon-Hee; Jung, Seung-Hyun; Jo, Jaeseong; Jee, Sun Ha; Chung, Yeun-Jun

2014-01-01

218

Identifying putative candidate genes and pathways involved in immune responses to porcine reproductive and respiratory syndrome virus (PRRSV) infection.  

PubMed

Differences in gene expression were compared between RNAs from lungs of high (HR) and low (LR) porcine reproductive and respiratory syndrome virus (PRRSV) burden pigs using the swine protein-annotated long oligonucleotide microarray, the Pigoligoarray. Pathway analyses were carried out to determine biological processes, pathways and networks that differ between the LR and HR responses. Differences existed between HR and LR pigs for 16 signalling pathways [P < 0.01/-log (P-value) >1.96]. Top canonical pathways included acute phase response signalling, crosstalk between dendritic cells and natural killer cells and tight junction signalling, with numerous immune response genes that were upregulated (SOCS1, SOD2, RBP4, HLA-B, HLA-G, PPP2R1A and TAP1) or downregulated (IL18, TF, C4BPA, C1QA, C1QB and TYROBP). One mechanism, regulation of complement activation, may have been blocked in HR (PRRSV-susceptible) pigs and could account for the poor clearance of PRRSV by infected macrophages. Multiple inhibiting signals may have prevented effective immune responses in susceptible HR pigs, although some protective genes were upregulated in these pigs. It is likely that in HR pigs, expression of genes associated with protection was delayed, so that the immune response was not stimulated early; thus, PRRSV infection prevented protective immune responses. PMID:22486506

Wysocki, M; Chen, H; Steibel, J P; Kuhar, D; Petry, D; Bates, J; Johnson, R; Ernst, C W; Lunney, J K

2012-06-01

219

Identifying developmental toxicity pathways for a subset of ToxCast chemicals using human embryonic stem cells and metabolomics  

SciTech Connect

Metabolomics analysis was performed on the supernatant of human embryonic stem (hES) cell cultures exposed to a blinded subset of 11 chemicals selected from the chemical library of EPA's ToxCast Trade-Mark-Sign chemical screening and prioritization research project. Metabolites from hES cultures were evaluated for known and novel signatures that may be indicative of developmental toxicity. Significant fold changes in endogenous metabolites were detected for 83 putatively annotated mass features in response to the subset of ToxCast chemicals. The annotations were mapped to specific human metabolic pathways. This revealed strong effects on pathways for nicotinate and nicotinamide metabolism, pantothenate and CoA biosynthesis, glutathione metabolism, and arginine and proline metabolism pathways. Predictivity for adverse outcomes in mammalian prenatal developmental toxicity studies used ToxRefDB and other sources of information, including Stemina Biomarker Discovery's predictive DevTox Registered-Sign model trained on 23 pharmaceutical agents of known developmental toxicity and differing potency. The model initially predicted developmental toxicity from the blinded ToxCast compounds in concordance with animal data with 73% accuracy. Retraining the model with data from the unblinded test compounds at one concentration level increased the predictive accuracy for the remaining concentrations to 83%. These preliminary results on a 11-chemical subset of the ToxCast chemical library indicate that metabolomics analysis of the hES secretome provides information valuable for predictive modeling and mechanistic understanding of mammalian developmental toxicity. -- Highlights: Black-Right-Pointing-Pointer We tested 11 environmental compounds in a hESC metabolomics platform. Black-Right-Pointing-Pointer Significant changes in secreted small molecule metabolites were observed. Black-Right-Pointing-Pointer Perturbed mass features map to pathways critical for normal development and pregnancy. Black-Right-Pointing-Pointer Arginine, proline, nicotinate, nicotinamide and glutathione pathways were affected.

Kleinstreuer, N.C., E-mail: kleinstreuer.nicole@epa.gov [NCCT, US EPA, RTP, NC 27711 (United States); Smith, A.M.; West, P.R.; Conard, K.R.; Fontaine, B.R. [Stemina Biomarker Discovery, Inc., Madison, WI 53719 (United States)] [Stemina Biomarker Discovery, Inc., Madison, WI 53719 (United States); Weir-Hauptman, A.M. [Covance, Inc., Madison, WI 53704 (United States)] [Covance, Inc., Madison, WI 53704 (United States); Palmer, J.A. [Stemina Biomarker Discovery, Inc., Madison, WI 53719 (United States)] [Stemina Biomarker Discovery, Inc., Madison, WI 53719 (United States); Knudsen, T.B.; Dix, D.J. [NCCT, US EPA, RTP, NC 27711 (United States)] [NCCT, US EPA, RTP, NC 27711 (United States); Donley, E.L.R. [Stemina Biomarker Discovery, Inc., Madison, WI 53719 (United States)] [Stemina Biomarker Discovery, Inc., Madison, WI 53719 (United States); Cezar, G.G. [Stemina Biomarker Discovery, Inc., Madison, WI 53719 (United States) [Stemina Biomarker Discovery, Inc., Madison, WI 53719 (United States); University of Wisconsin-Madison, Madison, WI 53706 (United States)

2011-11-15

220

The cGMP-PKG pathway is critical for inducing long-term sensitization of identified nociceptive sensory neurons  

Microsoft Academic Search

In various species, peripheral injury produces long-lasting sensitization of central and peripheral neurons representing the affected area. In Aplysia, memory-like traces (lasting days or weeks) of noxious peripheral stimulation include enhancement of central synaptic transmission and enhanced excitability of the central soma and peripheral branches of nociceptive sensory neurons. An important role for the cAMP-PKA-CREB pathway in consolidating long-term memory

Matthew Robert Lewin

1999-01-01

221

Whole-exome and targeted gene sequencing of gallbladder carcinoma identifies recurrent mutations in the ErbB pathway.  

PubMed

Individuals with gallbladder carcinoma (GBC), the most aggressive malignancy of the biliary tract, have a poor prognosis. Here we report the identification of somatic mutations for GBC in 57 tumor-normal pairs through a combination of exome sequencing and ultra-deep sequencing of cancer-related genes. The mutation pattern is defined by a dominant prevalence of C>T mutations at TCN sites. Genes with a significant frequency (false discovery rate (FDR) < 0.05) of non-silent mutations include TP53 (47.1%), KRAS (7.8%) and ERBB3 (11.8%). Moreover, ErbB signaling (including EGFR, ERBB2, ERBB3, ERBB4 and their downstream genes) is the most extensively mutated pathway, affecting 36.8% (21/57) of the GBC samples. Multivariate analyses further show that cases with ErbB pathway mutations have a worse outcome (P = 0.001). These findings provide insight into the somatic mutational landscape in GBC and highlight the key role of the ErbB signaling pathway in GBC pathogenesis. PMID:24997986

Li, Maolan; Zhang, Zhou; Li, Xiaoguang; Ye, Junyi; Wu, Xiangsong; Tan, Zhujun; Liu, Chang; Shen, Baiyong; Wang, Xu-An; Wu, Wenguang; Zhou, Daizhan; Zhang, Di; Wang, Ting; Liu, Bingya; Qu, Kai; Ding, Qichen; Weng, Hao; Ding, Qian; Mu, Jiasheng; Shu, Yijun; Bao, Runfa; Cao, Yang; Chen, Peizhan; Liu, Tianyu; Jiang, Lin; Hu, Yunping; Dong, Ping; Gu, Jun; Lu, Wei; Shi, Weibin; Lu, Jianhua; Gong, Wei; Tang, Zhaohui; Zhang, Yong; Wang, Xuefeng; Chin, Y Eugene; Weng, Xiaoling; Zhang, Hong; Tang, Wei; Zheng, Yonglan; He, Lin; Wang, Hui; Liu, Yun; Liu, Yingbin

2014-08-01

222

Multiplexing oscillatory biochemical signals  

NASA Astrophysics Data System (ADS)

In recent years it has been increasingly recognized that biochemical signals are not necessarily constant in time and that the temporal dynamics of a signal can be the information carrier. Moreover, it is now well established that the protein signaling network of living cells has a bow-tie structure and that components are often shared between different signaling pathways. Here we show by mathematical modeling that living cells can multiplex a constant and an oscillatory signal: they can transmit these two signals simultaneously through a common signaling pathway, and yet respond to them specifically and reliably. We find that information transmission is reduced not only by noise arising from the intrinsic stochasticity of biochemical reactions, but also by crosstalk between the different channels. Yet, under biologically relevant conditions more than 2 bits of information can be transmitted per channel, even when the two signals are transmitted simultaneously. These observations suggest that oscillatory signals are ideal for multiplexing signals.

de Ronde, Wiet; Rein ten Wolde, Pieter

2014-04-01

223

Biochemical Engineering.  

ERIC Educational Resources Information Center

Biochemical engineering as a scientific discipline is becoming accepted in England and is drawing many young men and women to its ranks. This article focuses on how engineering came to embrace the biological sciences. (Author/SA)

Dunnill, P.

1979-01-01

224

Transcript Profiling of Paoenia ostii during Artificial Chilling Induced Dormancy Release Identifies Activation of GA Pathway and Carbohydrate Metabolism  

PubMed Central

Endo-dormant flower buds must pass through a period of chilling to reinitiate growth and subsequent flowering, which is a major obstacle to the forcing culture of tree peony in winter. Customized cDNA microarray (8×15 K element) was used to investigate gene expression profiling in tree peony ‘Feng Dan Bai’ buds during 24 d chilling treatment at 0–4°C. According to the morphological changes after the whole plants were transferred to green house, endo-dormancy was released after 18 d chilling treatment, and prolonged chilling treatment increased bud break rate. Pearson correlation hierarchical clustering of sample groups was highly consistent with the dormancy transitions revealed by morphological changes. Totally 3,174 significantly differentially-expressed genes (P<0.05) were observed through endo-dormancy release process, of which the number of up-regulated (1,611) and that of down-regulated (1,563) was almost the same. Functional annotation of differentially-expressed genes revealed that cellular process, metabolic process, response to stimulus, regulation of biological process and development process were well-represented. Hierarchical clustering indicated that activation of genes involved in carbohydrate metabolism (Glycolysis, Citrate cycle and Pentose phosphate pathway), energy metabolism and cell growth. Based on the results of GO analysis, totally 51 probes presented in the microarray were associated with GA response and GA signaling pathway, and 22 of them were differently expressed. The expression profiles also revealed that the genes of GA biosynthesis, signaling and response involved in endo-dormancy release. We hypothesized that activation of GA pathway played a central role in the regulation of dormancy release in tree peony.

Liu, Chunying; Zhang, Yang; Zheng, Guosheng

2013-01-01

225

Transcript profiling of Paoenia ostii during artificial chilling induced dormancy release identifies activation of GA pathway and carbohydrate metabolism.  

PubMed

Endo-dormant flower buds must pass through a period of chilling to reinitiate growth and subsequent flowering, which is a major obstacle to the forcing culture of tree peony in winter. Customized cDNA microarray (8×15 K element) was used to investigate gene expression profiling in tree peony 'Feng Dan Bai' buds during 24 d chilling treatment at 0-4°C. According to the morphological changes after the whole plants were transferred to green house, endo-dormancy was released after 18 d chilling treatment, and prolonged chilling treatment increased bud break rate. Pearson correlation hierarchical clustering of sample groups was highly consistent with the dormancy transitions revealed by morphological changes. Totally 3,174 significantly differentially-expressed genes (P<0.05) were observed through endo-dormancy release process, of which the number of up-regulated (1,611) and that of down-regulated (1,563) was almost the same. Functional annotation of differentially-expressed genes revealed that cellular process, metabolic process, response to stimulus, regulation of biological process and development process were well-represented. Hierarchical clustering indicated that activation of genes involved in carbohydrate metabolism (Glycolysis, Citrate cycle and Pentose phosphate pathway), energy metabolism and cell growth. Based on the results of GO analysis, totally 51 probes presented in the microarray were associated with GA response and GA signaling pathway, and 22 of them were differently expressed. The expression profiles also revealed that the genes of GA biosynthesis, signaling and response involved in endo-dormancy release. We hypothesized that activation of GA pathway played a central role in the regulation of dormancy release in tree peony. PMID:23405132

Gai, Shupeng; Zhang, Yuxi; Liu, Chunying; Zhang, Yang; Zheng, Guosheng

2013-01-01

226

Drug intervention response predictions with paradigm (dirpp) identifies drug resistant cancer cell lines and pathway mechanisms of resistance.  

PubMed

The revolution in sequencing techniques in the past decade has provided an extensive picture of the molecular mechanisms behind complex diseases such as cancer. The Cancer Cell Line Encyclopedia (CCLE) and The Cancer Genome Project (CGP) have provided an unprecedented opportunity to examine copy number, gene expression, and mutational information for over 1000 cell lines of multiple tumor types alongside IC50 values for over 150 different drugs and drug related compounds. We present a novel pipeline called DIRPP, Drug Intervention Response Predictions with PARADIGM7, which predicts a cell line's response to a drug intervention from molecular data. PARADIGM (Pathway Recognition Algorithm using Data Integration on Genomic Models) is a probabilistic graphical model used to infer patient specific genetic activity by integrating copy number and gene expression data into a factor graph model of a cellular network. We evaluated the performance of DIRPP on endometrial, ovarian, and breast cancer related cell lines from the CCLE and CGP for nine drugs. The pipeline is sensitive enough to predict the response of a cell line with accuracy and precision across datasets as high as 80 and 88% respectively. We then classify drugs by the specific pathway mechanisms governing drug response. This classification allows us to compare drugs by cellular response mechanisms rather than simply by their specific gene targets. This pipeline represents a novel approach for predicting clinical drug response and generating novel candidates for drug repurposing and repositioning. PMID:24297540

Brubaker, Douglas; Difeo, Analisa; Chen, Yanwen; Pearl, Taylor; Zhai, Kaide; Bebek, Gurkan; Chance, Mark; Barnholtz-Sloan, Jill

2014-01-01

227

Method for identifying subsurface fluid migration and drainage pathways in and among oil and gas reservoirs using 3-D and 4-D seismic imaging  

DOEpatents

The invention utilizes 3-D and 4-D seismic surveys as a means of deriving information useful in petroleum exploration and reservoir management. The methods use both single seismic surveys (3-D) and multiple seismic surveys separated in time (4-D) of a region of interest to determine large scale migration pathways within sedimentary basins, and fine scale drainage structure and oil-water-gas regions within individual petroleum producing reservoirs. Such structure is identified using pattern recognition tools which define the regions of interest. The 4-D seismic data sets may be used for data completion for large scale structure where time intervals between surveys do not allow for dynamic evolution. The 4-D seismic data sets also may be used to find variations over time of small scale structure within individual reservoirs which may be used to identify petroleum drainage pathways, oil-water-gas regions and, hence, attractive drilling targets. After spatial orientation, and amplitude and frequency matching of the multiple seismic data sets, High Amplitude Event (HAE) regions consistent with the presence of petroleum are identified using seismic attribute analysis. High Amplitude Regions are grown and interconnected to establish plumbing networks on the large scale and reservoir structure on the small scale. Small scale variations over time between seismic surveys within individual reservoirs are identified and used to identify drainage patterns and bypassed petroleum to be recovered. The location of such drainage patterns and bypassed petroleum may be used to site wells.

Anderson, Roger N. (New York, NY); Boulanger, Albert (New York, NY); Bagdonas, Edward P. (Brookline, MA); Xu, Liqing (New Milford, NJ); He, Wei (New Milford, NJ)

1996-01-01

228

Method for identifying subsurface fluid migration and drainage pathways in and among oil and gas reservoirs using 3-D and 4-D seismic imaging  

DOEpatents

The invention utilizes 3-D and 4-D seismic surveys as a means of deriving information useful in petroleum exploration and reservoir management. The methods use both single seismic surveys (3-D) and multiple seismic surveys separated in time (4-D) of a region of interest to determine large scale migration pathways within sedimentary basins, and fine scale drainage structure and oil-water-gas regions within individual petroleum producing reservoirs. Such structure is identified using pattern recognition tools which define the regions of interest. The 4-D seismic data sets may be used for data completion for large scale structure where time intervals between surveys do not allow for dynamic evolution. The 4-D seismic data sets also may be used to find variations over time of small scale structure within individual reservoirs which may be used to identify petroleum drainage pathways, oil-water-gas regions and, hence, attractive drilling targets. After spatial orientation, and amplitude and frequency matching of the multiple seismic data sets, High Amplitude Event (HAE) regions consistent with the presence of petroleum are identified using seismic attribute analysis. High Amplitude Regions are grown and interconnected to establish plumbing networks on the large scale and reservoir structure on the small scale. Small scale variations over time between seismic surveys within individual reservoirs are identified and used to identify drainage patterns and bypassed petroleum to be recovered. The location of such drainage patterns and bypassed petroleum may be used to site wells. 22 figs.

Anderson, R.N.; Boulanger, A.; Bagdonas, E.P.; Xu, L.; He, W.

1996-12-17

229

Meta-analyses identify 13 loci associated with age at menopause and highlight DNA repair and immune pathways  

Microsoft Academic Search

To newly identify loci for age at natural menopause, we carried out a meta-analysis of 22 genome-wide association studies (GWAS) in 38,968 women of European descent, with replication in up to 14,435 women. In addition to four known loci, we identified 13 loci newly associated with age at natural menopause (at P < 5 × 10?8). Candidate genes located at

Lisette Stolk; John R B Perry; Daniel I Chasman; Chunyan He; Massimo Mangino; Patrick Sulem; Maja Barbalic; Linda Broer; Enda M Byrne; Florian Ernst; Tõnu Esko; Nora Franceschini; Daniel F Gudbjartsson; Jouke-Jan Hottenga; Peter Kraft; Patrick F McArdle; Eleonora Porcu; So-Youn Shin; Albert V Smith; Sophie van Wingerden; Guangju Zhai; Wei V Zhuang; Eva Albrecht; Behrooz Z Alizadeh; Thor Aspelund; Stefania Bandinelli; Lovorka Barac Lauc; Jacques S Beckmann; Mladen Boban; Eric Boerwinkle; Frank J Broekmans; Andrea Burri; Harry Campbell; Stephen J Chanock; Constance Chen; Marilyn C Cornelis; Tanguy Corre; Andrea D Coviello; Pio d'Adamo; Gail Davies; Ulf de Faire; Eco J C de Geus; Ian J Deary; George V Z Dedoussis; Panagiotis Deloukas; Shah Ebrahim; Gudny Eiriksdottir; Valur Emilsson; Johan G Eriksson; Bart C J M Fauser; Liana Ferreli; Luigi Ferrucci; Krista Fischer; Aaron R Folsom; Melissa E Garcia; Paolo Gasparini; Christian Gieger; Nicole Glazer; Diederick E Grobbee; Per Hall; Toomas Haller; Susan E Hankinson; Merli Hass; Caroline Hayward; Andrew C Heath; Albert Hofman; Erik Ingelsson; A Cecile J W Janssens; Andrew D Johnson; David Karasik; Sharon L R Kardia; Jules Keyzer; Douglas P Kiel; Ivana Kolcic; Zoltán Kutalik; Jari Lahti; Sandra Lai; Triin Laisk; Joop S E Laven; Debbie A Lawlor; Jianjun Liu; Lorna M Lopez; Yvonne V Louwers; Patrik K E Magnusson; Mara Marongiu; Nicholas G Martin; Irena Martinovic Klaric; Corrado Masciullo; Barbara McKnight; Sarah E Medland; David Melzer; Vincent Mooser; Pau Navarro; Anne B Newman; Dale R Nyholt; N Charlotte Onland-Moret; Aarno Palotie; Guillaume Paré; Alex N Parker; Nancy L Pedersen; Petra H M Peeters; Giorgio Pistis; Andrew S Plump; Ozren Polasek; Victor J M Pop; Bruce M Psaty; Katri Räikkönen; Emil Rehnberg; Jerome I Rotter; Igor Rudan; Cinzia Sala; Andres Salumets; Angelo Scuteri; Andrew Singleton; Jennifer A Smith; Harold Snieder; Nicole Soranzo; Simon N Stacey; John M Starr; Maria G Stathopoulou; Kathleen Stirrups; Ronald P Stolk; Unnur Styrkarsdottir; Yan V Sun; Albert Tenesa; Barbara Thorand; Daniela Toniolo; Laufey Tryggvadottir; Kim Tsui; Sheila Ulivi; Rob M van Dam; Yvonne T van der Schouw; Carla H van Gils; Peter van Nierop; Jacqueline M Vink; Peter M Visscher; Marlies Voorhuis; Gérard Waeber; Henri Wallaschofski; H Erich Wichmann; Elisabeth Widen; Colette J M Wijnands-van Gent; Gonneke Willemsen; James F Wilson; Bruce H R Wolffenbuttel; Alan F Wright; Laura M Yerges-Armstrong; Tatijana Zemunik; Lina Zgaga; M Carola Zillikens; Marek Zygmunt; Alice M Arnold; Dorret I Boomsma; Julie E Buring; Laura Crisponi; Ellen W Demerath; Vilmundur Gudnason; Tamara B Harris; Frank B Hu; David J Hunter; Lenore J Launer; Andres Metspalu; Grant W Montgomery; Ben A Oostra; Paul M Ridker; Serena Sanna; David Schlessinger; Tim D Spector; Kari Stefansson; Elizabeth A Streeten; Unnur Thorsteinsdottir; Manuela Uda; André G Uitterlinden; Cornelia M van Duijn; Henry Völzke; Joanne M Murabito; Jenny A Visser; Anna Murray; Kathryn L Lunetta

2012-01-01

230

Genome wide association study of SNP-, gene-, and pathway-based approaches to identify genes influencing susceptibility to Staphylococcus aureus infections  

PubMed Central

Background: We conducted a genome-wide association study (GWAS) to identify specific genetic variants that underlie susceptibility to diseases caused by Staphylococcus aureus in humans. Methods: Cases (n = 309) and controls (n = 2925) were genotyped at 508,921 single nucleotide polymorphisms (SNPs). Cases had at least one laboratory and clinician confirmed disease caused by S. aureus whereas controls did not. R-package (for SNP association), EIGENSOFT (to estimate and adjust for population stratification) and gene- (VEGAS) and pathway-based (DAVID, PANTHER, and Ingenuity Pathway Analysis) analyses were performed. Results: No SNP reached genome-wide significance. Four SNPs exceeded the p < 10?5 threshold including two (rs2455012 and rs7152530) reaching a p-value < 10?7. The nearby genes were PDE4B (rs2455012), TXNRD2 (rs3804047), VRK1 and BCL11B (rs7152530), and PNPLA5 (rs470093). The top two findings from the gene-based analysis were NMRK2 (pgene = 1.20E-05), which codes an integrin binding molecule (focal adhesion), and DAPK3 (pgene = 5.10E-05), a serine/threonine kinase (apoptosis and cytokinesis). The pathway analyses identified epithelial cell responses to mechanical and non-mechanical stress. Conclusion: We identified potential susceptibility genes for S. aureus diseases in this preliminary study but confirmation by other studies is needed. The observed associations could be relevant given the complexity of S. aureus as a pathogen and its ability to exploit multiple biological pathways to cause infections in humans.

Ye, Zhan; Vasco, Daniel A.; Carter, Tonia C.; Brilliant, Murray H.; Schrodi, Steven J.; Shukla, Sanjay K.

2014-01-01

231

Genome wide association study of SNP-, gene-, and pathway-based approaches to identify genes influencing susceptibility to Staphylococcus aureus infections.  

PubMed

Background: We conducted a genome-wide association study (GWAS) to identify specific genetic variants that underlie susceptibility to diseases caused by Staphylococcus aureus in humans. Methods: Cases (n = 309) and controls (n = 2925) were genotyped at 508,921 single nucleotide polymorphisms (SNPs). Cases had at least one laboratory and clinician confirmed disease caused by S. aureus whereas controls did not. R-package (for SNP association), EIGENSOFT (to estimate and adjust for population stratification) and gene- (VEGAS) and pathway-based (DAVID, PANTHER, and Ingenuity Pathway Analysis) analyses were performed. Results: No SNP reached genome-wide significance. Four SNPs exceeded the p < 10(-5) threshold including two (rs2455012 and rs7152530) reaching a p-value < 10(-7). The nearby genes were PDE4B (rs2455012), TXNRD2 (rs3804047), VRK1 and BCL11B (rs7152530), and PNPLA5 (rs470093). The top two findings from the gene-based analysis were NMRK2 (p gene = 1.20E-05), which codes an integrin binding molecule (focal adhesion), and DAPK3 (p gene = 5.10E-05), a serine/threonine kinase (apoptosis and cytokinesis). The pathway analyses identified epithelial cell responses to mechanical and non-mechanical stress. Conclusion: We identified potential susceptibility genes for S. aureus diseases in this preliminary study but confirmation by other studies is needed. The observed associations could be relevant given the complexity of S. aureus as a pathogen and its ability to exploit multiple biological pathways to cause infections in humans. PMID:24847357

Ye, Zhan; Vasco, Daniel A; Carter, Tonia C; Brilliant, Murray H; Schrodi, Steven J; Shukla, Sanjay K

2014-01-01

232

Proteomic Profiling Identifies Dysregulated Pathways in Small Cell Lung Cancer and Novel Therapeutic Targets Including PARP1  

PubMed Central

Small cell lung cancer (SCLC) is an aggressive malignancy distinct from non-small cell lung cancer (NSCLC) in its metastatic potential and treatment response. Using an integrative proteomic and transcriptomic analysis, we investigated molecular differences contributing to the distinct clinical behavior of SCLC and NSCLC. SCLC demonstrated lower levels of several receptor tyrosine kinases and decreased activation of PI3K and Ras/MEK pathways, but significantly increased levels of E2F1-regulated factors including EZH2, thymidylate synthase, apoptosis mediators, and DNA repair proteins. Additionally, poly (ADP-ribose) polymerase 1 (PARP1), a DNA repair protein and E2F1 co-activator, was highly expressed at the mRNA and protein levels in SCLC. SCLC growth was inhibited by PARP1 and EZH2 knockdown. Furthermore, SCLC was significantly more sensitive to PARP inhibitors than NSCLC, and PARP inhibition downregulated key components of the DNA repair machinery and enhanced the efficacy of chemotherapy.

Byers, Lauren Averett; Wang, Jing; Nilsson, Monique B.; Fujimoto, Junya; Saintigny, Pierre; Yordy, John; Giri, Uma; Peyton, Michael; Fan, You Hong; Diao, Lixia; Masrorpour, Fatemeh; Shen, Li; Liu, Wenbin; Duchemann, Boris; Tumula, Praveen; Bhardwaj, Vikas; Welsh, James; Weber, Stephanie; Glisson, Bonnie S.; Kalhor, Neda; Wistuba, Ignacio I.; Girard, Luc; Lippman, Scott M.; Mills, Gordon B.; Coombes, Kevin R.; Weinstein, John N.; Minna, John D.; Heymach, John V.

2013-01-01

233

An Arrayed RNA Interference Genome-Wide Screen Identifies Candidate Genes Involved in the MicroRNA 21 Biogenesis Pathway  

PubMed Central

Abstract MicroRNAs (miRNAs) are evolutionary conserved noncoding molecules that regulate gene expression. They influence a number of diverse biological functions, such as development and differentiation. However, their dysregulation has been shown to be associated with disease states, such as cancer. Genes and pathways regulating their biogenesis remain unknown and are highly sought after. For this purpose, we have validated a multiplexed high-content assay strategy to screen for such modulators. Here, we describe its implementation that makes use of a cell-based gain-of-function reporter assay monitoring enhanced green fluorescent protein expression under the control of miRNA 21 (miR-21); combined with measures of both cell metabolic activities through the use of Alamar Blue and cell death through imaged Hoechst-stained nuclei. The strategy was validated using a panel of known genes and enabled us to successfully progress to and complete an arrayed genome-wide short interfering RNA (siRNA) screen against the Ambion Silencer Select v4.0 library containing 64,755 siRNA duplexes covering 21,565 genes. We applied a high-stringency hit analysis method, referred to as the Bhinder–Djaballah analysis method, leading to the nomination of 1,273 genes as candidate inhibitors of the miR-21 biogenesis pathway; after several iterations eliminating those genes with only one active duplex and those enriched in seed sequence mediated off-target effects. Biological classifications revealed four major control junctions among them vesicular transport via clathrin-mediated endocytosis. Altogether, our screen has uncovered a number of novel candidate regulators that are potentially good druggable targets allowing for the discovery and development of small molecules for regulating miRNA function.

Shum, David; Bhinder, Bhavneet; Ramirez, Christina N.; Radu, Constantin; Calder, Paul A.; Beauchamp, Lesslie; Farazi, T.; Landthaler, M.; Tuschi, T.; Magdaleno, Susan

2013-01-01

234

An arrayed RNA interference genome-wide screen identifies candidate genes involved in the MicroRNA 21 biogenesis pathway.  

PubMed

MicroRNAs (miRNAs) are evolutionary conserved noncoding molecules that regulate gene expression. They influence a number of diverse biological functions, such as development and differentiation. However, their dysregulation has been shown to be associated with disease states, such as cancer. Genes and pathways regulating their biogenesis remain unknown and are highly sought after. For this purpose, we have validated a multiplexed high-content assay strategy to screen for such modulators. Here, we describe its implementation that makes use of a cell-based gain-of-function reporter assay monitoring enhanced green fluorescent protein expression under the control of miRNA 21 (miR-21); combined with measures of both cell metabolic activities through the use of Alamar Blue and cell death through imaged Hoechst-stained nuclei. The strategy was validated using a panel of known genes and enabled us to successfully progress to and complete an arrayed genome-wide short interfering RNA (siRNA) screen against the Ambion Silencer Select v4.0 library containing 64,755 siRNA duplexes covering 21,565 genes. We applied a high-stringency hit analysis method, referred to as the Bhinder-Djaballah analysis method, leading to the nomination of 1,273 genes as candidate inhibitors of the miR-21 biogenesis pathway; after several iterations eliminating those genes with only one active duplex and those enriched in seed sequence mediated off-target effects. Biological classifications revealed four major control junctions among them vesicular transport via clathrin-mediated endocytosis. Altogether, our screen has uncovered a number of novel candidate regulators that are potentially good druggable targets allowing for the discovery and development of small molecules for regulating miRNA function. PMID:23153064

Shum, David; Bhinder, Bhavneet; Ramirez, Christina N; Radu, Constantin; Calder, Paul A; Beauchamp, Lesslie; Farazi, T; Landthaler, M; Tuschi, T; Magdaleno, Susan; Djaballah, Hakim

2013-04-01

235

Loss of Niemann-Pick C1 or C2 Protein Results in Similar Biochemical Changes Suggesting That These Proteins Function in a Common Lysosomal Pathway  

PubMed Central

Niemann-Pick Type C (NPC) disease is a lysosomal storage disorder characterized by accumulation of unesterified cholesterol and other lipids in the endolysosomal system. NPC disease results from a defect in either of two distinct cholesterol-binding proteins: a transmembrane protein, NPC1, and a small soluble protein, NPC2. NPC1 and NPC2 are thought to function closely in the export of lysosomal cholesterol with both proteins binding cholesterol in vitro but they may have unrelated lysosomal roles. To investigate this possibility, we compared biochemical consequences of the loss of either protein. Analyses of lysosome-enriched subcellular fractions from brain and liver revealed similar decreases in buoyant densities of lysosomes from NPC1 or NPC2 deficient mice compared to controls. The subcellular distribution of both proteins was similar and paralleled a lysosomal marker. In liver, absence of either NPC1 or NPC2 resulted in similar alterations in the carbohydrate processing of the lysosomal protease, tripeptidyl peptidase I. These results highlight biochemical alterations in the lysosomal system of the NPC-mutant mice that appear secondary to lipid storage. In addition, the similarity in biochemical phenotypes resulting from either NPC1 or NPC2 deficiency supports models in which the function of these two proteins within lysosomes are linked closely.

Dixit, Sayali S.; Jadot, Michel; Sohar, Istvan; Sleat, David E.; Stock, Ann M.; Lobel, Peter

2011-01-01

236

Interactome mapping of the phosphatidylinositol 3-kinase-mammalian target of rapamycin pathway identifies deformed epidermal autoregulatory factor-1 as a new glycogen synthase kinase-3 interactor.  

PubMed

The phosphatidylinositol 3-kinase-mammalian target of rapamycin (PI3K-mTOR) pathway plays pivotal roles in cell survival, growth, and proliferation downstream of growth factors. Its perturbations are associated with cancer progression, type 2 diabetes, and neurological disorders. To better understand the mechanisms of action and regulation of this pathway, we initiated a large scale yeast two-hybrid screen for 33 components of the PI3K-mTOR pathway. Identification of 67 new interactions was followed by validation by co-affinity purification and exhaustive literature curation of existing information. We provide a nearly complete, functionally annotated interactome of 802 interactions for the PI3K-mTOR pathway. Our screen revealed a predominant place for glycogen synthase kinase-3 (GSK3) A and B and the AMP-activated protein kinase. In particular, we identified the deformed epidermal autoregulatory factor-1 (DEAF1) transcription factor as an interactor and in vitro substrate of GSK3A and GSK3B. Moreover, GSK3 inhibitors increased DEAF1 transcriptional activity on the 5-HT1A serotonin receptor promoter. We propose that DEAF1 may represent a therapeutic target of lithium and other GSK3 inhibitors used in bipolar disease and depression. PMID:20368287

Pilot-Storck, Fanny; Chopin, Emilie; Rual, Jean-François; Baudot, Anais; Dobrokhotov, Pavel; Robinson-Rechavi, Marc; Brun, Christine; Cusick, Michael E; Hill, David E; Schaeffer, Laurent; Vidal, Marc; Goillot, Evelyne

2010-07-01

237

Interactome Mapping of the Phosphatidylinositol 3-Kinase-Mammalian Target of Rapamycin Pathway Identifies Deformed Epidermal Autoregulatory Factor-1 as a New Glycogen Synthase Kinase-3 Interactor*  

PubMed Central

The phosphatidylinositol 3-kinase-mammalian target of rapamycin (PI3K-mTOR) pathway plays pivotal roles in cell survival, growth, and proliferation downstream of growth factors. Its perturbations are associated with cancer progression, type 2 diabetes, and neurological disorders. To better understand the mechanisms of action and regulation of this pathway, we initiated a large scale yeast two-hybrid screen for 33 components of the PI3K-mTOR pathway. Identification of 67 new interactions was followed by validation by co-affinity purification and exhaustive literature curation of existing information. We provide a nearly complete, functionally annotated interactome of 802 interactions for the PI3K-mTOR pathway. Our screen revealed a predominant place for glycogen synthase kinase-3 (GSK3) A and B and the AMP-activated protein kinase. In particular, we identified the deformed epidermal autoregulatory factor-1 (DEAF1) transcription factor as an interactor and in vitro substrate of GSK3A and GSK3B. Moreover, GSK3 inhibitors increased DEAF1 transcriptional activity on the 5-HT1A serotonin receptor promoter. We propose that DEAF1 may represent a therapeutic target of lithium and other GSK3 inhibitors used in bipolar disease and depression.

Pilot-Storck, Fanny; Chopin, Emilie; Rual, Jean-Francois; Baudot, Anais; Dobrokhotov, Pavel; Robinson-Rechavi, Marc; Brun, Christine; Cusick, Michael E.; Hill, David E.; Schaeffer, Laurent; Vidal, Marc; Goillot, Evelyne

2010-01-01

238

A sensitized genetic screen to identify novel regulators and components of the Drosophila janus kinase/signal transducer and activator of transcription pathway.  

PubMed Central

The JAK/STAT pathway exerts pleiotropic effects on a wide range of developmental processes in Drosophila. Four key components have been identified: Unpaired, a secreted ligand; Domeless, a cytokine-like receptor; Hopscotch, a JAK kinase; and Stat92E, a STAT transcription factor. The identification of additional components and regulators of this pathway remains an important issue. To this end, we have generated a transgenic line where we misexpress the upd ligand in the developing Drosophila eye. GMR-upd transgenic animals have dramatically enlarged eye-imaginal discs and compound eyes that are normally patterned. We demonstrate that the enlarged-eye phenotype is a result of an increase in cell number, and not cell volume, and arises from additional mitoses in larval eye discs. Thus, the GMR-upd line represents a system in which the proliferation and differentiation of eye precursor cells are separable. Removal of one copy of stat92E substantially reduces the enlarged-eye phenotype. We performed an F1 deficiency screen to identify dominant modifiers of the GMR-upd phenotype. We have identified 9 regions that enhance this eye phenotype and two specific enhancers: C-terminal binding protein and Daughters against dpp. We also identified 20 regions that suppress GMR-upd and 13 specific suppressors: zeste-white 13, pineapple eye, Dichaete, histone 2A variant, headcase, plexus, kohtalo, crumbs, hedgehog, decapentaplegic, thickveins, saxophone, and Mothers against dpp.

Bach, Erika A; Vincent, Stephane; Zeidler, Martin P; Perrimon, Norbert

2003-01-01

239

Pathway alignment: application to the comparative analysis of glycolytic enzymes.  

PubMed Central

Comparative analysis of metabolic pathways in different genomes yields important information on their evolution, on pharmacological targets and on biotechnological applications. In this study on glycolysis, three alternative ways of comparing biochemical pathways are combined: (1) analysis and comparison of biochemical data, (2) pathway analysis based on the concept of elementary modes, and (3) a comparative genome analysis of 17 completely sequenced genomes. The analysis reveals a surprising plasticity of the glycolytic pathway. Isoenzymes in different species are identified and compared; deviations from the textbook standard are detailed. Several potential pharmacological targets and by-passes (such as the Entner-Doudoroff pathway) to glycolysis are examined and compared in the different species. Archaean, bacterial and parasite specific adaptations are identified and described.

Dandekar, T; Schuster, S; Snel, B; Huynen, M; Bork, P

1999-01-01

240

Mutations in NMNAT1 cause Leber congenital amaurosis and identify a new disease pathway for retinal degeneration  

PubMed Central

Leber congenital amaurosis (LCA) is a blinding retinal disease that presents within the first year after birth. Using exome sequencing, we identified mutations in the nicotinamide adenine dinucleotide (NAD) synthase gene NMNAT1 encoding nicotinamide mononucleotide adenylyltransferase 1 in eight families with LCA, including the family in which LCA was originally linked to the LCA9 locus. Notably, all individuals with NMNAT1 mutations also have macular colobomas, which are severe degenerative entities of the central retina (fovea) devoid of tissue and photoreceptors. Functional assays of the proteins encoded by the mutant alleles identified in our study showed that the mutations reduce the enzymatic activity of NMNAT1 in NAD biosynthesis and affect protein folding. Of note, recent characterization of the slow Wallerian degeneration (Wlds) mouse model, in which prolonged axonal survival after injury is observed, identified NMNAT1 as a neuroprotective protein when ectopically expressed. Our findings identify a new disease mechanism underlying LCA and provide the first link between endogenous NMNAT1 dysfunction and a human nervous system disorder.

Koenekoop, Robert K.; Wang, Hui; Majewski, Jacek; Wang, Xia; Lopez, Irma; Ren, Huanan; Chen, Yiyun; Li, Yumei; Fishman, Gerald A.; Genead, Mohammed; Schwartzentruber, Jeremy; Solanki, Naimesh; Traboulsi, Elias I.; Cheng, Jingliang; Logan, Clare V.; McKibbin, Martin; Hayward, Bruce E.; Parry, David A.; Johnson, Colin A.; Nageeb, Mohammed; Poulter, James A.; Mohamed, Moin D.; Jafri, Hussain; Rashid, Yasmin; Taylor, Graham R.; Keser, Vafa; Mardon, Graeme; Xu, Huidan; Inglehearn, Chris F.; Fu, Qing; Toomes, Carmel; Chen, Rui

2013-01-01

241

An integrative multi-dimensional genetic and epigenetic strategy to identify aberrant genes and pathways in cancer  

PubMed Central

Background Genomics has substantially changed our approach to cancer research. Gene expression profiling, for example, has been utilized to delineate subtypes of cancer, and facilitated derivation of predictive and prognostic signatures. The emergence of technologies for the high resolution and genome-wide description of genetic and epigenetic features has enabled the identification of a multitude of causal DNA events in tumors. This has afforded the potential for large scale integration of genome and transcriptome data generated from a variety of technology platforms to acquire a better understanding of cancer. Results Here we show how multi-dimensional genomics data analysis would enable the deciphering of mechanisms that disrupt regulatory/signaling cascades and downstream effects. Since not all gene expression changes observed in a tumor are causal to cancer development, we demonstrate an approach based on multiple concerted disruption (MCD) analysis of genes that facilitates the rational deduction of aberrant genes and pathways, which otherwise would be overlooked in single genomic dimension investigations. Conclusions Notably, this is the first comprehensive study of breast cancer cells by parallel integrative genome wide analyses of DNA copy number, LOH, and DNA methylation status to interpret changes in gene expression pattern. Our findings demonstrate the power of a multi-dimensional approach to elucidate events which would escape conventional single dimensional analysis and as such, reduce the cohort sample size for cancer gene discovery.

2010-01-01

242

Specific inhibitors for identifying pathways for methane production from carbon monoxide by a nonadapted anaerobic mixed culture.  

PubMed

Specific inhibitors such as 2-bromoethanesulfonate (BES) and vancomycin were employed in activity batch tests to decipher metabolic pathways that are preferentially used by a mixed anaerobic consortium (sludge from an anaerobic digester) to transform carbon monoxide (CO) into methane (CH4). We first evaluated the inhibitory effect of both BES and vancomycin on the microbial community, as well as the efficiency and stability of vancomycin at 35 °C, over time. The activity tests with CO2-H2, CO, glucose, acetate, formate, propionate, butyrate, methanol, and ethanol showed that vancomycin does not inhibit some Gram-negative bacteria, and 50 mmol/L BES effectively blocks CH4 production in the sludge. However, when sludge was incubated with propionate, butyrate, methanol, or ethanol as the sole energy and carbon source, methanogenesis was only partially inhibited by BES. Separate tests showed that 0.07 mmol/L vancomycin is enough to maintain its inhibitory efficiency and stability in the population for at least 32 days at 35 °C. Using the inhibitors above, it was demonstrated that CO conversion to CH4 is an indirect, 2-step process, in which the CO is converted first to acetate and subsequently to CH4. PMID:24896194

Navarro, Silvia Sancho; Cimpoia, Ruxandra; Bruant, Guillaume; Guiot, Serge R

2014-06-01

243

Hierarchical modeling identifies novel lung cancer susceptibility variants in inflammation pathways among 10,140 cases and 11,012 controls  

PubMed Central

Recent evidence suggests that inflammation plays a pivotal role in the development of lung cancer. In this study, we used a two-stage approach to investigate associations between genetic variants in inflammation pathways and lung cancer risk based on genome-wide association study (GWAS) data. A total of 7,650 sequence variants from 720 genes relevant to inflammation pathways were identified using keyword and pathway searches from Gene Cards and Gene Ontology databases. In Stage 1, six GWAS datasets from the International Lung Cancer Consortium were pooled (4,441 cases and 5,094 controls of European ancestry), and a hierarchical modeling (HM) approach was used to incorporate prior information for each of the variants into the analysis. The prior matrix was constructed using (1) role of genes in the inflammation and immune pathways; (2) physical properties of the variants including the location of the variants, their conservation scores and amino acid coding; (3) LD with other functional variants and (4) measures of heterogeneity across the studies. HM affected the priority ranking of variants particularly among those having low prior weights, imprecise estimates and/or heterogeneity across studies. In Stage 2, we used an independent NCI lung cancer GWAS study (5,699 cases and 5,818 controls) for in silico replication. We identified one novel variant at the level corrected for multiple comparisons (rs2741354 in EPHX2 at 8q21.1 with p value = 7.4 × 10?6), and confirmed the associations between TERT (rs2736100) and the HLA region and lung cancer risk. HM allows for prior knowledge such as from bioinformatic sources to be incorporated into the analysis systematically, and it represents a complementary analytical approach to the conventional GWAS analysis.

Brenner, Darren R.; Brennan, Paul; Boffetta, Paolo; Amos, Christopher I.; Spitz, Margaret R.; Chen, Chu; Goodman, Gary; Heinrich, Joachim; Bickeboller, Heike; Rosenberger, Albert; Risch, Angela; Muley, Thomas; McLaughlin, John R.; Benhamou, Simone; Bouchardy, Christine; Lewinger, Juan Pablo; Witte, John S.; Chen, Gary; Bull, Shelley

2013-01-01

244

Massive analysis of cDNA Ends (MACE) and miRNA expression profiling identifies proatherogenic pathways in chronic kidney disease.  

PubMed

Epigenetic dysregulation contributes to the high cardiovascular disease burden in chronic kidney disease (CKD) patients. Although microRNAs (miRNAs) are central epigenetic regulators, which substantially affect the development and progression of cardiovascular disease (CVD), no data on miRNA dysregulation in CKD-associated CVD are available until now. We now performed high-throughput miRNA sequencing of peripheral blood mononuclear cells from ten clinically stable hemodialysis (HD) patients and ten healthy controls, which allowed us to identify 182 differentially expressed miRNAs (e.g., miR-21, miR-26b, miR-146b, miR-155). To test biological relevance, we aimed to connect miRNA dysregulation to differential gene expression. Genome-wide gene expression profiling by MACE (Massive Analysis of cDNA Ends) identified 80 genes to be differentially expressed between HD patients and controls, which could be linked to cardiovascular disease (e.g., KLF6, DUSP6, KLF4), to infection / immune disease (e.g., ZFP36, SOCS3, JUND), and to distinct proatherogenic pathways such as the Toll-like receptor signaling pathway (e.g., IL1B, MYD88, TICAM2), the MAPK signaling pathway (e.g., DUSP1, FOS, HSPA1A), and the chemokine signaling pathway (e.g., RHOA, PAK1, CXCL5). Formal interaction network analysis proved biological relevance of miRNA dysregulation, as 68 differentially expressed miRNAs could be connected to 47 reciprocally expressed target genes. Our study is the first comprehensive miRNA analysis in CKD that links dysregulated miRNA expression with differential expression of genes connected to inflammation and CVD. After recent animal data suggested that targeting miRNAs is beneficial in experimental CVD, our data may now spur further research in the field of CKD-associated human CVD. PMID:24184689

Zawada, Adam M; Rogacev, Kyrill S; Müller, Sören; Rotter, Björn; Winter, Peter; Fliser, Danilo; Heine, Gunnar H

2014-01-01

245

Multigene pathway-based analyses identify nasopharyngeal carcinoma risk associations for cumulative adverse effects of TERT-CLPTM1L and DNA double-strand breaks repair.  

PubMed

The genetic etiology of nasopharyngeal carcinoma (NPC) and mechanisms for inherited susceptibility remain unclear. To examine genetic risk factors for NPC, we hypothesized that heritable risk is attributable to cumulative effects of multiple common low-risk variants. With the premise that individual SNPs only confer subtle effects for cancer risk, a multigenic pathway-based approach was used to systematically examine associations between NPC genetic susceptibility with SNPs in genes in DNA repair pathways and from previously identified cancer genome-wide association study analyses. This case-control study covers 161 genes/loci and focuses on pathway-based analyses in 2,349 Hong Kong individuals, allowing stratification according to NPC familial status for meaningful association analysis. Three SNPs (rs401681, rs6774494 and rs3757318) corresponding to TERT/CLPTM1L (OR 95% CI?=?0.77, 0.68-0.88), MDS1-EVI1 (OR 95% CI=0.79 0.69-0.89) and CCDC170 (OR 95% CI?=?0.76, 0.66-0.86) conferred modest protective effects individually for NPC risk by the logistic regression analysis after multiple testing adjustment (pBonferroni ?identified rs2380165 in BLM (OR 95% CI?=?1.49, 1.20-1.86, pBonferroni ?pathway-based analysis revealed that the combined gene dosage effects for increasing numbers of unfavorable genotypes in TERT-CLPTM1L and double-strand break repair (DSBR) conferred elevated risk in FH+ and sporadic NPC patients (ORs per allele, 95% CIs?=?1.37, 1.22-1.55, pBonferroni ?=?5.00 × 10(-6) ; 1.17, 1.09-1.26, pBonferroni ?=?4.58 × 10(-4) , respectively, in TERT/NHEJ pathways). Our data suggested cumulative increased NPC risk associations with TERT-CLPTM1L and DSBR pathways contribute to genetic susceptibility to NPC and have potential translational relevance for patient stratification and therapeutics. PMID:24615621

Yee Ko, Josephine Mun; Dai, Wei; Wun Wong, Elibe Hiu; Kwong, Dora; Tong Ng, Wai; Lee, Anne; Kai Cheong Ngan, Roger; Chung Yau, Chun; Tung, Stewart; Li Lung, Maria

2014-10-01

246

Using genome-context data to identify specific types of functional associations in pathway\\/genome databases  

Microsoft Academic Search

Background: Hundreds of genes lacking homology to any protein of known function are sequenced every day. Genome-context methods have proved useful in providing clues about functional annotations for many proteins. However, genome-context methods detect many biological types of functional associations, and do not identify which type of functional association they have found. Results: We have developed two new genome-context-based algorithms.

Michelle L. Green; Peter D. Karp

2007-01-01

247

Genome-wide association study and mouse model identify interaction between RET and EDNRB pathways in Hirschsprung disease  

Microsoft Academic Search

Genetic studies of Hirschsprung disease, a common congenital malformation, have identified eight genes with mutations that can be associated with this condition. Mutations at individual loci are, however, neither necessary nor sufficient to cause clinical disease. We conducted a genome-wide association study in 43 Mennonite family trios using 2,083 microsatellites and single-nucleotide polymorphisms and a new multipoint linkage disequilibrium method

Minerva M. Carrasquillo; Andrew S. McCallion; Erik G. Puffenberger; Carl S. Kashuk; Nassim Nouri; Aravinda Chakravarti

2002-01-01

248

Studies toward the Unique Pederin Family Member Psymberin: Structure Activity Relationships, Biochemical Studies and Genetics Identify the Mode of Action of Psymberin  

PubMed Central

Psymberin is the only member of the pederin natural product family that contains a dihydroisocoumarin side chain. Structural modifications of psymberin uncoupled inhibition of protein translation from cytotoxicity, suggesting that psymberin has more than one bioactivity. A forward genetic screen in Caenorhabditis elegans was conducted to identify the molecular target(s) of psymberin. Multiple independent psymberin-resistant mutants were isolated, each containing the same point mutation in a gene encoding a ribosomal protein. However, a psymberin-resistant mutant strain bearing this mutation was not cross-resistant to the pederin family member mycalamide A, which binds to the archaeal form of the same protein. Thus, two pederin family members likely differ in how they bind the same molecular target. The accumulation of psymberin in cells was sensitive to the stereochemistry of the amide side chain at C4 or C8 and the presence of the dihydroisocoumarin side chain. The observation that psymberin diastereomers or dihydroisocoumarin-truncated analogs lose all cytotoxic activity while retaining the ability to inhibit protein translation in a cell-free in vitro assay can be explained in the context of these differential cell uptake issues. Finally, we also demonstrate that the blistering activity associated with pederin and other members of the family is not due to their protein synthesis inhibiting activity. Unlike pederin and mycalamide, psymberin does not display irritant or blistering activity.

Wu, Cheng-Yang; Feng, Yu; Cardenas, Eduardo R.; Williams, Noelle; Floreancig, Paul E.; De Brabander, Jef K.; Roth, Michael G.

2012-01-01

249

Identifying common pressure pathways from a complex network of human activities to support ecosystem-based management.  

PubMed

The marine environment is heavily exploited, but unintentional consequences cause wide-ranging negative effects to its characteristics. Linkage frameworks (e.g., DPSIR [driver-pressure-state-impact-response]) are commonly used to describe an interaction between human activities and ecological characteristics of the ecosystem, but as each linkage is viewed independently, the diversity of pressures that affect those characteristics may not be identified or managed effectively. Here we demonstrate an approach for using linkages to build a simple network to capture the complex relationships arising from multiple sectors and their activities. Using data-analysis tools common to ecology, we show how linkages can be placed into mechanistically similar groups. Management measures can be combined into fewer and more simplified measures that target groups of pressures rather than individual pressures, which is likely to increase compliance and the success of the measure while reducing the cost of enforcement. Given that conservation objectives (regional priorities) can vary, we also demonstrate by way of a case study example from the Marine Strategy Framework Directive, how management priorities might change, and illustrate how the approach can be used to identify sectors for control that best support the conservation objectives. PMID:23865227

Knights, Antony M; Koss, Rebecca S; Robinson, Leonie A

2013-06-01

250

Biochemical analysis of a highly specific, pH stable xylanase gene identified from a bovine rumen-derived metagenomic library.  

PubMed

A metagenomic library was generated using microbial DNA extracted from the rumen contents of a grass hay-fed dairy cow using a bacterial artificial chromosome-based vector system. Functional screening of the library identified a gene encoding a potent glycoside hydrolase, xyn10N18, localised within a xylanolytic gene cluster consisting of four open-reading frames (ORFs). The ORF, xyn10N18, encodes an endo-?-1,4-xylanase with a glycosyl hydrolase family 10 (GH10) catalytic domain, adopts a canonical ?8/ß8-fold and possesses conserved catalytic glutamate residues typical of GH10 xylanases. Xyn10N18 exhibits optimal catalytic activity at 35 °C and pH 6.5 and was highly stable to pH changes retaining at least 85 % relative catalytic activity over a broad pH range (4.0-12.0). It retained 25 % of its relative activity at both low (4 °C) and high (55 °C) temperatures, however the stability of the enzyme rapidly decreased at temperatures of >40 °C. The specific activity of Xyn10N18 is enhanced by the divalent cations Mn(2+) and Co(2+) and is dramatically reduced by Hg(2+) and Cu(2+). Interestingly, EDTA had little effect on specific activity indicating that divalent cations do not function mechanistically. The enzyme was highly specific for xylan containing substrates and showed no catalytic activity against cellulose. Analysis of the hydrolysis products indicated that Xyn10N18 was an endoxylanase. Through a combination of structural modelling and in vitro enzyme characterisation this study provides an understanding of the mechanism and the substrate specificity of this enzyme serving as a starting point for directed evolution of Xyn10N18 and subsequent downstream use in industry. PMID:22534823

Gong, X; Gruniniger, R J; Forster, R J; Teather, R M; McAllister, T A

2013-03-01

251

Genome-Wide siRNA-Based Functional Genomics of Pigmentation Identifies Novel Genes and Pathways That Impact Melanogenesis in Human Cells  

PubMed Central

Melanin protects the skin and eyes from the harmful effects of UV irradiation, protects neural cells from toxic insults, and is required for sound conduction in the inner ear. Aberrant regulation of melanogenesis underlies skin disorders (melasma and vitiligo), neurologic disorders (Parkinson's disease), auditory disorders (Waardenburg's syndrome), and opthalmologic disorders (age related macular degeneration). Much of the core synthetic machinery driving melanin production has been identified; however, the spectrum of gene products participating in melanogenesis in different physiological niches is poorly understood. Functional genomics based on RNA-mediated interference (RNAi) provides the opportunity to derive unbiased comprehensive collections of pharmaceutically tractable single gene targets supporting melanin production. In this study, we have combined a high-throughput, cell-based, one-well/one-gene screening platform with a genome-wide arrayed synthetic library of chemically synthesized, small interfering RNAs to identify novel biological pathways that govern melanin biogenesis in human melanocytes. Ninety-two novel genes that support pigment production were identified with a low false discovery rate. Secondary validation and preliminary mechanistic studies identified a large panel of targets that converge on tyrosinase expression and stability. Small molecule inhibition of a family of gene products in this class was sufficient to impair chronic tyrosinase expression in pigmented melanoma cells and UV-induced tyrosinase expression in primary melanocytes. Isolation of molecular machinery known to support autophagosome biosynthesis from this screen, together with in vitro and in vivo validation, exposed a close functional relationship between melanogenesis and autophagy. In summary, these studies illustrate the power of RNAi-based functional genomics to identify novel genes, pathways, and pharmacologic agents that impact a biological phenotype and operate outside of preconceived mechanistic relationships.

Bodemann, Brian; Petersen, Sean; Aruri, Jayavani; Koshy, Shiney; Richardson, Zachary; Le, Lu Q.; Krasieva, Tatiana; Roth, Michael G.; Farmer, Pat; White, Michael A.

2008-01-01

252

Large-scale association analyses identify new loci influencing glycemic traits and provide insight into the underlying biological pathways  

PubMed Central

Through genome-wide association meta-analyses of up to 133,010 individuals of European ancestry without diabetes, including individuals newly genotyped using the Metabochip, we have raised the number of confirmed loci influencing glycemic traits to 53, of which 33 also increase type 2 diabetes risk (q < 0.05). Loci influencing fasting insulin showed association with lipid levels and fat distribution, suggesting impact on insulin resistance. Gene-based analyses identified further biologically plausible loci, suggesting that additional loci beyond those reaching genome-wide significance are likely to represent real associations. This conclusion is supported by an excess of directionally consistent and nominally significant signals between discovery and follow-up studies. Functional follow-up of these newly discovered loci will further improve our understanding of glycemic control.

Scott, Robert A; Lagou, Vasiliki; Welch, Ryan P; Wheeler, Eleanor; Montasser, May E; Luan, Jian'an; Magi, Reedik; Strawbridge, Rona J; Rehnberg, Emil; Gustafsson, Stefan; Kanoni, Stavroula; Rasmussen-Torvik, Laura J; Yengo, Loic; Lecoeur, Cecile; Shungin, Dmitry; Sanna, Serena; Sidore, Carlo; Johnson, Paul C D; Jukema, J Wouter; Johnson, Toby; Mahajan, Anubha; Verweij, Niek; Thorleifsson, Gudmar; Hottenga, Jouke-Jan; Shah, Sonia; Smith, Albert V; Sennblad, Bengt; Gieger, Christian; Salo, Perttu; Perola, Markus; Timpson, Nicholas J; Evans, David M; Pourcain, Beate St; Wu, Ying; Andrews, Jeanette S; Hui, Jennie; Bielak, Lawrence F; Zhao, Wei; Horikoshi, Momoko; Navarro, Pau; Isaacs, Aaron; O'Connell, Jeffrey R; Stirrups, Kathleen; Vitart, Veronique; Hayward, Caroline; Esko, Tonu; Mihailov, Evelin; Fraser, Ross M; Fall, Tove; Voight, Benjamin F; Raychaudhuri, Soumya; Chen, Han; Lindgren, Cecilia M; Morris, Andrew P; Rayner, Nigel W; Robertson, Neil; Rybin, Denis; Liu, Ching-Ti; Beckmann, Jacques S; Willems, Sara M; Chines, Peter S; Jackson, Anne U; Kang, Hyun Min; Stringham, Heather M; Song, Kijoung; Tanaka, Toshiko; Peden, John F; Goel, Anuj; Hicks, Andrew A; An, Ping; Muller-Nurasyid, Martina; Franco-Cereceda, Anders; Folkersen, Lasse; Marullo, Letizia; Jansen, Hanneke; Oldehinkel, Albertine J; Bruinenberg, Marcel; Pankow, James S; North, Kari E; Forouhi, Nita G; Loos, Ruth J F; Edkins, Sarah; Varga, Tibor V; Hallmans, Goran; Oksa, Heikki; Antonella, Mulas; Nagaraja, Ramaiah; Trompet, Stella; Ford, Ian; Bakker, Stephan J L; Kong, Augustine; Kumari, Meena; Gigante, Bruna; Herder, Christian; Munroe, Patricia B; Caulfield, Mark; Antti, Jula; Mangino, Massimo; Small, Kerrin; Miljkovic, Iva; Liu, Yongmei; Atalay, Mustafa; Kiess, Wieland; James, Alan L; Rivadeneira, Fernando; Uitterlinden, Andre G; Palmer, Colin N A; Doney, Alex S F; Willemsen, Gonneke; Smit, Johannes H; Campbell, Susan; Polasek, Ozren; Bonnycastle, Lori L; Hercberg, Serge; Dimitriou, Maria; Bolton, Jennifer L; Fowkes, Gerard R; Kovacs, Peter; Lindstrom, Jaana; Zemunik, Tatijana; Bandinelli, Stefania; Wild, Sarah H; Basart, Hanneke V; Rathmann, Wolfgang; Grallert, Harald; Maerz, Winfried; Kleber, Marcus E; Boehm, Bernhard O; Peters, Annette; Pramstaller, Peter P; Province, Michael A; Borecki, Ingrid B; Hastie, Nicholas D; Rudan, Igor; Campbell, Harry; Watkins, Hugh; Farrall, Martin; Stumvoll, Michael; Ferrucci, Luigi; Waterworth, Dawn M; Bergman, Richard N; Collins, Francis S; Tuomilehto, Jaakko; Watanabe, Richard M; de Geus, Eco J C; Penninx, Brenda W; Hofman, Albert; Oostra, Ben A; Psaty, Bruce M; Vollenweider, Peter; Wilson, James F; Wright, Alan F; Hovingh, G Kees; Metspalu, Andres; Uusitupa, Matti; Magnusson, Patrik K E; Kyvik, Kirsten O; Kaprio, Jaakko; Price, Jackie F; Dedoussis, George V; Deloukas, Panos; Meneton, Pierre; Lind, Lars; Boehnke, Michael; Shuldiner, Alan R; van Duijn, Cornelia M; Morris, Andrew D; Toenjes, Anke; Peyser, Patricia A; Beilby, John P; Korner, Antje; Kuusisto, Johanna; Laakso, Markku; Bornstein, Stefan R; Schwarz, Peter E H; Lakka, Timo A; Rauramaa, Rainer; Adair, Linda S; Smith, George Davey; Spector, Tim D; Illig, Thomas; de Faire, Ulf; Hamsten, Anders; Gudnason, Vilmundur; Kivimaki, Mika; Hingorani, Aroon; Keinanen-Kiukaanniemi, Sirkka M; Saaristo, Timo E; Boomsma, Dorret I; Stefansson, Kari; van der Harst, Pim; Dupuis, Josee; Pedersen, Nancy L; Sattar, Naveed; Harris, Tamara B; Cucca, Francesco; Ripatti, Samuli; Salomaa, Veikko; Mohlke, Karen L; Balkau, Beverley; Froguel, Philippe; Pouta, Anneli; Jarvelin, Marjo-Riitta; Wareham, Nicholas J; Bouatia-Naji, Nabila; McCarthy, Mark I; Franks, Paul W; Meigs, James B; Teslovich, Tanya M; Florez, Jose C; Langenberg, Claudia; Ingelsson, Erik; Prokopenko, Inga; Barroso, Ines

2012-01-01

253

Multiple biomarker tissue arrays: A computational approach to identifying protein-protein interactions in the EGFR/ERK signalling pathway  

PubMed Central

Background Many studies have demonstrated genetic and environmental factors that lead to renal cell carcinoma (RCC) and that occur during a protracted period of tumourigenesis. It appears suitable to identify and characterise potential molecular markers that appear during tumourigenesis and that might provide rapid and effective possibilities for the early detection of RCC. EGFR activation induces cell cycle progression, inhibition of apoptosis and angiogenesis, promotion of invasion/metastasis, and other tumour promoting activities. Over-expression of EGFR is thought to play an important role in tumour initiation and progression of RCC because up-regulation of EGFR has been associated with high grade cancers and a worse prognosis. Methods Characterisation of the protein profile interacting with EGFR was performed using the following: an immunohistochemical (IHC) study of EGFR, a comprehensive computational study of EGFR protein-protein interactions, an analysis correlating the expression levels of EGFR with other significant markers in the tumourigenicity of RCC, and finally, an analysis of the utility of EGFR for prognosis in a cohort of patients with renal cell carcinoma. Results The cases that showed a higher level of this protein fell within the clear cell histological subtype (p?=?0.001). The EGFR significance statistic was found with respect to a worse prognosis. In vivo significant correlations were found with PDGFR-?, Flk-1, Hif1-?, proteins related to differentiation (such as DLL3 and DLL4 ligands), and certain metabolic proteins such as Glut5. In silico significant associations gave us a panel of 32 EGFR-interacting proteins (EIP) using the APID and STRING databases. Conclusions This work summarises the multifaceted role of EGFR in the pathology of RCC, and it identifies EIPs that could help to provide mechanistic explanations for the different behaviours observed in tumours.

2012-01-01

254

Global transcriptome analysis identifies regulated transcripts and pathways activated during oogenesis and early embryogenesis in atlantic cod.  

PubMed

The molecular mechanisms underlying oogenesis and maternally controlled embryogenesis in fish are not fully understood, especially in marine species. Our aim was to study the egg and embryo transcriptome during oogenesis and early embryogenesis in Atlantic cod. Follicles from oogenesis stages (pre-, early-, and late-vitellogenic), ovulated eggs, and two embryonic stages (blastula, gastrula) were collected from broodstock fish and fertilized eggs. Gene expression profiles were measured in a 44?K oligo microarray consisting of 23,000 cod genes. Hundreds of differentially expressed genes (DEGs) were identified in the follicle stages investigated, implicating a continuous accumulation and degradation of polyadenylated transcripts throughout oogenesis. Very few DEGs were identified from ovulated egg to blastula, showing a more stable maternal RNA pool in early embryonic stages. The highest induction of expression was observed between blastula and gastrula, signifying the onset of zygotic transcription. During early vitellogenesis, several of the most upregulated genes are linked to nervous system signaling, suggesting increasing requirements for ovarian synaptic signaling to stimulate the rapid growth of oocytes. Highly upregulated genes during late vitellogenesis are linked to protein processing, fat metabolism, osmoregulation, and arrested meiosis. One of the genes with the highest upregulation in the ovulated egg is involved in oxidative phosphorylation, reflecting increased energy requirements during fertilization and the first rapid cell divisions of early embryogenesis. In conclusion, this study provides a large-scale presentation of the Atlantic cod's maternally controlled transcriptome in ovarian follicles through oogenesis, ovulated eggs, and early embryos. Mol. Reprod. Dev. 81: 619-635, 2014. © 2014 Wiley Periodicals, Inc. PMID:24687555

Kleppe, Lene; Edvardsen, Rolf Brudvik; Furmanek, Tomasz; Taranger, Geir Lasse; Wargelius, Anna

2014-07-01

255

Combined transcriptome and proteome analysis identifies pathways and markers associated with the establishment of rapeseed microspore-derived embryo development.  

PubMed

Microspore-derived embryo (MDE) cultures are used as a model system to study plant cell totipotency and as an in vitro system to study embryo development. We characterized and compared the transcriptome and proteome of rapeseed (Brassica napus) MDEs from the few-celled stage to the globular/heart stage using two MDE culture systems: conventional cultures in which MDEs initially develop as unorganized clusters that usually lack a suspensor, and a novel suspensor-bearing embryo culture system in which the embryo proper originates from the distal cell of a suspensor-like structure and undergoes the same ordered cell divisions as the zygotic embryo. Improved histodifferentiation of suspensor-bearing MDEs suggests a new role for the suspensor in driving embryo cell identity and patterning. An MDE culture cDNA array and two-dimensional gel electrophoresis and protein sequencing were used to compile global and specific expression profiles for the two types of MDE cultures. Analysis of the identities of 220 candidate embryo markers, as well as the identities of 32 sequenced embryo up-regulated protein spots, indicate general roles for protein synthesis, glycolysis, and ascorbate metabolism in the establishment of MDE development. A collection of 135 robust markers for the transition to MDE development was identified, a number of which may be coregulated at the gene and protein expression level. Comparison of the expression profiles of preglobular-stage conventional MDEs and suspensor-bearing MDEs identified genes whose differential expression may reflect improved histodifferentiation of suspensor-bearing embryos. This collection of early embryo-expressed genes and proteins serves as a starting point for future marker development and gene function studies aimed at understanding the molecular regulation of cell totipotency and early embryo development in plants. PMID:17384159

Joosen, Ronny; Cordewener, Jan; Supena, Ence Darmo Jaya; Vorst, Oscar; Lammers, Michiel; Maliepaard, Chris; Zeilmaker, Tieme; Miki, Brian; America, Twan; Custers, Jan; Boutilier, Kim

2007-05-01

256

SFGD: a comprehensive platform for mining functional information from soybean transcriptome data and its use in identifying acyl-lipid metabolism pathways  

PubMed Central

Background Soybean (Glycine max L.) is one of the world’s most important leguminous crops producing high-quality protein and oil. Increasing the relative oil concentration in soybean seeds is many researchers’ goal, but a complete analysis platform of functional annotation for the genes involved in the soybean acyl-lipid pathway is still lacking. Following the success of soybean whole-genome sequencing, functional annotation has become a major challenge for the scientific community. Whole-genome transcriptome analysis is a powerful way to predict genes with biological functions. It is essential to build a comprehensive analysis platform for integrating soybean whole-genome sequencing data, the available transcriptome data and protein information. This platform could also be used to identify acyl-lipid metabolism pathways. Description In this study, we describe our construction of the Soybean Functional Genomics Database (SFGD) using Generic Genome Browser (Gbrowse) as the core platform. We integrated microarray expression profiling with 255 samples from 14 groups’ experiments and mRNA-seq data with 30 samples from four groups’ experiments, including spatial and temporal transcriptome data for different soybean development stages and environmental stresses. The SFGD includes a gene co-expression regulatory network containing 23,267 genes and 1873 miRNA-target pairs, and a group of acyl-lipid pathways containing 221 enzymes and more than 1550 genes. The SFGD also provides some key analysis tools, i.e. BLAST search, expression pattern search and cis-element significance analysis, as well as gene ontology information search and single nucleotide polymorphism display. Conclusion The SFGD is a comprehensive database integrating genome and transcriptome data, and also for soybean acyl-lipid metabolism pathways. It provides useful toolboxes for biologists to improve the accuracy and robustness of soybean functional genomics analysis, further improving understanding of gene regulatory networks for effective crop improvement. The SFGD is publically accessible at http://bioinformatics.cau.edu.cn/SFGD/, with all data available for downloading.

2014-01-01

257

Putative Pathway of Sex Pheromone Biosynthesis and Degradation by Expression Patterns of Genes Identified from Female Pheromone Gland and Adult Antenna of Sesamia inferens (Walker).  

PubMed

The general pathway of biosynthesis and degradation for Type-I sex pheromones in moths is well established, but some genes involved in this pathway remain to be characterized. The purple stem borer, Sesamia inferens, employs a pheromone blend containing components with three different terminal functional groups (Z11-16:OAc, Z11-16:OH, and Z11-16:Ald) of Type-I sex pheromones. Thus, it provides a good model to study the diversity of genes involved in pheromone biosynthesis and degradation pathways. By analyzing previously obtained transcriptomic data of the sex pheromone glands and antennae, we identified 73 novel genes that are possibly related to pheromone biosynthesis (46 genes) or degradation (27 genes). Gene expression patterns and phylogenetic analysis revealed that one desaturase (SinfDes4), one fatty acid reductase (SinfFAR2), and one fatty acid xtransport protein (SinfFATP1) genes were predominantly expressed in pheromone glands, and clustered with genes involved in pheromone synthesis in other moth species. Ten genes including five carboxylesterases (SinfCXE10, 13, 14, 18, and 20), three aldehyde oxidases (SinfAOX1, 2 and 3), and two alcohol dehydrogenases (SinfAD1 and 3) were expressed specifically or predominantly in antennae, and could be candidate genes involved in pheromone degradation. SinfAD1 and 3 are the first reported alcohol dehydrogenase genes with antennae-biased expression. Based on these results we propose a pathway involving these potential enzyme-encoding gene candidates in sex pheromone biosynthesis and degradation in S. inferens. This study provides robust background information for further elucidation of the genetic basis of sex pheromone biosynthesis and degradation, and ultimately provides potential targets to disrupt sexual communication in S. inferens for control purposes. PMID:24817326

Zhang, Ya-Nan; Xia, Yi-Han; Zhu, Jia-Yao; Li, Sheng-Yun; Dong, Shuang-Lin

2014-05-01

258

YlxM Is a Newly Identified Accessory Protein That Influences the Function of Signal Recognition Particle Pathway Components in Streptococcus mutans.  

PubMed

Streptococcus mutans is a cariogenic oral pathogen whose virulence is determined largely by its membrane composition. The signal recognition particle (SRP) protein-targeting pathway plays a pivotal role in membrane biogenesis. S. mutans SRP pathway mutants demonstrate growth defects, cannot contend with environmental stress, and exhibit multiple changes in membrane composition. This study sought to define a role for ylxM, which in S. mutans and numerous other bacteria resides directly upstream of the ffh gene, encoding a major functional element of the bacterial SRP. YlxM was observed as a produced protein in S. mutans. Its predicted helix-turn-helix motif suggested that it has a role as a transcriptional regulator of components within the SRP pathway; however, no evidence of transcriptional regulation was found. Instead, capture enzyme-linked immunosorbent assay (ELISA), affinity chromatography, and bio-layer interferometry (BLI) demonstrated that S. mutans YlxM interacts with the SRP components Ffh and small cytoplasmic RNA (scRNA) but not with the SRP receptor FtsY. In the absence of FtsY, YlxM increased the GTP hydrolysis activity of Ffh alone and in complex with scRNA. However, in the presence of FtsY, YlxM caused an overall diminution of net GTPase activity. Thus, YlxM appears to modulate GTP hydrolysis, a process necessary for proper recycling of SRP pathway components. The presence of YlxM conferred a significant competitive growth advantage under nonstress and acid stress conditions when wild-type and ylxM mutant strains were cultured together. Our results identify YlxM as a component of the S. mutans SRP and suggest a regulatory function affecting GTPase activity. PMID:24659773

Williams, Matthew L; Crowley, Paula J; Hasona, Adnan; Brady, L Jeannine

2014-06-01

259

Translational research in brain metastasis is identifying molecular pathways that may lead to the development of new therapeutic strategies  

PubMed Central

Central nervous system (CNS) or brain metastasis is an emerging area of interest in organ-specific metastasis research. Lung and breast cancers are the most common types of primary tumors to develop brain metastases. This disease complication contributes significantly to the morbidity and mortality of both of these common cancers; as such, brain metastasis is designated an unmet medical need by the US Food and Drug Administration. Recently, an increase in incidence of CNS disease has been noted in the literature for breast cancer, while it has been an ongoing major complication from lung cancer. Progress in treating brain metastases has been hampered by a lack of model systems, a lack of human tissue samples, and the exclusion of brain metastatic patients from many clinical trials. While each of those is significant, the major impediment to effectively treating brain metastatic disease is the blood–brain barrier (BBB). This barrier excludes most chemotherapeutics from the brain and creates a sanctuary site for metastatic tumors. Recent findings on the biology of this disease and translational leads identified by molecular studies are discussed in this article.

Gril, Brunilde; Evans, Lynda; Palmieri, Diane; Steeg, Patricia S.

2010-01-01

260

Analysis of ?SMA-labeled progenitor cell commitment identifies notch signaling as an important pathway in fracture healing.  

PubMed

Fracture healing is a regenerative process that involves coordinated responses of many cell types, but characterization of the roles of specific cell populations in this process has been limited. We have identified alpha smooth muscle actin (?SMA) as a marker of a population of mesenchymal progenitor cells in the periosteum that contributes to osteochondral elements during fracture healing. Using a lineage tracing approach, we labeled ?SMA-expressing cells, and characterized changes in the periosteal population during the early stages of fracture healing by histology, flow cytometry, and gene expression profiling. In response to fracture, the ?SMA-labeled population expanded and began to differentiate toward the osteogenic and chondrogenic lineages. The frequency of mesenchymal progenitor cell markers such as Sca1 and PDGFR? increased after fracture. By 6 days after fracture, genes involved in matrix production and remodeling were elevated. In contrast, genes associated with muscle contraction and Notch signaling were downregulated after fracture. We confirmed that activating Notch signaling in ?SMA-labeled cells inhibited differentiation into osteogenic and adipogenic lineages in vitro and ectopic bone formation in vivo. By characterizing changes in a selected ?SMA-labeled progenitor cell population during fracture callus formation, we have shown that modulation of Notch signaling may determine osteogenic potential of ?SMA-expressing progenitor cells during bone healing. PMID:24190076

Matthews, Brya G; Grcevic, Danka; Wang, Liping; Hagiwara, Yusuke; Roguljic, Hrvoje; Joshi, Pujan; Shin, Dong-Guk; Adams, Douglas J; Kalajzic, Ivo

2014-05-01

261

A System Biology Approach to Identify Regulatory Pathways Underlying the Neuroendocrine Control of Female Puberty in Rats and Nonhuman Primates  

PubMed Central

Puberty is a major developmental milestone controlled by the interaction of genetic factors and environmental cues of mostly metabolic and circadian nature. An increased pulsatile release of the decapeptide gonadotropin releasing hormone (GnRH) from hypothalamic neurosecretory neurons is required for both the initiation and progression of the pubertal process. This increase is brought about by coordinated changes that occur in neuronal and glial networks associated with GnRH neurons. These changes ultimately result in increased neuronal and glial stimulatory inputs to the GnRH neuronal network and a reduction of transsynaptic inhibitory influences. While some of the major players controlling pubertal GnRH secretion have been identified using gene-centric approaches, much less is known about the system-wide control of the overall process. Because the pubertal activation of GnRH release involves a diversity of cellular phenotypes, and a myriad of intracellular and cell-to-cell signaling molecules, it appears that the overall process is controlled by a highly coordinated and interactive regulatory system involving hundreds, if not thousands, of gene products. In this article we will discuss emerging evidence suggesting that these genes are arranged as functionally connected networks organized, both internally and across sub-networks, in a hierarchical fashion. According to this concept, the core of these networks is composed of transcriptional regulators that, by directing expression of downstream subordinate genes, provide both stability and coordination to the cellular networks involved in initiating the pubertal process. The integrative response of these gene networks to external inputs is postulated to be coordinated by epigenetic mechanisms.

Lomniczi, Alejandro; Wright, Hollis; Castellano, Juan Manuel; Sonmez, Kemal; Ojeda, Sergio R.

2014-01-01

262

Biochemical and functional characterization of phosphoserine aminotransferase from Entamoeba histolytica, which possesses both phosphorylated and non-phosphorylated serine metabolic pathways  

Microsoft Academic Search

The enteric protozoan parasite Entamoeba histolytica is a unicellular eukaryote that possesses both phosphorylated and non-phosphorylated serine metabolic pathways. In the present study, we described enzymological and functional characterization of phosphoserine aminotransferase (PSAT) from E. histolytica. E. histolytica PSAT (EhPSAT) showed maximum activity for the forward reaction at basic pH, dissimilar to mammalian PSAT, which showed sharp neutral optimum pH.

Vahab Ali; Tomoyoshi Nozaki

2006-01-01

263

Microarray Analysis in a Cell Death Resistant Glioma Cell Line to Identify Signaling Pathways and Novel Genes Controlling Resistance and Malignancy  

PubMed Central

Glioblastoma multiforme (GBM) is a lethal type of cancer mainly resistant to radio- and chemotherapy. Since the tumor suppressor p53 functions as a transcription factor regulating the expression of genes involved in growth inhibition, DNA repair and apoptosis, we previously assessed whether specific differences in the modulation of gene expression are responsible for the anti-tumor properties of a dominant positive p53, chimeric tumor suppressor (CTS)-1. CTS-1 is based on the sequence of p53 and designed to resist various mechanisms of inactivation which limit the activity of p53. To identify CTS-1-regulated cell death-inducing genes, we generated a CTS-1-resistant glioma cell line (229R). We used Affymetrix whole-genome microarray expression analysis to analyze alterations in gene expression and identified a variety of CTS-1 regulated genes involved in cancer-linked processes. 313 genes were differentially expressed in Adeno-CTS-1 (Ad-CTS-1)-infected and 700 genes in uninfected 229R cells compared to matching parental cells. Ingenuity Pathway Analysis (IPA) determined a variety of differentially expressed genes in Ad-CTS-1-infected cells that were members of the intracellular networks with central tumor-involved players such as nuclear factor kappa B (NF-?B), protein kinase B (PKB/AKT) or transforming growth factor beta (TGF-?). Differentially regulated genes include secreted factors as well as intracellular proteins and transcription factors regulating not only cell death, but also processes such as tumor cell motility and immunity. This work gives an overview of the pathways differentially regulated in the resistant versus parental glioma cells and might be helpful to identify candidate genes which could serve as targets to develop novel glioma specific therapy strategies.

Seznec, Janina; Naumann, Ulrike

2011-01-01

264

Epidermal Growth Factor Receptor Pathway Analysis Identifies Amphiregulin as a Key Factor for Cisplatin Resistance of Human Breast Cancer Cells*S  

PubMed Central

The use of platinum complexes for the therapy of breast cancer is an emerging new treatment modality. To gain insight into the mechanisms underlying cisplatin resistance in breast cancer, we used estrogen receptor-positive MCF-7 cells as a model system. We generated cisplatin-resistant MCF-7 cells and determined the functional status of epidermal growth factor receptor (EGFR), MAPK, and AKT signaling pathways by phosphoreceptor tyrosine kinase and phospho-MAPK arrays. The cisplatin-resistant MCF-7 cells are characterized by increased EGFR phosphorylation, high levels of AKT1 kinase activity, and ERK1 phosphorylation. In contrast, the JNK and p38 MAPK modules of the MAPK signaling pathway were inactive. These conditions were associated with inactivation of the p53 pathway and increased BCL-2 expression. We investigated the expression of genes encoding the ligands for the ERBB signaling cascade and found a selective up-regulation of amphiregulin expression, which occurred at later stages of cisplatin resistance development. Amphiregulin is a specific ligand of the EGFR (ERBB1) and a potent mitogen for epithelial cells. After exposure to cisplatin, the resistant MCF-7 cells secreted amphiregulin protein over extended periods of time, and knockdown of amphiregulin expression by specific short interfering RNA resulted in a nearly complete reversion of the resistant phenotype. To demonstrate the generality and importance of our findings, we examined amphiregulin expression and cisplatin resistance in a variety of human breast cancer cell lines and found a highly significant correlation. In contrast, amphiregulin levels did not significantly correlate with cisplatin resistance in a panel of lung cancer cell lines. We have thus identified a novel function of amphiregulin for cisplatin resistance in human breast cancer cells.

Eckstein, Niels; Servan, Kati; Girard, Luc; Cai, Di; von Jonquieres, Georg; Jaehde, Ulrich; Kassack, Matthias U.; Gazdar, Adi F.; Minna, John D.; Royer, Hans-Dieter

2009-01-01

265

Pharmacogenomic profiling and pathway analyses identify MAPK-dependent migration as an acute response to SN38 in p53 null and mutant colorectal cancer cells  

PubMed Central

The topoisomerase I inhibitor irinotecan is used to treat advanced colorectal cancer and has been shown to have p53-independent anti-cancer activity. The aim of this study was to identify the p53-independent signalling mechanisms activated by irinotecan. Transcriptional profiling of isogenic HCT116 p53 wild-type and p53 null cells was carried out following treatment with the active metabolite of irinotecan, SN38. Unsupervised analysis methods demonstrated that p53 status had a highly significant impact on gene expression changes in response to SN38. Pathway analysis indicated that pathways involved in cell motility (adherens junction, focal adhesion, MAPK and regulation of the actin cytoskeleton) were significantly activated in p53 null cells, but not p53 wild-type cells, following SN38 treatment. In functional assays, SN38 treatment increased the migratory potential of p53 null and mutant colorectal cancer cell lines, but not p53 wild-type lines. Moreover, p53 null SN38-resistant cells were found to migrate at a faster rate than parental drug-sensitive p53 null cells, whereas p53 wild-type SN38-resistant cells failed to migrate. Notably, co-treatment with inhibitors of the MAPK pathway inhibited the increased migration observed following SN38 treatment in p53 null and mutant cells. Thus, in the absence of wild-type p53, SN38 promotes migration of colorectal cancer cells, and inhibiting MAPK blocks this potentially pro-metastatic adaptive response to this anti-cancer drug.

Allen, Wendy L.; Turkington, Richard C.; Stevenson, Leanne; Carson, Gail; Coyle, Vicky M.; Hector, Suzanne; Dunne, Philip; Van Schaeybroeck, Sandra; Longley, Daniel B.; Johnston, Patrick G.

2012-01-01

266

A Genetic Screen for Novel Components of the Ras\\/Mitogen-Activated Protein Kinase Signaling Pathway That Interact With the yan Gene of Drosophila Identifies split ends, a New RNA Recognition Motif-Containing Protein  

Microsoft Academic Search

The receptor tyrosine kinase (RTK) signaling pathway is used reiteratively during the development of all multicellular organisms. While the core RTK\\/Ras\\/MAPK signaling cassette has been studied extensively, little is known about the nature of the downstream targets of the pathway or how these effectors regulate the specificity of cellular responses. Drosophila yan is one of a few downstream components identified

Ilaria Rebay; Fangli Chen; Francis Hsiao; Peter A. Kolodziej; Bing H. Kuang; Todd Laverty; Chris Suh; Matthew Voas; Andrina Williams; Gerald M. Rubin

2000-01-01

267

A Novel Peptide-Based SILAC Method to Identify the Posttranslational Modifications Provides Evidence for Unconventional Ubiquitination in the ER-Associated Degradation Pathway  

PubMed Central

The endoplasmic reticulum-associated degradation (ERAD) pathway is responsible for disposing misfolded proteins from the endoplasmic reticulum by inducing their ubiquitination and degradation. Ubiquitination is conventionally observed on lysine residues and has been demonstrated on cysteine residues and protein N-termini. Ubiquitination is fundamental to the ERAD process; however, a mutant T-cell receptor ? (TCR?) lacking lysine residues is targeted for the degradation by the ERAD pathway. We have shown that ubiquitination of lysine-less TCR? occurs on internal, non-lysine residues and that the same E3 ligase conjugates ubiquitin to TCR? in the presence or absence of lysine residues. Mass-spectrometry indicates that WT-TCR? is ubiquitinated on multiple lysine residues. Recent publications have provided indirect evidence that serine and threonine residues may be modified by ubiquitin. Using a novel peptide-based stable isotope labeling in cell culture (SILAC) approach, we show that specific lysine-less TCR? peptides become modified. In this study, we demonstrate that it is possible to detect both ester and thioester based ubiquitination events, although the exact linkage on lysine-less TCR? remains elusive. These findings demonstrate that SILAC can be used as a tool to identify modified peptides, even those with novel modifications that may not be detected using conventional proteomic work flows or informatics algorithms.

Anania, Veronica G.; Bustos, Daisy J.; Lill, Jennie R.; Kirkpatrick, Donald S.; Coscoy, Laurent

2013-01-01

268

In vivo RNAi screen for BMI1 targets identifies TGF-?/BMP-ER stress pathways as key regulators of neural- and malignant glioma-stem cell homeostasis.  

PubMed

In mouse and human neural progenitor and glioblastoma "stem-like" cells, we identified key targets of the Polycomb-group protein BMI1 by combining ChIP-seq with in vivo RNAi screening. We discovered that Bmi1 is important in the cellular response to the transforming growth factor-?/bone morphogenetic protein (TGF-?/BMP) and endoplasmic reticulum (ER) stress pathways, in part converging on the Atf3 transcriptional repressor. We show that Atf3 is a tumor-suppressor gene inactivated in human glioblastoma multiforme together with Cbx7 and a few other candidates. Acting downstream of the ER stress and BMP pathways, ATF3 binds to cell-type-specific accessible chromatin preloaded with AP1 and participates in the inhibition of critical oncogenic networks. Our data support the feasibility of combining ChIP-seq and RNAi screens in solid tumors and highlight multiple p16(INK4a)/p19(ARF)-independent functions for Bmi1 in development and cancer. PMID:23680149

Gargiulo, Gaetano; Cesaroni, Matteo; Serresi, Michela; de Vries, Nienke; Hulsman, Danielle; Bruggeman, Sophia W; Lancini, Cesare; van Lohuizen, Maarten

2013-05-13

269

siRNA screening identifies differences in the Fanconi anemia pathway in BALB/c-Trp53+/- with susceptibility versus C57BL/6-Trp53+/- mice with resistance to mammary tumors  

PubMed Central

BALB/c mice heterozygous for Trp53 develop a high proportion of spontaneous mammary tumors, a phenotype distinct from other mouse strains. BALB/c-Trp53+/? female mice, thus, resemble the hereditary Li-Fraumeni syndrome (LFS) characterized by early-onset of breast cancer, even though LFS involves TP53 mutations, which may involve not only loss- but also gain-of-function. Previous analysis of tumors in BALB/c-Trp53+/? females showed frequent loss of heterozygosity involving the wild-type allele of Trp53 and displayed characteristics indicative of mitotic recombination. Critical involvement of DNA double-strand break (DSB) repair dysfunction, particularly of homologous recombination (HR), was also noticed in the etiology of human breast cancer. To better define functional alterations in BALB/c-Trp53+/? mice, we applied a fluorescence-based DSB repair assay on mouse embryonic fibroblasts (MEFs) from BALB/c-Trp53+/? versus C57BL/6J-Trp53+/? mice. This approach revealed deregulation of HR but not non-homologous end-joining (NHEJ) in BALB/c-Trp53+/?, which was further confirmed for mammary epithelial cells. Screening of a small interfering RNA-library targeting DSB repair, recombination, replication and signaling genes, identified 25 genes causing differences between homologous DSB repair in the two strains upon silencing. Interactome analysis of the hits revealed clustering of replication-related and fanconi anemia (FA)/breast cancer susceptibility (BRCA) genes. Further dissection of the functional change in BALB/c-Trp53+/? by immunofluorescence microscopy of nuclear 53BP1, Replication protein A (RPA) and Rad51 foci uncovered differences in crosslink and replication-associated repair. Chromosome breakage, G2 arrest and biochemical analyses indicated a FA pathway defect downstream of FancD2 associated with reduced levels of BRCA2. Consistent with polygenic models for BRCA, mammary carcinogenesis in BALB/c-Trp53+/? mice may, therefore, be promoted by a BRCA modifier allele in the FA pathway in the context of partial p53 loss-of-function.

Bohringer, M; Obermeier, K; Griner, N; Waldraff, D; Dickinson, E; Eirich, K; Schindler, D; Hagen, M; Jerry, D J; Wiesmuller, L

2013-01-01

270

Biochemical and functional characterization of phosphoserine aminotransferase from Entamoeba histolytica, which possesses both phosphorylated and non-phosphorylated serine metabolic pathways.  

PubMed

The enteric protozoan parasite Entamoeba histolytica is a unicellular eukaryote that possesses both phosphorylated and non-phosphorylated serine metabolic pathways. In the present study, we described enzymological and functional characterization of phosphoserine aminotransferase (PSAT) from E. histolytica. E. histolytica PSAT (EhPSAT) showed maximum activity for the forward reaction at basic pH, dissimilar to mammalian PSAT, which showed sharp neutral optimum pH. EhPSAT activity was significantly inhibited by substrate analogs, O-phospho-d-serine, O-phospho-l-threonine, and O-acetylserine, suggesting possible regulation of the amoebic PSAT by these metabolic intermediates. Fractionation of the whole parasite lysate and rEhPSAT by anion exchange chromatography verified that EhPSAT represents a dominant PSAT activity. EhPSAT showed a close kinship to PSAT from bacteroides based on amino acid alignment and phylogenetic analyses, suggesting that E. histolytica gained this gene from bacteroides by lateral gene transfer. Comparisons of kinetic properties of recombinant PSAT from E. histolytica and Arabidopsis thaliana showed that EhPSAT possesses significantly higher affinity toward glutamate than the A. thaliana counterpart, which may be explained by significant differences in the isoelectric point and the substitution of arginine, which is involved the binding to the gamma-carboxylate moiety of glutamate, in Escherichia coli PSAT, to serine or threonine in E. histolytica or A. thaliana PSAT, respectively. Heterologous expression of EhPSAT successfully rescued growth defect of a serine-auxotrophic E. coli strain KL282, where serC was deleted, confirming its in vivo role in serine biosynthesis. Together with our previous demonstration of phosphoglycerate dehydrogenase, the present study reinforces physiological significance of the phosphorylated pathway in amoeba. PMID:16289358

Ali, Vahab; Nozaki, Tomoyoshi

2006-01-01

271

Genome-scale RNAi on living-cell microarrays identifies novel regulators of Drosophila melanogaster TORC1-S6K pathway signaling  

PubMed Central

The evolutionarily conserved target of rapamycin complex 1 (TORC1) controls cell growth in response to nutrient availability and growth factors. TORC1 signaling is hyperactive in cancer, and regulators of TORC1 signaling may represent therapeutic targets for human diseases. To identify novel regulators of TORC1 signaling, we performed a genome-scale RNA interference screen on microarrays of Drosophila melanogaster cells expressing human RPS6, a TORC1 effector whose phosphorylated form we detected by immunofluorescence. Our screen revealed that the TORC1–S6K–RPS6 signaling axis is regulated by many subcellular components, including the Class I vesicle coat (COPI), the spliceosome, the proteasome, the nuclear pore, and the translation initiation machinery. Using additional RNAi reagents, we confirmed 70 novel genes as significant on-target regulators of RPS6 phosphorylation, and we characterized them with extensive secondary assays probing various arms of the TORC1 pathways, identifying functional relationships among those genes. We conclude that cell-based microarrays are a useful platform for genome-scale and secondary screening in Drosophila, revealing regulators that may represent drug targets for cancers and other diseases of deregulated TORC1 signaling.

Lindquist, Robert A.; Ottina, Kathleen A.; Wheeler, Douglas B.; Hsu, Peggy P.; Thoreen, Carson C.; Guertin, David A.; Ali, Siraj M.; Sengupta, Shomit; Shaul, Yoav D.; Lamprecht, Michael R.; Madden, Katherine L.; Papallo, Adam R.; Jones, Thouis R.; Sabatini, David M.; Carpenter, Anne E.

2011-01-01

272

Lesch-Nyhan syndrome: the synthesis of inosine 5'-phosphate in the hypoxanthine-guanine phosphoribosyltransferase-deficient erythrocyte by alternate biochemical pathways.  

PubMed

Erythrocytes, obtained from a normal adult male and from a patient with Lesch-Nyhan syndrome, were incubated with [8-14C]adenine and [8-14C]hypoxanthine (Table 1). The labeled adenine was utilized to about the same extent for the synthesis of AMP by the normal subject's and the patient's erythrocytes. Deamination of AMP to IMP occurred to about the same extent in both samples. In contrast, hypoxanthine was utilized extensively for IMP synthesis in the normal erythrocyte only. The amount of total label in the IMP was about 100 times that of the Lesch-Nyhan erythrocyte, a consequence of the deficiency of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity in the syndrome. No significant labeling of the AMP occurred. When aliquots of erythrocytes from both sources were incubated with 4-amino-5-imidazolecarboxamide (AICA) and sodium [14C]formate, extensive labeling of the IMP occurred in normal and in Lesch-Nyhan erythrocytes. The data suggest that AICA serves as a substrate for the adenine phosphoribosyltransferase (APRT) of the Lesch-Nyhan erythrocyte and that the ribotide of AICA, 5'-phosphoribosyl-5-aminoimidazole-4-carboxamide (AICAR), undergoes formylation by labeled N10-formyl tetrahydrofolic acid formed from the reaction of sodium [14C]formate with the tetrahydrofolic acid of the cell. The formyl-AICAR undergoes ring closure to IMP by a series of reactions comparable to those described for the normal erythrocyte. When 5-amino-1-ribosyl-4-imidazolecarboxamide (rAICA) and sodium [14C]formate were incubated with erythrocyte suspensions, extensive utilization for IMP synthesis was also observed in normal erythrocytes and in erythrocytes from Lesch-Nyhan patients (Table 2). The reaction sequence is somewhat different from that of AICA. AICA is not a substrate for the purine nucleoside phosphorylase of rabbit or human erythrocytes. The mechanism of rAICA utilization is visualized as a direct phosphorylation of the ribosyl compound, possibly by the adenosine kinase of the human cell. The ribotide, AICAR, formed by this mechanism, undergoes formylation and ring closure, yielding IMP. The glutamine antagonist, diazooxonorleucine (DON), was added to aliquots of patients' cells incubated with rAICA and sodium [14C]formate. DON is an effective inhibitor of the conversion of IMP to GMP and its presence in an incubation suspension resulted in a somewhat greater radioactivity of the total cellular IMP. The extension of the current studies to Lesch-Nyhan cells in culture may serve to assist in the direct evaluation of the regulatory role of IMP in the de novo pathway of purine nucleotide biosynthesis. Because of the substrate requirements of the reactions, the metabolism of AICA and rAICA may also serve to differentiate the roles of purine nucleotides and of phosphoribosylpyrophosphate (PRPP) in the pathway regulation. The findings presented also offer a possible therapeutic approach to the early treatment of the disease in the afflicted neonate... PMID:870876

Lowy, B A; Williams, M K

1977-05-01

273

Lesions from patients with sporadic cerebral cavernous malformations harbor somatic mutations in the CCM genes: evidence for a common biochemical pathway for CCM pathogenesis.  

PubMed

Cerebral cavernous malformations (CCMs) are vascular lesions affecting the central nervous system. CCM occurs either sporadically or in an inherited, autosomal dominant manner. Constitutional (germline) mutations in any of three genes, KRIT1, CCM2 and PDCD10, can cause the inherited form. Analysis of CCM lesions from inherited cases revealed biallelic somatic mutations, indicating that CCM follows a Knudsonian two-hit mutation mechanism. It is still unknown, however, if the sporadic cases of CCM also follow this genetic mechanism. We extracted DNA from 11 surgically excised lesions from sporadic CCM patients, and sequenced the three CCM genes in each specimen using a next-generation sequencing approach. Four sporadic CCM lesion samples (36%) were found to contain novel somatic mutations. Three of the lesions contained a single somatic mutation, and one lesion contained two biallelic somatic mutations. Herein, we also describe evidence of somatic mosaicism in a patient presenting with over 130 CCM lesions localized to one hemisphere of the brain. Finally, in a lesion regrowth sample, we found that the regrown CCM lesion contained the same somatic mutation as the original lesion. Together, these data bolster the idea that all forms of CCM have a genetic underpinning of the two-hit mutation mechanism in the known CCM genes. Recent studies have found aberrant Rho kinase activation in inherited CCM pathogenesis, and we present evidence that this pathway is activated in sporadic CCM patients. These results suggest that all CCM patients, including those with the more common sporadic form, are potentially amenable to the same therapy. PMID:24698976

McDonald, David A; Shi, Changbin; Shenkar, Robert; Gallione, Carol J; Akers, Amy L; Li, Stephanie; De Castro, Nicholas; Berg, Michel J; Corcoran, David L; Awad, Issam A; Marchuk, Douglas A

2014-08-15

274

The Use of Genome-Wide eQTL Associations in Lymphoblastoid Cell Lines to Identify Novel Genetic Pathways Involved in Complex Traits  

PubMed Central

The integrated analysis of genotypic and expression data for association with complex traits could identify novel genetic pathways involved in complex traits. We profiled 19,573 expression probes in Epstein-Barr virus-transformed lymphoblastoid cell lines (LCLs) from 299 twins and correlated these with 44 quantitative traits (QTs). For 939 expressed probes correlating with more than one QT, we investigated the presence of eQTL associations in three datasets of 57 CEU HapMap founders and 86 unrelated twins. Genome-wide association analysis of these probes with 2.2 m SNPs revealed 131 potential eQTLs (1,989 eQTL SNPs) overlapping between the HapMap datasets, five of which were in cis (58 eQTL SNPs). We then tested 535 SNPs tagging the eQTL SNPs, for association with the relevant QT in 2,905 twins. We identified nine potential SNP-QT associations (P<0.01) but none significantly replicated in five large consortia of 1,097–16,129 subjects. We also failed to replicate previous reported eQTL associations with body mass index, plasma low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and triglycerides levels derived from lymphocytes, adipose and liver tissue. Our results and additional power calculations suggest that proponents may have been overoptimistic in the power of LCLs in eQTL approaches to elucidate regulatory genetic effects on complex traits using the small datasets generated to date. Nevertheless, larger tissue-specific expression data sets relevant to specific traits are becoming available, and should enable the adoption of similar integrated analyses in the near future.

Min, Josine L.; Taylor, Jennifer M.; Richards, J. Brent; Watts, Tim; Pettersson, Fredrik H.; Broxholme, John; Ahmadi, Kourosh R.; Surdulescu, Gabriela L.; Lowy, Ernesto; Gieger, Christian; Newton-Cheh, Chris; Perola, Markus; Soranzo, Nicole; Surakka, Ida; Lindgren, Cecilia M.; Ragoussis, Jiannis; Morris, Andrew P.

2011-01-01

275

Gene expression profiling and candidate gene re-sequencing identifies pathways and mutations important for malignant transformation caused by leukemogenic fusion genes  

PubMed Central

NUP98-HOXD13 (NHD13) and CALM-AF10 (CA10) are oncogenic fusion proteins produced by recurrent chromosomal translocations in patients with acute myeloid leukemia (AML). Transgenic mice that express these fusions develop AML with a long latency and incomplete penetrance, suggesting that collaborating genetic events are required for leukemic transformation. We employed genetic techniques to identify both pre-leukemic abnormalities in healthy transgenic mice as well as collaborating events leading to leukemic transformation. Candidate gene resequencing revealed that 6 out of 27 (22%) CA10 AMLs spontaneously acquired a Ras pathway mutation and 8 out of 27 (30%) acquired a Flt3 mutation. Two CA10 AMLs acquired a Flt3 internal-tandem duplication, demonstrating that these mutations can be acquired in murine as well as human AML. Gene expression profiles revealed a marked upregulation of Hox genes, particularly Hoxa5, Hoxa9, and Hoxa10 in both NHD13 and CA10 mice. Furthermore, mir196b, which is embedded within the Hoxa locus, was overexpressed in both CA10 and NHD13 samples. In contrast, the Hox cofactors Meis1 and Pbx3 were differentially expressed; Meis1 was increased in CA10 AMLs but not NHD13 AMLs, whereas Pbx3 was consistently increased in NHD13 but not CA10 AMLs. Silencing of Pbx3 in NHD13 cells led to decreased proliferation, increased apoptosis, and decreased colony formation in vitro, suggesting a previously unexpected role for Pbx3 in leukemic transformation.

Novak, Rachel L.; Harper, David P.; Caudell, David; Slape, Christopher; Beachy, Sarah H.; Aplan, Peter D.

2012-01-01

276

Microarray-Assisted Pathway Analysis Identifies MT1X & NF?B as Mediators of TCRP1-Associated Resistance to Cisplatin in Oral Squamous Cell Carcinoma  

PubMed Central

We recently reported that TCRP1, a novel multidrug-resistance associated human gene, can mediate cisplatin resistance in OSCC cells. However, the molecular mechanism underlying this role of TCRP1 remained to be elucidated. In this study, by using Human Toxicology and Drug Resistance Microarray, we identified 30 genes with significantly different expression levels between Tca/PYM and TCRP1 knockdown cell lines. Co-immunoprecipitation experiments and GST-pull down assays showed that metallothionein1X (MT1X) and Akt interact with TCRP1. siRNA-mediated knockdown of TCRP1 and MT1X was found to sensitize cells to cisplatin, leading to increased apoptosis and inhibition of cell proliferation. These functions of TCRP1 may be caused at least in part via activation of the PI3K/Akt/NF-?B signaling pathway. Taken together, our findings indicate that TCRP1 may be an important drug target for improvement of the treatment and survival of patients with oral squamous cell carcinoma.

Peng, Bo; Gu, Yixue; Xiong, Yan; Zheng, Guopei; He, Zhimin

2012-01-01

277

Integrating Bayesian variable selection with Modular Response Analysis to infer biochemical network topology  

PubMed Central

Background Recent advancements in genetics and proteomics have led to the acquisition of large quantitative data sets. However, the use of these data to reverse engineer biochemical networks has remained a challenging problem. Many methods have been proposed to infer biochemical network topologies from different types of biological data. Here, we focus on unraveling network topologies from steady state responses of biochemical networks to successive experimental perturbations. Results We propose a computational algorithm which combines a deterministic network inference method termed Modular Response Analysis (MRA) and a statistical model selection algorithm called Bayesian Variable Selection, to infer functional interactions in cellular signaling pathways and gene regulatory networks. It can be used to identify interactions among individual molecules involved in a biochemical pathway or reveal how different functional modules of a biological network interact with each other to exchange information. In cases where not all network components are known, our method reveals functional interactions which are not direct but correspond to the interaction routes through unknown elements. Using computer simulated perturbation responses of signaling pathways and gene regulatory networks from the DREAM challenge, we demonstrate that the proposed method is robust against noise and scalable to large networks. We also show that our method can infer network topologies using incomplete perturbation datasets. Consequently, we have used this algorithm to explore the ERBB regulated G1/S transition pathway in certain breast cancer cells to understand the molecular mechanisms which cause these cells to become drug resistant. The algorithm successfully inferred many well characterized interactions of this pathway by analyzing experimentally obtained perturbation data. Additionally, it identified some molecular interactions which promote drug resistance in breast cancer cells. Conclusions The proposed algorithm provides a robust, scalable and cost effective solution for inferring network topologies from biological data. It can potentially be applied to explore novel pathways which play important roles in life threatening disease like cancer.

2013-01-01

278

Stability of open pathways  

PubMed Central

We consider the steady state of an open biochemical pathway, with controlled flow. Previously we have shown that the steady state of open enzyme catalysed reactions may be unstable, which discourages the application of the quasi-steady-state approximation (QSSA) (IEE Proc. Syst. Biol. 153 (2006) 187). Here we examine basic open biochemical pathway structures, to see the stability of their steady states. Following De Leenheer et al. (J. Math. Chem. 41 (2007) 295), we employ the Gershgorin circle theorem, which elegantly assesses stability. This is the key tool for our analysis. Once we have the linear stability matrix laid out in a suitable form, the application of the method is straightforward. We find that in open biochemical pathways, simple chains, branches and loops always have stable steady states. We conclude that simple open pathways are stable.

Flach, Edward H.; Schnell, Santiago

2010-01-01

279

Biochemical transformations  

SciTech Connect

Maximum solar energy conversion efficiency for plant growth is about 5 to 6%, but the highest annual yields of biomass when translated into conversion efficiency generally are less than 3% for C/sub 4/ plants and 2% for C/sub 3/ plants. C/sub 3/ plant efficiency falls off drastically due to photorespiration under otherwise optimal conditions. C/sub 4/ metabolism is less efficient than C/sub 3/ under limiting light, but avoidance of photorespiration allows C/sub 4/ plants to have a higher efficiency under optimal growing conditions. Annual growth is severely limited by growing season length, water availability and other agronomic factors. Ideal energy conversion efficiencies might be approached under controlled environment conditions. Despite many advantages for the future, this is not presently economic in most cases. Plants will have to be developed to gain maximum advantage of such systems. Conversion of storage and structural carbohydrates to more valuable, energy-rich chemicals and fuels can be accomplished outside the plant or by biosynthesis in the plant. Use of plants such as Euphorbia species for production of ''hydrocarbons'' may be economically attractive in areas such as those with high solar energy and limited, but efficient rainfall. Economics depend on yield of desired products per unit of biomass. Examination of biosynthetic pathways from carbohydrates to ''hydrocarbons'' suggests possibilities for improving yield of desired products through plant breeding and plant cell tissue culture.

Bassham, J.A.

1980-07-01

280

A Genome-Wide RNAi Screen Identifies FOXO4 as a Metastasis-Suppressor through Counteracting PI3K/AKT Signal Pathway in Prostate Cancer  

PubMed Central

Activation of the PI3K/AKT signal pathway is a known driving force for the progression to castration-recurrent prostate cancer (CR-CaP), which constitutes the major lethal phenotype of CaP. Here, we identify using a genomic shRNA screen the PI3K/AKT-inactivating downstream target, FOXO4, as a potential CaP metastasis suppressor. FOXO4 protein levels inversely correlate with the invasive potential of a panel of human CaP cell lines, with decreased mRNA levels correlating with increased incidence of clinical metastasis. Knockdown (KD) of FOXO4 in human LNCaP cells causes increased invasion in vitro and lymph node (LN) metastasis in vivo without affecting indices of proliferation or apoptosis. Increased Matrigel invasiveness was found by KD of FOXO1 but not FOXO3. Comparison of differentially expressed genes affected by FOXO4-KD in LNCaP cells in culture, in primary tumors and in LN metastases identified a panel of upregulated genes, including PIP, CAMK2N1, PLA2G16 and PGC, which, if knocked down by siRNA, could decrease the increased invasiveness associated with FOXO4 deficiency. Although only some of these genes encode FOXO promoter binding sites, they are all RUNX2-inducible, and RUNX2 binding to the PIP promoter is increased in FOXO4-KD cells. Indeed, the forced expression of FOXO4 reversed the increased invasiveness of LNCaP/shFOXO4 cells; the forced expression of FOXO4 did not alter RUNX2 protein levels, yet it decreased RUNX2 binding to the PIP promoter, resulting in PIP downregulation. Finally, there was a correlation between FOXO4, but not FOXO1 or FOXO3, downregulation and decreased metastasis-free survival in human CaP patients. Our data strongly suggest that increased PI3K/AKT-mediated metastatic invasiveness in CaP is associated with FOXO4 loss, and that mechanisms to induce FOXO4 re-expression might suppress CaP metastatic aggressiveness.

Su, Bing; Gao, Lingqiu; Baranowski, Catherine; Gillard, Bryan; Wang, Jianmin; Ransom, Ryan; Ko, Hyun-Kyung; Gelman, Irwin H.

2014-01-01

281

Transcript Profiling Identifies Dynamic Gene Expression Patterns and an Important Role for Nrf2/Keap1 Pathway in the Developing Mouse Esophagus  

PubMed Central

Background and Aims Morphological changes during human and mouse esophageal development have been well characterized. However, changes at the molecular level in the course of esophageal morphogenesis remain unclear. This study aims to globally profile critical genes and signaling pathways during the development of mouse esophagus. By using microarray analysis this study also aims to determine how the Nrf2/Keap1 pathway regulates the morphogenesis of the esophageal epithelium. Methods Gene expression microarrays were used to survey gene expression in the esophagus at three critical phases: specification, metaplasia and maturation. The esophagi were isolated from wild-type, Nrf2?/?, Keap1?/?, or Nrf2?/?Keap1?/? embryos or young adult mice. Array data were statistically analyzed for differentially expressed genes and pathways. Histochemical and immunohistochemical staining were used to verify potential involvement of the Wnt pathway, Ppar?/? and the PI3K/Akt pathway in the development of esophageal epithelium. Results Dynamic gene expression patterns accompanied the morphological changes of the developing esophagus at critical phases. Particularly, the Nrf2/Keap1 pathway had a baseline activity in the metaplasia phase and was further activated in the maturation phase. The Wnt pathway was active early and became inactive later in the metaplasia phase. In addition, Keap1?/? mice showed increased expression of Nrf2 downstream targets and genes involved in keratinization. Microarray and immunostaining data also suggested that esophageal hyperkeratosis in the Keap1?/? mice was due to activation of Ppar?/? and the PI3K/Akt pathway. Conclusions Morphological changes of the esophageal epithelium are associated with dynamic changes in gene expression. Nrf2/Keap1 pathway activity is required for maturation of mouse esophageal epithelium.

Li, Haiyan; Hu, Yuhui; Tevebaugh, Whitney; Yamamoto, Masayuki; Que, Jianwen; Chen, Xiaoxin

2012-01-01

282

A new dynamical layout algorithm for complex biochemical reaction networks  

PubMed Central

Background To study complex biochemical reaction networks in living cells researchers more and more rely on databases and computational methods. In order to facilitate computational approaches, visualisation techniques are highly important. Biochemical reaction networks, e.g. metabolic pathways are often depicted as graphs and these graphs should be drawn dynamically to provide flexibility in the context of different data. Conventional layout algorithms are not sufficient for every kind of pathway in biochemical research. This is mainly due to certain conventions to which biochemists/biologists are used to and which are not in accordance to conventional layout algorithms. A number of approaches has been developed to improve this situation. Some of these are used in the context of biochemical databases and make more or less use of the information in these databases to aid the layout process. However, visualisation becomes also more and more important in modelling and simulation tools which mostly do not offer additional connections to databases. Therefore, layout algorithms used in these tools have to work independently of any databases. In addition, all of the existing algorithms face some limitations with respect to the number of edge crossings when it comes to larger biochemical systems due to the interconnectivity of these. Last but not least, in some cases, biochemical conventions are not met properly. Results Out of these reasons we have developed a new algorithm which tackles these problems by reducing the number of edge crossings in complex systems, taking further biological conventions into account to identify and visualise cycles. Furthermore the algorithm is independent from database information in order to be easily adopted in any application. It can also be tested as part of the SimWiz package (free to download for academic users at [1]). Conclusion The new algorithm reduces the complexity of pathways, as well as edge crossings and edge length in the resulting graphical representation. It also considers existing and further biological conventions to create a drawing most biochemists are familiar with. A lot of examples can be found on [2].

Wegner, Katja; Kummer, Ursula

2005-01-01

283

Alterations in metabolic pathways and networks in Alzheimer's disease.  

PubMed

The pathogenic mechanisms of Alzheimer's disease (AD) remain largely unknown and clinical trials have not demonstrated significant benefit. Biochemical characterization of AD and its prodromal phase may provide new diagnostic and therapeutic insights. We used targeted metabolomics platform to profile cerebrospinal fluid (CSF) from AD (n=40), mild cognitive impairment (MCI, n=36) and control (n=38) subjects; univariate and multivariate analyses to define between-group differences; and partial least square-discriminant analysis models to classify diagnostic groups using CSF metabolomic profiles. A partial correlation network was built to link metabolic markers, protein markers and disease severity. AD subjects had elevated methionine (MET), 5-hydroxyindoleacetic acid (5-HIAA), vanillylmandelic acid, xanthosine and glutathione versus controls. MCI subjects had elevated 5-HIAA, MET, hypoxanthine and other metabolites versus controls. Metabolite ratios revealed changes within tryptophan, MET and purine pathways. Initial pathway analyses identified steps in several pathways that appear altered in AD and MCI. A partial correlation network showed total tau most directly related to norepinephrine and purine pathways; amyloid-? (Ab42) was related directly to an unidentified metabolite and indirectly to 5-HIAA and MET. These findings indicate that MCI and AD are associated with an overlapping pattern of perturbations in tryptophan, tyrosine, MET and purine pathways, and suggest that profound biochemical alterations are linked to abnormal Ab42 and tau metabolism. Metabolomics provides powerful tools to map interlinked biochemical pathway perturbations and study AD as a disease of network failure. PMID:23571809

Kaddurah-Daouk, R; Zhu, H; Sharma, S; Bogdanov, M; Rozen, S G; Matson, W; Oki, N O; Motsinger-Reif, A A; Churchill, E; Lei, Z; Appleby, D; Kling, M A; Trojanowski, J Q; Doraiswamy, P M; Arnold, S E

2013-01-01

284

Biochemical Engineering of Natural Product Biosynthesis Pathways  

Microsoft Academic Search

Metabolic engineering of natural products is a science that has been built on the goals of traditional strain improvement with the availability of modern molecular biological technologies. In the past 15 years, the state of the art in metabolic engineering of natural products has advanced from the first proof-of-principle experiment based on minimal known genetics to a commonplace event using

William R. Strohl

2001-01-01

285

Pathway Projector: Web-Based Zoomable Pathway Browser Using KEGG Atlas and Google Maps API  

PubMed Central

Background Biochemical pathways provide an essential context for understanding comprehensive experimental data and the systematic workings of a cell. Therefore, the availability of online pathway browsers will facilitate post-genomic research, just as genome browsers have contributed to genomics. Many pathway maps have been provided online as part of public pathway databases. Most of these maps, however, function as the gateway interface to a specific database, and the comprehensiveness of their represented entities, data mapping capabilities, and user interfaces are not always sufficient for generic usage. Methodology/Principal Findings We have identified five central requirements for a pathway browser: (1) availability of large integrated maps showing genes, enzymes, and metabolites; (2) comprehensive search features and data access; (3) data mapping for transcriptomic, proteomic, and metabolomic experiments, as well as the ability to edit and annotate pathway maps; (4) easy exchange of pathway data; and (5) intuitive user experience without the requirement for installation and regular maintenance. According to these requirements, we have evaluated existing pathway databases and tools and implemented a web-based pathway browser named Pathway Projector as a solution. Conclusions/Significance Pathway Projector provides integrated pathway maps that are based upon the KEGG Atlas, with the addition of nodes for genes and enzymes, and is implemented as a scalable, zoomable map utilizing the Google Maps API. Users can search pathway-related data using keywords, molecular weights, nucleotide sequences, and amino acid sequences, or as possible routes between compounds. In addition, experimental data from transcriptomic, proteomic, and metabolomic analyses can be readily mapped. Pathway Projector is freely available for academic users at http://www.g-language.org/PathwayProjector/.

Kono, Nobuaki; Arakawa, Kazuharu; Ogawa, Ryu; Kido, Nobuhiro; Oshita, Kazuki; Ikegami, Keita; Tamaki, Satoshi; Tomita, Masaru

2009-01-01

286

Soya bean G? proteins with distinct biochemical properties exhibit differential ability to complement Saccharomyces cerevisiae gpa1 mutant.  

PubMed

Signalling pathways mediated by heterotrimeric G-proteins are common to all eukaryotes. Plants have a limited number of each of the G-protein subunits, with the most elaborate G-protein network discovered so far in soya bean (Glycine max, also known as soybean) which has four G?, four G? and ten G? proteins. Biochemical characterization of G? proteins from plants suggests significant variation in their properties compared with the well-characterized non-plant proteins. Furthermore, the four soya bean G? (GmG?) proteins exhibit distinct biochemical activities among themselves, but the extent to which such biochemical differences contribute to their in vivo function is also not known. We used the yeast gpa1 mutant which displays constitutive signalling and growth arrest in the pheromone-response pathway as an in vivo model to evaluate the effect of distinct biochemical activities of GmG? proteins. We showed that specific GmG? proteins can be activated during pheromone-dependent receptor-mediated signalling in yeast and they display different strengths towards complementation of yeast gpa1 phenotypes. We also identified amino acids that are responsible for differential complementation abilities of specific G? proteins. These data establish that specific plant G? proteins are functional in the receptor-mediated pheromone-response pathway in yeast and that the subtle biochemical differences in their activity are physiologically relevant. PMID:24694027

Roy Choudhury, Swarup; Wang, Yuqi; Pandey, Sona

2014-07-01

287

BIOSYNTHETIC PATHWAYS: Biosynthesis Meets Bioinformatics  

NSDL National Science Digital Library

Access to the article is free, however registration and sign-in are required. In his Perspective, Cane discusses the increasing importance of the study of biosynthetic pathways in the discovery of biochemical reactions and pathways. Two recent papers (Rohdich et al. and Khaleeli et al.) highlight the increasing role of molecular biology and genomics in the study of biosynthetic pathways.

David E. Cane (Brown University;Department of Chemistry)

2000-02-04

288

A genetic screen for novel components of the Ras/Mitogen-activated protein kinase signaling pathway that interact with the yan gene of Drosophila identifies split ends, a new RNA recognition motif-containing protein.  

PubMed Central

The receptor tyrosine kinase (RTK) signaling pathway is used reiteratively during the development of all multicellular organisms. While the core RTK/Ras/MAPK signaling cassette has been studied extensively, little is known about the nature of the downstream targets of the pathway or how these effectors regulate the specificity of cellular responses. Drosophila yan is one of a few downstream components identified to date, functioning as an antagonist of the RTK/Ras/MAPK pathway. Previously, we have shown that ectopic expression of a constitutively active protein (yan(ACT)) inhibits the differentiation of multiple cell types. In an effort to identify new genes functioning downstream in the Ras/MAPK/yan pathway, we have performed a genetic screen to isolate dominant modifiers of the rough eye phenotype associated with eye-specific expression of yan(ACT). Approximately 190,000 mutagenized flies were screened, and 260 enhancers and 90 suppressors were obtained. Among the previously known genes we recovered are four RTK pathway components, rolled (MAPK), son-of-sevenless, Star, and pointed, and two genes, eyes absent and string, that have not been implicated previously in RTK signaling events. We also isolated mutations in five previously uncharacterized genes, one of which, split ends, we have characterized molecularly and have shown to encode a member of the RRM family of RNA-binding proteins.

Rebay, I; Chen, F; Hsiao, F; Kolodziej, P A; Kuang, B H; Laverty, T; Suh, C; Voas, M; Williams, A; Rubin, G M

2000-01-01

289

Basic Sciences - Biochemical Pathology  

Cancer.gov

Cell-cell and cell-matrix interactions are important regulators of normal cell growth and differentiation and play essential roles in pathological conditions such as tumor metastasis and infection by pathogens. We are defining functions of adhesion molecules, their cell surface and matrix receptors, and the signal transduction pathways that regulate their activities in specific diseases. These studies will identify new molecular targets and could provide a basis for designing novel therapeutic agents.

290

Activation and Inhibition of Cellular Calcium and Tyrosine Kinase Signaling Pathways Identify Targets of the HBx Protein Involved in Hepatitis B Virus Replication  

Microsoft Academic Search

Human hepatitis B virus (HBV) HBx protein is a multifunctional protein that activates cellular signaling pathways and is thought to be essential for viral infection. Woodchuck HBV mutants that lack HBx are unable to replicate in vivo or are severely impaired. HBV replication in HepG2 cells, a human hepatoblastoma cell line, is stimulated 5- to 10-fold by HBx protein. We

Michael J. Bouchard; Robyn J. Puro; Lihua Wang; Robert J. Schneider

2003-01-01

291

Epistasis analysis between homologous recombination genes in Saccharomyces cerevisiae identifies multiple repair pathways for Sgs1, Mus81-Mms4 and RNase H2.  

PubMed

The DNA repair genes SGS1 and MUS81 of Saccharomyces cerevisiae are thought to control alternative pathways for the repair of toxic recombination intermediates based on the fact that sgs1? mus81? synthetic lethality is suppressed in the absence of homologous recombination (HR). Although these genes appear to functionally overlap in yeast and other model systems, the specific pathways controlled by SGS1 and MUS81 are poorly defined. Epistasis analyses based on DNA damage sensitivity previously indicated that SGS1 functioned primarily downstream of RAD51, and that MUS81 was independent of RAD51. To further define these genetic pathways, we carried out a systematic epistasis analysis between the RAD52-epistasis group genes and SGS1, MUS81, and RNH202, which encodes a subunit of RNase H2. Based on synthetic-fitness interactions and DNA damage sensitivities, we find that RAD52 is epistatic to MUS81 but not SGS1. In contrast, RAD54, RAD55 and RAD57 are epistatic to SGS1, MUS81 and RNH202. As expected, SHU2 is epistatic to SGS1, while both SHU1 and SHU2 are epistatic to MUS81. Importantly, loss of any RNase H2 subunit on its own resulted in increased recombination using a simple marker-excision assay. RNase H2 is thus needed to maintain genome stability consistent with the sgs1? rnh202? synthetic fitness defect. We conclude that SGS1 and MUS81 act in parallel pathways downstream of RAD51 and RAD52, respectively. The data further indicate these pathways share common components and display complex interactions. PMID:21741981

Ii, Miki; Ii, Tatsuya; Mironova, Larisa I; Brill, Steven J

2011-09-01

292

Epistasis analysis between homologous recombination genes in Saccharomyces cerevisiae identifies multiple repair pathways for Sgs1, Mus81-Mms4 and RNase H2  

PubMed Central

The DNA repair genes SGS1 and MUS81 of Saccharomyces cerevisiae are thought to control alternative pathways for the repair of toxic recombination intermediates based on the fact that sgs1? mus81? synthetic lethality is suppressed in the absence of homologous recombination (HR). Although these genes appear to functionally overlap in yeast and other model systems, the specific pathways controlled by SGS1 and MUS81 are poorly defined. Epistasis analyses based on DNA damage sensitivity previously indicated that SGS1 functioned primarily downstream of RAD51, and that MUS81 was independent of RAD51. To further define these genetic pathways, we carried out a systematic epistasis analysis between the RAD52-epistasis group genes and SGS1, MUS81, and RNH202, which encodes a subunit of RNase H2. Based on synthetic-fitness interactions and DNA damage sensitivities, we find that RAD52 is epistatic to MUS81 but not SGS1. In contrast, RAD54, RAD55 and RAD57 are epistatic to SGS1, MUS81 and RNH202. As expected, SHU2 is epistatic to SGS1, while both SHU1 and SHU2 are epistatic to MUS81. Importantly, loss of any RNase H2 subunit on its own resulted in increased recombination using a simple marker-excision assay. RNase H2 is thus needed to maintain genome stability consistent with the sgs1? rnh202? synthetic fitness defect. We conclude that SGS1 and MUS81 act in parallel pathways downstream of RAD51 and RAD52, respectively. The data further indicate these pathways share common components and display complex interactions.

Ii, Miki; Ii, Tatsuya; Mironova, Larisa I.; Brill, Steven J.

2011-01-01

293

Method for identifying subsurface fluid migration and drainage pathways in and among oil and gas reservoirs using 3-D and 4-D seismic imaging  

Microsoft Academic Search

The invention utilizes 3-D and 4-D seismic surveys as a means of deriving information useful in petroleum exploration and reservoir management. The methods use both single seismic surveys (3-D) and multiple seismic surveys separated in time (4-D) of a region of interest to determine large scale migration pathways within sedimentary basins, and fine scale drainage structure and oil-water-gas regions within

R. N. Anderson; A. Boulanger; E. P. Bagdonas; L. Xu; W. He

1996-01-01

294

Integrated Analyses of Genome-Wide DNA Occupancy and Expression Profiling Identify Key Genes and Pathways Involved in Cellular Transformation by a Marek's Disease Virus Oncoprotein, Meq  

PubMed Central

Marek's disease (MD) is an economically significant disease in chickens that is caused by the highly oncogenic Marek's disease virus (MDV). A major unanswered question is the mechanism of MDV-induced tumor formation. Meq, a bZIP transcription factor discovered in the 1990s, is critically involved in viral oncogenicity, but only a few of its host target genes have been described, impeding our understanding of MDV-induced tumorigenesis. Using chromatin immunoprecipitation-sequencing (ChIP-seq) and microarray analysis, a high-confidence list of Meq binding sites in the chicken genome and a global transcriptome of Meq-responsive genes were generated. Meq binding sites were found to be enriched in the promoter regions of upregulated genes but not in those of downregulated genes. ChIP-seq was also performed for c-Jun, a known heterodimeric partner of Meq. The close location of binding sites of Meq and c-Jun was noted, suggesting cooperativity between these two factors in modulating transcription. Pathway analysis indicated that Meq transcriptionally regulates many genes that are part of several signaling pathways including the extracellular signal-regulated kinase /mitogen-activated protein kinase (ERK/MAPK), Jak-STAT, and ErbB pathways, which are critical for oncogenesis and/or include signaling mediators involved in apoptosis. Meq activates oncogenic signaling cascades by transcriptionally activating major kinases in the ERK/MAPK pathway and simultaneously repressing phosphatases, as verified using inhibitors of MEK and ERK1/2 in a cell proliferation assay. This study provides significant insights into the mechanistic basis of Meq-dependent cell transformation.

Subramaniam, Sugalesini; Johnston, John; Preeyanon, Likit; Brown, C. Titus; Kung, Hsing-Jien

2013-01-01

295

Pathways of the North Pacific Intermediate Water identified through the tangent linear and adjoint models of an ocean general circulation model  

NASA Astrophysics Data System (ADS)

This study develops a strategy for tracing a target water mass, and applies it to analyzing the pathway of the North Pacific Intermediate Water (NPIW) from the subarctic gyre to the northwestern part of the subtropical gyre south of Japan in a simulation of an ocean general circulation model. This strategy estimates the pathway of the water mass that travels from an origin to a destination area during a specific period using a conservation property concerning tangent linear and adjoint models. In our analysis, a large fraction of the low salinity origin water mass of NPIW initially comes from the Okhotsk or Bering Sea, flows through the southeastern side of the Kuril Islands, and is advected to the Mixed Water Region (MWR) by the Oyashio current. It then enters the Kuroshio Extension (KE) at the first KE ridge, and is advected eastward by the KE current. However, it deviates southward from the KE axis around 158°E over the Shatsky Rise, or around 170°E on the western side of the Emperor Seamount Chain, and enters the subtropical gyre. It is finally transported westward by the recirculation flow. This pathway corresponds well to the shortcut route of NPIW from MWR to the region south of Japan inferred from analysis of the long-term freshening trend of NPIW observation. Copyright 2013 John Wiley & Sons, Ltd.

Fujii, Yosuke; Nakano, Toshiya; Usui, Norihisa; Matsumoto, Satoshi; Tsujino, Hiroyuki; Kamachi, Masafumi

2013-04-01

296

A screen for genes involved in the anaphase proteolytic pathway identifies tsm1(+), a novel Schizosaccharomyces pombe gene important for microtubule integrity.  

PubMed Central

The growth of several mitotic mutants of Schizosaccharomyces pombe, including nuc2-663, is inhibited by the protease inhibitor N-Tosyl-L-Phenylalanine Chloromethyl Ketone (TPCK). Because nuc2(+) encodes a presumptive component of the Anaphase Promoting Complex, which is required for the ubiquitin-dependent proteolysis of certain proteins during exit from mitosis, we have used sensitivity to TPCK as a criterion by which to search for novel S. pombe mutants defective in the anaphase-promoting pathway. In a genetic screen for temperature-sensitive mitotic mutants that were also sensitive to TPCK at a permissive temperature, we isolated three tsm (TPCK-sensitive mitotic) strains. Two of these are alleles of cut1(+), but tsm1-512 maps to a novel genetic location. The tsm1-512 mutation leads to delayed nuclear division at restrictive temperatures, apparently as a result of an impaired ability to form a metaphase spindle. After shift of early G2 cells to 36 degrees, tsm1-512 arrests transiently in the second mitotic division and then exits mitosis, as judged by spindle elongation and septation. The chromosomes, however, often fail to segregate properly. Genetic interactions between tsm1-512 and components of the anaphase proteolytic pathway suggest a functional involvement of the Tsm1 protein in this pathway.

Grishchuk, E L; Howe, J L; McIntosh, J R

1998-01-01

297

Large-Scale Comparative Phenotypic and Genomic Analyses Reveal Ecological Preferences of Shewanella Species and Identify Metabolic Pathways Conserved at the Genus Level ? †  

PubMed Central

The use of comparative genomics for the study of different microbiological species has increased substantially as sequence technologies become more affordable. However, efforts to fully link a genotype to its phenotype remain limited to the development of one mutant at a time. In this study, we provided a high-throughput alternative to this limiting step by coupling comparative genomics to the use of phenotype arrays for five sequenced Shewanella strains. Positive phenotypes were obtained for 441 nutrients (C, N, P, and S sources), with N-based compounds being the most utilized for all strains. Many genes and pathways predicted by genome analyses were confirmed with the comparative phenotype assay, and three degradation pathways believed to be missing in Shewanella were confirmed as missing. A number of previously unknown gene products were predicted to be parts of pathways or to have a function, expanding the number of gene targets for future genetic analyses. Ecologically, the comparative high-throughput phenotype analysis provided insights into niche specialization among the five different strains. For example, Shewanella amazonensis strain SB2B, isolated from the Amazon River delta, was capable of utilizing 60 C compounds, whereas Shewanella sp. strain W3-18-1, isolated from deep marine sediment, utilized only 25 of them. In spite of the large number of nutrient sources yielding positive results, our study indicated that except for the N sources, they were not sufficiently informative to predict growth phenotypes from increasing evolutionary distances. Our results indicate the importance of phenotypic evaluation for confirming genome predictions. This strategy will accelerate the functional discovery of genes and provide an ecological framework for microbial genome sequencing projects.

Rodrigues, Jorge L. M.; Serres, Margrethe H.; Tiedje, James M.

2011-01-01

298

Large-scale comparative phenotypic and genomic analyses reveal ecological preferences of shewanella species and identify metabolic pathways conserved at the genus level.  

PubMed

The use of comparative genomics for the study of different microbiological species has increased substantially as sequence technologies become more affordable. However, efforts to fully link a genotype to its phenotype remain limited to the development of one mutant at a time. In this study, we provided a high-throughput alternative to this limiting step by coupling comparative genomics to the use of phenotype arrays for five sequenced Shewanella strains. Positive phenotypes were obtained for 441 nutrients (C, N, P, and S sources), with N-based compounds being the most utilized for all strains. Many genes and pathways predicted by genome analyses were confirmed with the comparative phenotype assay, and three degradation pathways believed to be missing in Shewanella were confirmed as missing. A number of previously unknown gene products were predicted to be parts of pathways or to have a function, expanding the number of gene targets for future genetic analyses. Ecologically, the comparative high-throughput phenotype analysis provided insights into niche specialization among the five different strains. For example, Shewanella amazonensis strain SB2B, isolated from the Amazon River delta, was capable of utilizing 60 C compounds, whereas Shewanella sp. strain W3-18-1, isolated from deep marine sediment, utilized only 25 of them. In spite of the large number of nutrient sources yielding positive results, our study indicated that except for the N sources, they were not sufficiently informative to predict growth phenotypes from increasing evolutionary distances. Our results indicate the importance of phenotypic evaluation for confirming genome predictions. This strategy will accelerate the functional discovery of genes and provide an ecological framework for microbial genome sequencing projects. PMID:21642407

Rodrigues, Jorge L M; Serres, Margrethe H; Tiedje, James M

2011-08-01

299

Combined miRNA and mRNA Signature Identifies Key Molecular Players and Pathways Involved in Chikungunya Virus Infection in Human Cells  

PubMed Central

Since its discovery, Chikungunya fever caused by a virus (CHIKV) has ravaged most of Africa and Southeast Asia. Despite there being more than a million reported cases in India alone and the seriousness of the disease in the chronic phase, a clear understanding of the disease pathogenesis and host response remains elusive. Here, we use microarray technology and quantitative PCR method to establish the complete miRNA, snoRNA and mRNA signature of host response upon CHIKV infection in human cell line infection model, HEK293T. The results were further validated in human primary cells (dermal fibroblasts). miRNA expression profiling revealed regulation of 152 miRNAs post CHIKV infection. An interesting overlap in miRNA signature was seen majorly with HCV, HPV and HIV1 virus. The microarray data further validated by qRT-PCR revealed induction of miR-744, miR-638, miR-503 and others among the top upregulated miRNAs. Notably, we found induction of snoRNAs belonging to C/D cluster including close paralogs of U3, U44, U76 and U78 snoRNAs. Genes were found to be differentially expressed along 3 major pathways; TGF-?, endocytosis and the cell cycle pathways. qRT-PCR data confirmed strong induction of TGF-? (SMAD6, JUN, SKIL) and endocytosis pathway (CXCR4, HSPA8, ADRB1) genes while downregulation of cell cycle genes (CDC27 and CDC23). Interestingly, use of TGF-? inhibitor, SB-431542, increased CHIKV mediated cell death. Overall, this study aims at providing the first complete transcriptome signature of host response upon CHIKV infection to aid identification of possible biomarkers and therapeutic targets.

Saxena, Tanvi; Tandon, Bhavna; Sharma, Shivani; Chameettachal, Shibu; Ray, Pratima; Ray, Alok R.; Kulshreshtha, Ritu

2013-01-01

300

Combined miRNA and mRNA signature identifies key molecular players and pathways involved in chikungunya virus infection in human cells.  

PubMed

Since its discovery, Chikungunya fever caused by a virus (CHIKV) has ravaged most of Africa and Southeast Asia. Despite there being more than a million reported cases in India alone and the seriousness of the disease in the chronic phase, a clear understanding of the disease pathogenesis and host response remains elusive. Here, we use microarray technology and quantitative PCR method to establish the complete miRNA, snoRNA and mRNA signature of host response upon CHIKV infection in human cell line infection model, HEK293T. The results were further validated in human primary cells (dermal fibroblasts). miRNA expression profiling revealed regulation of 152 miRNAs post CHIKV infection. An interesting overlap in miRNA signature was seen majorly with HCV, HPV and HIV1 virus. The microarray data further validated by qRT-PCR revealed induction of miR-744, miR-638, miR-503 and others among the top upregulated miRNAs. Notably, we found induction of snoRNAs belonging to C/D cluster including close paralogs of U3, U44, U76 and U78 snoRNAs. Genes were found to be differentially expressed along 3 major pathways; TGF-?, endocytosis and the cell cycle pathways. qRT-PCR data confirmed strong induction of TGF-? (SMAD6, JUN, SKIL) and endocytosis pathway (CXCR4, HSPA8, ADRB1) genes while downregulation of cell cycle genes (CDC27 and CDC23). Interestingly, use of TGF-? inhibitor, SB-431542, increased CHIKV mediated cell death. Overall, this study aims at providing the first complete transcriptome signature of host response upon CHIKV infection to aid identification of possible biomarkers and therapeutic targets. PMID:24278205

Saxena, Tanvi; Tandon, Bhavna; Sharma, Shivani; Chameettachal, Shibu; Ray, Pratima; Ray, Alok R; Kulshreshtha, Ritu

2013-01-01

301

1+1 = 3: a fusion of 2 enzymes in the methionine salvage pathway of Tetrahymena thermophila creates a trifunctional enzyme that catalyzes 3 steps in the pathway.  

PubMed

The methionine salvage pathway is responsible for regenerating methionine from its derivative, methylthioadenosine. The complete set of enzymes of the methionine pathway has been previously described in bacteria. Despite its importance, the pathway has only been fully described in one eukaryotic organism, yeast. Here we use a computational approach to identify the enzymes of the methionine salvage pathway in another eukaryote, Tetrahymena thermophila. In this organism, the pathway has two fused genes, MTNAK and MTNBD. Each of these fusions involves two different genes whose products catalyze two different single steps of the pathway in other organisms. One of the fusion proteins, mtnBD, is formed by enzymes that catalyze non-consecutive steps in the pathway, mtnB and mtnD. Interestingly the gene that codes for the intervening enzyme in the pathway, mtnC, is missing from the genome of Tetrahymena. We used complementation tests in yeast to show that the fusion of mtnB and mtnD from Tetrahymena is able to do in one step what yeast does in three, since it can rescue yeast knockouts of mtnB, mtnC, or mtnD. Fusion genes have proved to be very useful in aiding phylogenetic reconstructions and in the functional characterization of genes. Our results highlight another characteristic of fusion proteins, namely that these proteins can serve as biochemical shortcuts, allowing organisms to completely bypass steps in biochemical pathways. PMID:19851454

Salim, Hannah M W; Negritto, Maria Cristina; Cavalcanti, Andre R O

2009-10-01

302

1+1 = 3: A Fusion of 2 Enzymes in the Methionine Salvage Pathway of Tetrahymena thermophila Creates a Trifunctional Enzyme That Catalyzes 3 Steps in the Pathway  

PubMed Central

The methionine salvage pathway is responsible for regenerating methionine from its derivative, methylthioadenosine. The complete set of enzymes of the methionine pathway has been previously described in bacteria. Despite its importance, the pathway has only been fully described in one eukaryotic organism, yeast. Here we use a computational approach to identify the enzymes of the methionine salvage pathway in another eukaryote, Tetrahymena thermophila. In this organism, the pathway has two fused genes, MTNAK and MTNBD. Each of these fusions involves two different genes whose products catalyze two different single steps of the pathway in other organisms. One of the fusion proteins, mtnBD, is formed by enzymes that catalyze non-consecutive steps in the pathway, mtnB and mtnD. Interestingly the gene that codes for the intervening enzyme in the pathway, mtnC, is missing from the genome of Tetrahymena. We used complementation tests in yeast to show that the fusion of mtnB and mtnD from Tetrahymena is able to do in one step what yeast does in three, since it can rescue yeast knockouts of mtnB, mtnC, or mtnD. Fusion genes have proved to be very useful in aiding phylogenetic reconstructions and in the functional characterization of genes. Our results highlight another characteristic of fusion proteins, namely that these proteins can serve as biochemical shortcuts, allowing organisms to completely bypass steps in biochemical pathways.

Salim, Hannah M. W.; Negritto, Maria Cristina; Cavalcanti, Andre R. O.

2009-01-01

303

The Leaf Epidermome of Catharanthus roseus Reveals Its Biochemical Specialization[W][OA  

PubMed Central

Catharanthus roseus is the sole commercial source of the monoterpenoid indole alkaloids (MIAs), vindoline and catharanthine, components of the commercially important anticancer dimers, vinblastine and vincristine. Carborundum abrasion technique was used to extract leaf epidermis–enriched mRNA, thus sampling the epidermome, or complement, of proteins expressed in the leaf epidermis. Random sequencing of the derived cDNA library established 3655 unique ESTs, composed of 1142 clusters and 2513 singletons. Virtually all known MIA pathway genes were found in this remarkable set of ESTs, while only four known genes were found in the publicly available Catharanthus EST data set. Several novel MIA pathway candidate genes were identified, as demonstrated by the cloning and functional characterization of loganic acid O-methyltransferase involved in secologanin biosynthesis. The pathways for triterpene biosynthesis were also identified, and metabolite analysis showed that oleanane-type triterpenes were localized exclusively to the cuticular wax layer. The pathways for flavonoid and very-long-chain fatty acid biosynthesis were also located in this cell type. The results illuminate the biochemical specialization of Catharanthus leaf epidermis for the production of multiple classes of metabolites. The value and versatility of this EST data set for biochemical and biological analysis of leaf epidermal cells is also discussed.

Murata, Jun; Roepke, Jonathon; Gordon, Heather; De Luca, Vincenzo

2008-01-01

304

Robust simplifications of multiscale biochemical networks  

PubMed Central

Background Cellular processes such as metabolism, decision making in development and differentiation, signalling, etc., can be modeled as large networks of biochemical reactions. In order to understand the functioning of these systems, there is a strong need for general model reduction techniques allowing to simplify models without loosing their main properties. In systems biology we also need to compare models or to couple them as parts of larger models. In these situations reduction to a common level of complexity is needed. Results We propose a systematic treatment of model reduction of multiscale biochemical networks. First, we consider linear kinetic models, which appear as "pseudo-monomolecular" subsystems of multiscale nonlinear reaction networks. For such linear models, we propose a reduction algorithm which is based on a generalized theory of the limiting step that we have developed in [1]. Second, for non-linear systems we develop an algorithm based on dominant solutions of quasi-stationarity equations. For oscillating systems, quasi-stationarity and averaging are combined to eliminate time scales much faster and much slower than the period of the oscillations. In all cases, we obtain robust simplifications and also identify the critical parameters of the model. The methods are demonstrated for simple examples and for a more complex model of NF-?B pathway. Conclusion Our approach allows critical parameter identification and produces hierarchies of models. Hierarchical modeling is important in "middle-out" approaches when there is need to zoom in and out several levels of complexity. Critical parameter identification is an important issue in systems biology with potential applications to biological control and therapeutics. Our approach also deals naturally with the presence of multiple time scales, which is a general property of systems biology models.

Radulescu, Ovidiu; Gorban, Alexander N; Zinovyev, Andrei; Lilienbaum, Alain

2008-01-01

305

Manganese Catalysts for C-H activation: An Experimental/Theoretical Study Identifies the Stereoelectronic Factor that Controls the Switch between Hydroxylation and Desaturation Pathways  

PubMed Central

We describe competitive C–H activation chemistry of two types, desaturation and hydroxylation, using synthetic manganese catalysts with several substrates. 9,10-dihydrophenanthrene (DHP) gives the highest desaturation activity, the final products being phenanthrene (P1) and phenanthrene-9,10-oxide (P3), the latter being thought to arise from epoxidation of some of the phenanthrene. The hydroxylase pathway also occurs as suggested by the presence of the dione product, phenanthrene-9,10-dione (P2), thought to arise from further oxidation of hydroxylation intermediate 9-hydroxy-9,10-dihydrophenanthrene. The experimental work together with the DFT calculations shows that the postulated Mn oxo active species, [Mn(O)(tpp)(Cl)] (tpp = tetraphenyl porphyrin), can promote the oxidation of dihydrophenanthrene by either desaturation or hydroxylation pathways. The calculations show that these two competing reactions have a common initial step – radical H abstraction from one of the DHP sp3 C–H bonds. The resulting Mn hydroxo intermediate is capable of promoting not only OH rebound (hydroxylation) but also a second H abstraction adjacent to the first (desaturation). Like the active MnV=O species, this MnIV-OH species also has radical character on oxygen and can thus give H abstraction. Both steps have very low and therefore very similar energy barriers, leading to a product mixture. Since the radical character of the catalyst is located on the oxygen p orbital perpendicular to the MnIV-OH plane, the orientation of the organic radical with respect to this plane determines which reaction, desaturation or hydroxylation, will occur. Stereoelectronic factors such as the rotational orientation of the OH in the enzyme active site is thus likely to constitute the switch between hydroxylation and desaturation behavior.

Hull, Jonathan F.; Balcells, David; Sauer, Effiette L. O.; Raynaud, Christophe; Brudvig, Gary W.; Crabtree, Robert H.; Eisenstein, Odile

2010-01-01

306

Arabidopsis Ethylene Signaling Pathway  

NSDL National Science Digital Library

In plants, ethylene gas functions as a potent endogenous growth regulator. In the model system Arabidopsis thaliana, the molecular mechanisms that underlie perception and transduction of the ethylene signal to the nucleus, where the transcription of hundreds of genes is altered, are being elucidated. In the current view, ethylene is sensed by a family of five receptors that show similarity to the bacterial two-component histidine kinases, and in plants function as negative regulators of the pathway. Binding of the ethylene gas turns off the receptors, resulting in the inactivation of another negative regulator of ethylene signaling, CTR1, a Raf-like protein kinase that directly interacts with the receptors. EIN2, a protein of unknown biochemical activity that functions as a positive regulator of the pathway, acts downstream of CTR. Derepression of EIN2 by ethylene upon disabling of the receptors and CTR1 leads to the activation of EIN3 and EIN3-like transcription factors. In the absence of ethylene, the levels of EIN3 protein are extremely low because of the function of two F-box-containing proteins, EBF1 and EBF2, that target EIN3 for proteosome-mediated degradation. In the presence of ethylene, the EIN3 protein accumulates in the nucleus and initiates a transcriptional cascade, resulting in the activation and repression of hundreds of genes. To date, the only empirically demonstrated direct target of EIN3 is the APETALA2 (AP2)-domain–containing transcription factor gene ERF1. The coregulation of ERF1 by another plant hormone, jasmonic acid, illustrates how a transcriptional cascade could be utilized in a combinatorial fashion to generate a large diversity of responses using a limited number of input signals. As new components and points of intersection with other pathways are identified, the Connections Map will be updated.

Anna N. Stepanova (North Carolina State University;Department of Genetics REV); Jose M. Alonso (North Carolina State University;Department of Genetics REV)

2005-03-22

307

An SNP-guided microRNA map of fifteen common human disorders identifies a consensus disease phenocode aiming at principal components of the nuclear import pathway.  

PubMed

Recent large-scale genome-wide association (GWA) studies of SNP variations captured many thousands individual genetic profiles of H. sapiens and facilitated identification of significant genetic traits which are highly likely to influence the pathogenesis of several major human diseases. Here we apply the integrative genomics principles to interrogate relationships between structural features and gene expression patterns of disease-linked SNPs, microRNAs and mRNAs of protein-coding genes in association to phenotypes of 15 major human disorders, namely bipolar disease (BD); rheumatoid arthritis (RA); coronary artery disease (CAD); Crohn's disease (CD); type 1 diabetes (T1D); type 2 diabetes (T2D); hypertension (HT); ankylosing spondylitis (AS); Graves' disease (autoimmune thyroid disease; AITD); multiple sclerosis (MS); breast cancer (BC); prostate cancer (PC); systemic lupus erythematosus (SLE); vitiligo-associated multiple autoimmune disease (VIT); and ulcerative colitis (UC). We selected for sequence homology profiling a set of approximately 250 SNPs which were unequivocally associated with common human disorders based on multiple independent studies of 220,124 individual samples comprising 85,077 disease cases and 129,506 controls. Our analysis reveals a systematic primary sequence homology/complementarity-driven pattern of associations between disease-linked SNPs, microRNAs and protein-coding mRNAs defined here as a human disease phenocode. We utilize this approach to draw SNP-guided microRNA maps of major human diseases and define a consensus disease phenocode for fifteen major human disorders. A consensus disease phenocode comprises 72 SNPs and 18 microRNAs with an apparent propensity to target mRNA sequences derived from a single protein-coding gene, KPNA1. Each of microRNAs in this elite set appears linked to at least three common human diseases and has potential protein-coding mRNA targets among the principal components of the nuclear import pathway. We confirmed the validity of our findings by analyzing independent sets of most significant disease-linked SNPs and demonstrating statistically significant KPNA1-gene expression phenotypes associated with human genotypes of CD, BD, T2D and RA populations. Our analysis supports the idea that variations in DNA sequences associated with multiple human diseases may affect phenotypes in trans via non-protein-coding RNA intermediaries interfering with functions of microRNAs and defines the nuclear import pathway as a potential major target in 15 common human disorders. PMID:18719369

Glinsky, Gennadi V

2008-08-15

308

Metabolomics tools for identifying biomarkers for neuropsychiatric diseases.  

PubMed

The repertoire of biochemicals (or small molecules) present in cells, tissue, and body fluids is known as the metabolome. Today, clinicians utilize only a very small part of the information contained in the metabolome, as revealed by the quantification of a limited set of analytes to gain information on human health. Examples include measuring glucose or cholesterol to monitor diabetes and cardiovascular health, respectively. With a focus on comprehensively studying the metabolome, the rapidly growing field of metabolomics captures the metabolic state of organisms at the global or "-omics" level. Given that the overall health status of an individual is captured by his or her metabolic state, which is a reflection of what has been encoded by the genome and modified by environmental factors, metabolomics has the potential to have a great impact upon medical practice by providing a wealth of relevant biochemical data. Metabolomics promises to improve current, single metabolites-based clinical assessments by identifying metabolic signatures (biomarkers) that embody global biochemical changes in disease, predict responses to treatment or medication side effects (pharmachometabolomics). State of the art metabolomic analytical platforms and informatics tools are being used to map potential biomarkers for a multitude of disorders including those of the central nervous system (CNS). Indeed, CNS disorders are linked to disturbances in metabolic pathways related to neurotransmitter systems (dopamine, serotonin, GABA and glutamate); fatty acids such as arachidonic acid-cascade; oxidative stress and mitochondrial function. Metabolomics tools are enabling us to map in greater detail perturbations in many biochemical pathways and links among these pathways this information is key for development of biomarkers that are disease-specific. In this review, we elaborate on some of the concepts and technologies used in metabolomics and its promise for biomarker discovery. We also highlight early findings from metabolomic studies in CNS disorders such as schizophrenia, Major Depressive Disorder (MDD), Bipolar Disorder (BD), Amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). PMID:19303440

Quinones, Marlon P; Kaddurah-Daouk, Rima

2009-08-01

309

``Nanocavity'' Biochemical Sensor  

Microsoft Academic Search

A biochemical sensor, based on a coaxial ``nanocavity,'' is described. The sensor is capable of detecting small changes in complex impedance, resulting from the presence of target entities in, and near, the device. Its nanoscale dimensions can be tuned for size-specificity, and its constituent components functionalized, for biochemical specificity. The measured capacitance of the bare sensor is in good agreement

T. Kirkpatrick; J. I. Oh; P. Dhakal; D. Cai; H. Z. Zhao; A. Cimeno; L. Ren; K. Kempa; Z. F. Ren; T. C. Chiles; M. J. Naughton

2009-01-01

310

Biochemical transformation of coals  

DOEpatents

A method of biochemically transforming macromolecular compounds found in solid carbonaceous materials, such as coal is provided. The preparation of new microorganisms, metabolically weaned through challenge growth processes to biochemically transform solid carbonaceous materials at extreme temperatures, pressures, pH, salt and toxic metal concentrations is also disclosed. 7 figs.

Lin, M.S.; Premuzic, E.T.

1999-03-23

311

Thermodynamic analysis of trinitrotoluene biodegradation and mineralization pathways  

SciTech Connect

Biodegradation of 2,4,6-trinitrotoluene (TNT) proceeds through several different metabolic pathways. However, the reaction steps which are considered rate-controlling have not been fully determined. Glycolysis and other biological pathways contain biochemical reactions which are acutely rate-limiting due to enzyme control. These rate-limiting steps also have large negative Gibbs free energy changes. Because xenobiotic compounds such as TNT can be used by biological systems as nitrogen, carbon, and energy sources, it is likely that their degradation pathways also contain acutely rate-limiting steps. Identification of these rate-controlling reactions will enhance and better direct genetic engineering techniques to increase specific enzyme levels. This article identifies likely rate-controlling steps (or sets of steps) in reported TNT biodegradation pathways by estimating the Gibbs free energy change for each step and for the overall pathways. The biological standard Gibbs free energy change of reaction was calculated for each pathway step using a group contribution method specifically tailored for biomolecules. The method was also applied to hypothetical pathways constructed to mineralize TNT using several different microorganisms. Pathways steps that have large negative Gibbs free energy changes are postulated to be potentially rate-controlling. The microorganisms which utilize degradation pathways with the largest overall (from TNT to citrate) negative Gibbs free energy changes were also determined.

Shelley, M.D.; Autenrieth, R.L.; Wild, J.R.; Dale, B.E. [Texas A and M Univ., College Station, TX (United States)] [Texas A and M Univ., College Station, TX (United States)

1996-07-20

312

Mutations in Escherichia coli ExbB Transmembrane Domains Identify Scaffolding and Signal Transduction Functions and Exclude Participation in a Proton Pathway  

PubMed Central

The TonB system couples cytoplasmic membrane proton motive force (pmf) to active transport of diverse nutrients across the outer membrane. Current data suggest that cytoplasmic membrane proteins ExbB and ExbD harness pmf energy. Transmembrane domain (TMD) interactions between TonB and ExbD allow the ExbD C terminus to modulate conformational rearrangements of the periplasmic TonB C terminus in vivo. These conformational changes somehow allow energization of high-affinity TonB-gated transporters by direct interaction with TonB. While ExbB is essential for energy transduction, its role is not well understood. ExbB has N-terminus-out, C-terminus-in topology with three TMDs. TMDs 1 and 2 are punctuated by a cytoplasmic loop, with the C-terminal tail also occupying the cytoplasm. We tested the hypothesis that ExbB TMD residues play roles in proton translocation. Reassessment of TMD boundaries based on hydrophobic character and residue conservation among distantly related ExbB proteins brought earlier widely divergent predictions into congruence. All TMD residues with potentially function-specific side chains (Lys, Cys, Ser, Thr, Tyr, Glu, and Asn) and residues with probable structure-specific side chains (Trp, Gly, and Pro) were substituted with Ala and evaluated in multiple assays. While all three TMDs were essential, they had different roles: TMD1 was a region through which ExbB interacted with the TonB TMD. TMD2 and TMD3, the most conserved among the ExbB/TolQ/MotA/PomA family, played roles in signal transduction between cytoplasm and periplasm and the transition from ExbB homodimers to homotetramers. Consideration of combined data excludes ExbB TMD residues from direct participation in a proton pathway.

Baker, Kristin R.

2013-01-01

313

MicroRNA profiling identifies miR-29 as a regulator of disease-associated pathways in experimental biliary atresia  

PubMed Central

Biliary atresia is a pediatric liver disease of unknown underlying etiology, in which fibro-inflammatory destruction of the extrahepatic biliary system leads to obstructive cholestasis. MicroRNAs are a class of short (18–23 nucleotide), non-coding RNA molecules which act as negative regulators of target mRNA stability and translation. The importance of these molecules in normal and diseased liver has been demonstrated, but their potential role in the pathogenesis of biliary atresia has not been addressed. We have profiled changes in liver microRNA levels in an established mouse model of the disease, identified significantly altered transcripts, and defined the spatial expression patterns of selected microRNAs. Two of these, miR-29a/29b1 are up-regulated in experimental biliary atresia. Using antisense oligonucleotide-mediated inhibition in mice, we have delineated the full set of hepatic genes regulated by miR-29 and identified two mRNA targets of potential pathological relevance in experimental biliary atresia, Igf1 and Il1RAP. We have used reporter assays to confirm that Igf1 and Il1RAP are direct targets of miR-29.

Hand, Nicholas J.; Horner, Amber M.; Master, Zankhana R.; Boateng, LaTasha A.; LeGuen, Claire; Uvaydova, Marina

2012-01-01

314

Heterologous protein production using the twin arginine translocation pathway  

DOEpatents

Provided are means for evaluating and identifying putative substrates of the twin arginine translocation (Tat) secretory pathway in Streptomyces and other bacterial species. Also provided, therefore, are simple ways to express, secrete and purify correctly folded heterologous proteins on a large scale using host microorganisms, such as, Streptomyces and the Tat pathway therein. Many of the thus-produced proteins are of significant therapeutic value in the pharmaceutical and biochemical industries, particularly when they can be secreted from the host in fully-folded active form. Accordingly, there are further provided the heterologous proteins produced by the Tat secretion pathway using the foregoing methods, and the computer algorithm used to identify the Tat signal sequence and putative substrates.

Pohlschroder, Mechtild (Philadelphia, PA); Kissinger, Jessica C (Athens, GA); Rose, R. Wesley (Glenside, PA); Brueser, Thomas (Halle, DE); Dilks, Kieran (Collingswood, NJ)

2008-11-04

315

Phosphoproteomic analysis of anaplastic lymphoma kinase (ALK) downstream signaling pathways identifies signal transducer and activator of transcription 3 as a functional target of activated ALK in neuroblastoma cells.  

PubMed

Activation of the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase is a key oncogenic mechanism in a growing number of tumor types. In the majority of cases, ALK is activated by fusion with a dimerizing partner protein as a result of chromosomal translocation events, most studied in the case of the nucleophosmin-ALK and echinoderm microtubule-associated protein-like 4-ALK oncoproteins. It is now also appreciated that the full-length ALK receptor can be activated by point mutations and by deletions within the extracellular domain, such as those observed in neuroblastoma. Several studies have employed phosphoproteomics approaches to find substrates of ALK fusion proteins. In this study, we used MS-based phosphotyrosine profiling to characterize phosphotyrosine signaling events associated with the full-length ALK receptor. A number of previously identified and novel targets were identified. One of these, signal transducer and activator of transcription 3 (STAT3), has previously been observed to be activated in response to oncogenic ALK signaling, but the significance of this in signaling from the full-length ALK receptor has not been explored further. We show here that activated ALK robustly activates STAT3 on Tyr705 in a number of independent neuroblastoma cell lines. Furthermore, knockdown of STAT3 by RNA interference resulted in a reduction in myelocytomatosis neuroblastom (MYCN) protein levels downstream of ALK signaling. These observations, together with a decreased level of MYCN and inhibition of neuroblastoma cell growth in the presence of STAT3 inhibitors, suggest that activation of STAT3 is important for ALK signaling activity in neuroblastoma. PMID:23889739

Sattu, Kamaraj; Hochgräfe, Falko; Wu, Jianmin; Umapathy, Ganesh; Schönherr, Christina; Ruuth, Kristina; Chand, Damini; Witek, Barbara; Fuchs, James; Li, Pui-Kai; Hugosson, Fredrik; Daly, Roger J; Palmer, Ruth H; Hallberg, Bengt

2013-11-01

316

Putative cold acclimation pathways in Arabidopsis thaliana identified by a combined analysis of mRNA co-expression patterns, promoter motifs and transcription factors  

PubMed Central

Background With the advent of microarray technology, it has become feasible to identify virtually all genes in an organism that are induced by developmental or environmental changes. However, relying solely on gene expression data may be of limited value if the aim is to infer the underlying genetic networks. Development of computational methods to combine microarray data with other information sources is therefore necessary. Here we describe one such method. Results By means of our method, previously published Arabidopsis microarray data from cold acclimated plants at six different time points, promoter motif sequence data extracted from ~24,000 Arabidopsis promoters and known transcription factor binding sites were combined to construct a putative genetic regulatory interaction network. The inferred network includes both previously characterised and hitherto un-described regulatory interactions between transcription factor (TF) genes and genes that encode other TFs or other proteins. Part of the obtained transcription factor regulatory network is presented here. More detailed information is available in the additional files. Conclusion The rule-based method described here can be used to infer genetic networks by combining data from microarrays, promoter sequences and known promoter binding sites. This method should in principle be applicable to any biological system. We tested the method on the cold acclimation process in Arabidopsis and could identify a more complex putative genetic regulatory network than previously described. However, it should be noted that information on specific binding sites for individual TFs were in most cases not available. Thus, gene targets for the entire TF gene families were predicted. In addition, the networks were built solely by a bioinformatics approach and experimental verifications will be necessary for their final validation. On the other hand, since our method highlights putative novel interactions, more directed experiments could now be performed.

Chawade, Aakash; Brautigam, Marcus; Lindlof, Angelica; Olsson, Olof; Olsson, Bjorn

2007-01-01

317

Phosphoproteomic analysis of anaplastic lymphoma kinase (ALK) downstream signaling pathways identifies signal transducer and activator of transcription 3 as a functional target of activated ALK in neuroblastoma cells  

PubMed Central

Activation of the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase is a key oncogenic mechanism in a growing number of tumor types. In the majority of cases, ALK is activated by fusion with a dimerizing partner protein as a result of chromosomal translocation events, most studied in the case of the nucleophosmin–ALK and echinoderm microtubule-associated protein-like?4–ALK oncoproteins. It is now also appreciated that the full-length ALK receptor can be activated by point mutations and by deletions within the extracellular domain, such as those observed in neuroblastoma. Several studies have employed phosphoproteomics approaches to find substrates of ALK fusion proteins. In this study, we used MS-based phosphotyrosine profiling to characterize phosphotyrosine signaling events associated with the full-length ALK receptor. A number of previously identified and novel targets were identified. One of these, signal transducer and activator of transcription?3 (STAT3), has previously been observed to be activated in response to oncogenic ALK signaling, but the significance of this in signaling from the full-length ALK receptor has not been explored further. We show here that activated ALK robustly activates STAT3 on Tyr705 in a number of independent neuroblastoma cell lines. Furthermore, knockdown of STAT3 by RNA interference resulted in a reduction in myelocytomatosis neuroblastom (MYCN) protein levels downstream of ALK signaling. These observations, together with a decreased level of MYCN and inhibition of neuroblastoma cell growth in the presence of STAT3 inhibitors, suggest that activation of STAT3 is important for ALK signaling activity in neuroblastoma.

Sattu, Kamaraj; Hochgrafe, Falko; Wu, Jianmin; Umapathy, Ganesh; Schonherr, Christina; Ruuth, Kristina; Chand, Damini; Witek, Barbara; Fuchs, James; Li, Pui-Kai; Hugosson, Fredrik; Daly, Roger J; Palmer, Ruth H; Hallberg, Bengt

2013-01-01

318

Comparison of the Gene Expression Profiles of Human Fetal Cortical Astrocytes with Pluripotent Stem Cell Derived Neural Stem Cells Identifies Human Astrocyte Markers and Signaling Pathways and Transcription Factors Active in Human Astrocytes  

PubMed Central

Astrocytes are the most abundant cell type in the central nervous system (CNS) and have a multitude of functions that include maintenance of CNS homeostasis, trophic support of neurons, detoxification, and immune surveillance. It has only recently been appreciated that astrocyte dysfunction is a primary cause of many neurological disorders. Despite their importance in disease very little is known about global gene expression for human astrocytes. We have performed a microarray expression analysis of human fetal astrocytes to identify genes and signaling pathways that are important for astrocyte development and maintenance. Our analysis confirmed that the fetal astrocytes express high levels of the core astrocyte marker GFAP and the transcription factors from the NFI family which have been shown to play important roles in astrocyte development. A group of novel markers were identified that distinguish fetal astrocytes from pluripotent stem cell-derived neural stem cells (NSCs) and NSC-derived neurons. As in murine astrocytes, the Notch signaling pathway appears to be particularly important for cell fate decisions between the astrocyte and neuronal lineages in human astrocytes. These findings unveil the repertoire of genes expressed in human astrocytes and serve as a basis for further studies to better understand astrocyte biology, especially as it relates to disease.

Malik, Nasir; Wang, Xiantao; Shah, Sonia; Efthymiou, Anastasia G.; Yan, Bin; Heman-Ackah, Sabrina; Zhan, Ming; Rao, Mahendra

2014-01-01

319

Mechanism of Salinity Tolerance in Plants: Physiological, Biochemical, and Molecular Characterization  

PubMed Central

Salinity is a major abiotic stress limiting growth and productivity of plants in many areas of the world due to increasing use of poor quality of water for irrigation and soil salinization. Plant adaptation or tolerance to salinity stress involves complex physiological traits, metabolic pathways, and molecular or gene networks. A comprehensive understanding on how plants respond to salinity stress at different levels and an integrated approach of combining molecular tools with physiological and biochemical techniques are imperative for the development of salt-tolerant varieties of plants in salt-affected areas. Recent research has identified various adaptive responses to salinity stress at molecular, cellular, metabolic, and physiological levels, although mechanisms underlying salinity tolerance are far from being completely understood. This paper provides a comprehensive review of major research advances on biochemical, physiological, and molecular mechanisms regulating plant adaptation and tolerance to salinity stress.

Huang, Bingru

2014-01-01

320

Biochemical and genetic analyses of childhood attention deficit/hyperactivity disorder.  

PubMed

Attention deficit/hyperactivity disorder (ADHD) in children is a neurobehavioral disorder characterized by inattention, hyperactivity, and/or impulsivity. The biochemical abnormalities and genetic factors play significant roles in the etiology of ADHD. These symptoms affect the behavior performance and social relationships of children in school and at home. Recently, many studies about biochemical abnormalities in ADHD have been published. Several research groups have also suggested the genetic contribution to ADHD, and attempted to identify susceptibility and candidate genes for this disorder through the genetic linkage and association studies. To date, these studies have reported substantial evidence implicating several genes (dopaminergic: DRD4, DAT1, DRD5, COMT; noradrenergic: DBH, ADRA2A; serotonergic: 5-HTT, HTR1B, HTR2A; cholinergic: CHRNA4, and central nervous system development pathway: SNAP25, BDNF) in the etiology of ADHD. Understanding the biochemistry and genetics of ADHD will allow us to provide a useful addition with other treatment procedures for ADHD. PMID:22825876

Caylak, Emrah

2012-09-01

321

Biochemical Engineering Fundamentals  

ERIC Educational Resources Information Center

Discusses a biochemical engineering course that is offered as part of a chemical engineering curriculum and includes topics that influence the behavior of man-made or natural microbial or enzyme reactors. (MLH)

Bailey, J. E.; Ollis, D. F.

1976-01-01

322

Clerkship pathway  

PubMed Central

Abstract Objective To identify factors that help predict success for international medical graduates (IMGs) who train in Canadian residency programs and pass the Canadian certification examinations. Design A retrospective analysis of 58 variables in the files of IMGs who applied to the Collège des médecins du Québec between 2000 and 2008. Setting Quebec. Participants Eight hundred ten IMGs who applied to the Collège des médecins du Québec through either the “equivalency pathway” (ie, starting training at a residency level) or the “clerkship pathway” (ie, relearning at the level of a medical student in the last 2 years of the MD diploma). Main outcome measures Success factors in achieving certification. Data were analyzed using descriptive statistics and ANOVA (analysis of variance). Results International medical graduates who chose the “clerkship pathway” had greater success on certification examinations than those who started at the residency level did. Conclusion There are several factors that influence IMGs’ success on certification examinations, including integration issues, the acquisition of clinical decision-making skills, and the varied educational backgrounds. These factors perhaps can be better addressed by a regular clerkship pathway, in which IMGs benefit from learner-centred teaching and have more time for reflection on and understanding of the North American approach to medical education. The clerkship pathway is a useful strategy for assuring the integration of IMGs in the North American health care system. A 2-year relearning period in medical school at a clinical clerkship level deserves careful consideration.

MacLellan, Anne-Marie; Brailovsky, Carlos; Miller, Francois; Leboeuf, Sylvie

2012-01-01

323

Adverse Outcome Pathways: From Definition to Application  

EPA Science Inventory

A challenge for both human health and ecological toxicologists is the transparent application of mechanistic (e.g., molecular, biochemical, histological) data to risk assessments. The adverse outcome pathway (AOP) is a conceptual framework designed to meet this need. Specifical...

324

Biochemical analysis of TssK, a core component of the bacterial Type VI secretion system, reveals distinct oligomeric states of TssK and identifies a TssK-TssFG subcomplex  

PubMed Central

Gram-negative bacteria use the Type VI secretion system (T6SS) to inject toxic proteins into rival bacteria or eukaryotic cells. However, the mechanism of the T6SS is incompletely understood. In the present study, we investigated a conserved component of the T6SS, TssK, using the antibacterial T6SS of Serratia marcescens as a model system. TssK was confirmed to be essential for effector secretion by the T6SS. The native protein, although not an integral membrane protein, appeared to localize to the inner membrane, consistent with its presence within a membrane-anchored assembly. Recombinant TssK purified from S. marcescens was found to exist in several stable oligomeric forms, namely trimer, hexamer and higher-order species. Native-level purification of TssK identified TssF and TssG as interacting proteins. TssF and TssG, conserved T6SS components of unknown function, were required for T6SS activity, but not for correct localization of TssK. A complex containing TssK, TssF and TssG was subsequently purified in vitro, confirming that these three proteins form a new subcomplex within the T6SS. Our findings provide new insight into the T6SS assembly, allowing us to propose a model whereby TssK recruits TssFG into the membrane-associated T6SS complex and different oligomeric states of TssK may contribute to the dynamic mechanism of the system.

English, Grant; Byron, Olwyn; Cianfanelli, Francesca R.; Prescott, Alan R.; Coulthurst, Sarah J.

2014-01-01

325

Biochemical analysis of TssK, a core component of the bacterial Type VI secretion system, reveals distinct oligomeric states of TssK and identifies a TssK-TssFG subcomplex.  

PubMed

Gram-negative bacteria use the Type VI secretion system (T6SS) to inject toxic proteins into rival bacteria or eukaryotic cells. However, the mechanism of the T6SS is incompletely understood. In the present study, we investigated a conserved component of the T6SS, TssK, using the antibacterial T6SS of Serratia marcescens as a model system. TssK was confirmed to be essential for effector secretion by the T6SS. The native protein, although not an integral membrane protein, appeared to localize to the inner membrane, consistent with its presence within a membrane-anchored assembly. Recombinant TssK purified from S. marcescens was found to exist in several stable oligomeric forms, namely trimer, hexamer and higher-order species. Native-level purification of TssK identified TssF and TssG as interacting proteins. TssF and TssG, conserved T6SS components of unknown function, were required for T6SS activity, but not for correct localization of TssK. A complex containing TssK, TssF and TssG was subsequently purified in vitro, confirming that these three proteins form a new subcomplex within the T6SS. Our findings provide new insight into the T6SS assembly, allowing us to propose a model whereby TssK recruits TssFG into the membrane-associated T6SS complex and different oligomeric states of TssK may contribute to the dynamic mechanism of the system. PMID:24779861

English, Grant; Byron, Olwyn; Cianfanelli, Francesca R; Prescott, Alan R; Coulthurst, Sarah J

2014-07-15

326

Relationship between extreme pathways and structurally minimal pathways.  

PubMed

The determination of reaction pathways is one of the most important functions that should be performed in exploring the kinetics of catalyzed chemical reactions or biochemical reactions, the latter being generally catalyzed by enzymes. It is proven that the terms, "type-I extreme pathway" and "structurally minimal pathway", both introduced to characterize the kinetics of a catalyzed reaction are equivalent. These two terms are based on two distinct methodologies, one mainly rooted in convex analysis and the other in graph theory. The equivalence promises further even more effective methods for reaction-pathway identification by synergistic integration of existing ones. PMID:23142845

Barany, Mate; Bertok, Botond; Fan, L T; Friedler, Ferenc

2013-09-01

327

Molecular aspects of the endocytic pathway.  

PubMed Central

Observation of the flow of material along the endocytic pathway has lead to the description of the basic architecture of the pathway and provided insight into the relationship between compartments. Significant advances have been made in the study of endocytic transport steps at the molecular level, of which studies of cargo selection, vesicle budding and membrane fusion events comprise the major part. Progress in this area has been driven by two approaches, yeast genetics and in vitro or cell-free assays, which reconstitute particular transport steps and allow biochemical manipulation. The complex protein machineries that control vesicle budding and fusion are significantly conserved between the secretory and endocytic pathways such that proteins that regulate particular steps are often part of a larger family of proteins which exercise a conserved function at other locations within the cell. Well characterized examples include vesicle coat proteins, rabs (small GTPases) and soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (SNAREs). Intracompartmental pH, lipid composition and cytoskeletal organization have also been identified as important determinants of the orderly flow of material within the endocytic pathway.

Clague, M J

1998-01-01

328

Biochemical upgrading of oils  

DOEpatents

A process for biochemical conversion of heavy crude oils is provided. The process includes contacting heavy crude oils with adapted biocatalysts. The resulting upgraded oil shows, a relative increase in saturated hydrocarbons, emulsions and oxygenates and a decrease in compounds containing organic sulfur, organic nitrogen and trace metals. Adapted microorganisms which have been modified under challenged growth processes are also disclosed. 121 figs.

Premuzic, E.T.; Lin, M.S.

1999-01-12

329

Measures of Biochemical Sociology  

ERIC Educational Resources Information Center

In a previous article, the authors introduced a new sub field in sociology that we labeled "biochemical sociology." We introduced the definition of a sociology that encompasses sociological measures, psychological measures, and biological indicators Snell & Marsh (2003). In this article, we want to demonstrate a research strategy that would assess…

Snell, Joel; Marsh, Mitchell

2008-01-01

330

Biochemical engineering and industry  

Microsoft Academic Search

The use of biochemical engineering in an industrial setting is presented by examples from the author's own field of expertise: process development for industrial enzymes. Examples given cover the fields bioreactor performance, measurement and control on fermentation, downstream operations and development and optimization of production strains. The use of engineering principles is placed in the industrial context of a high

Anders Gram

1997-01-01

331

Biochemical findings in sarcoidosis  

Microsoft Academic Search

The majority of patients with sarcoidosis in this large series have had a number of biochemical investigations performed. Abnormal calcium metabolism was demonstrated in 40% of the patients but permanent renal damage due to nephrocalcinosis as a result of persistent derangement of calcium metabolism was rare. Raised immunoglobulin levels were seen. Half the white and two-thirds of the West Indian

P R Studdy; R Bird; E Neville; D G James

1980-01-01

332

Genome-wide association study identifies novel loci associated with concentrations of four plasma phospholipid fatty acids in the de novo lipogenesis pathway: results from the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium.  

PubMed

BACKGROUND- Palmitic acid (16:0), stearic acid (18:0), palmitoleic acid (16:1n-7), and oleic acid (18:1n-9) are major saturated and monounsaturated fatty acids that affect cellular signaling and metabolic pathways. They are synthesized via de novo lipogenesis and are the main saturated and monounsaturated fatty acids in the diet. Levels of these fatty acids have been linked to diseases including type 2 diabetes mellitus and coronary heart disease. METHODS AND RESULTS- Genome-wide association studies were conducted in 5 population-based cohorts comprising 8961 participants of European ancestry to investigate the association of common genetic variation with plasma levels of these 4 fatty acids. We identified polymorphisms in 7 novel loci associated with circulating levels of ?1 of these fatty acids. ALG14 (asparagine-linked glycosylation 14 homolog) polymorphisms were associated with higher 16:0 (P=2.7×10(-11)) and lower 18:0 (P=2.2×10(-18)). FADS1 and FADS2 (desaturases) polymorphisms were associated with higher 16:1n-7 (P=6.6×10(-13)) and 18:1n-9 (P=2.2×10(-32)) and lower 18:0 (P=1.3×10(-20)). LPGAT1 (lysophosphatidylglycerol acyltransferase) polymorphisms were associated with lower 18:0 (P=2.8×10(-9)). GCKR (glucokinase regulator; P=9.8×10(-10)) and HIF1AN (factor inhibiting hypoxia-inducible factor-1; P=5.7×10(-9)) polymorphisms were associated with higher 16:1n-7, whereas PKD2L1 (polycystic kidney disease 2-like 1; P=5.7×10(-15)) and a locus on chromosome 2 (not near known genes) were associated with lower 16:1n-7 (P=4.1×10(-8)). CONCLUSIONS- Our findings provide novel evidence that common variations in genes with diverse functions, including protein-glycosylation, polyunsaturated fatty acid metabolism, phospholipid modeling, and glucose- and oxygen-sensing pathways, are associated with circulating levels of 4 fatty acids in the de novo lipogenesis pathway. These results expand our knowledge of genetic factors relevant to de novo lipogenesis and fatty acid biology. PMID:23362303

Wu, Jason H Y; Lemaitre, Rozenn N; Manichaikul, Ani; Guan, Weihua; Tanaka, Toshiko; Foy, Millennia; Kabagambe, Edmond K; Djousse, Luc; Siscovick, David; Fretts, Amanda M; Johnson, Catherine; King, Irena B; Psaty, Bruce M; McKnight, Barbara; Rich, Stephen S; Chen, Yii-Der I; Nettleton, Jennifer A; Tang, Weihong; Bandinelli, Stefania; Jacobs, David R; Browning, Brian L; Laurie, Cathy C; Gu, Xiangjun; Tsai, Michael Y; Steffen, Lyn M; Ferrucci, Luigi; Fornage, Myriam; Mozaffarian, Dariush

2013-04-01

333

In vitro microarray analysis identifies genes in acute-phase response pathways that are down-regulated in the liver of chicken embryos exposed in ovo to PFUdA.  

PubMed

Perfluoroundecanoic acid (PFUdA) is one of the most highly detected perfluoroalkyl compounds in wild bird tissues and eggs. Although PFUdA does not affect hatching success, many PFCs are known to impair post-hatch development and survival. Here we use microarrays to survey the transcriptional response of cultured chicken embryonic hepatocytes (CEH) to PFUdA for potential targets of PFUdA action that could lead to developmental deficiencies in exposed birds. At 1 ?M and 10 ?M PFUdA significantly altered the expression of 346 and 676 transcripts, respectively (fold-change>1.5, p<0.05, false discovery rate-corrected). Using functional, pathway and interactome analysis we identified several potentially important targets of PFUdA exposure, including the suppression of the acute-phase response (APR). We then measured the expression of five APR genes, fibrinogen alpha (fga), fibrinogen gamma (fgg), thrombin (f2), plasminogen (plg), and protein C (proC), in the liver of chicken embryos exposed in ovo to PFUdA. The expression of fga, f2, and proC were down-regulated in embryo livers (100 or 1000 ng/g, p<0.1) as predicted from microarray analysis, whereas fibrinogen gamma (fgg) was up-regulated and plg was not significantly affected. Our results demonstrate the utility of CEH coupled with transcriptome analysis as an in vitro screening tool for identifying novel effects of toxicant exposure. Additionally, we identified APR suppression as a potentially important and environmentally relevant target of PFUdA. These findings suggest in ovo exposure of birds to PFUdA may lead to post-hatch developmental deficiencies, such as impaired inflammatory response. PMID:23602845

O'Brien, Jason M; Williams, Andrew; Yauk, Carole L; Crump, Doug; Kennedy, Sean W

2013-09-01

334

Genetic analysis of photoreceptor action pathways in Arabidopsis thaliana  

SciTech Connect

The specific strategies and long-term goals of this proposal remain intact relative to the original proposal. We continue to isolate and characterize photomorphogenic mutants of Arabidopsis thaliana. The molecular and biochemical characterization of one of these mutants, det1, has led to one publication of original data and to one Society for Experimental Biology Symposium paper (see below). The phenotype of a second mutant, det2, has also been studied during this funding period. In addition, we have continued work on a general strategy to isolate mutations in trans-acting regulatory factors that mediate light-regulated gene expression, and have identified several potentially interesting regulatory mutants. In the third funding period, we will concentrate on the genetical, biochemical, and molecular characterization of these new mutants. Construction of double mutants between the new mutants and the previously characterized morphological mutants should allow us to construct a pathway for light-regulated seedling development in Arabidopsis.

Not Available

1991-01-01

335

Identifying positive selection candidate loci for high-altitude adaptation in Andean populations  

PubMed Central

High-altitude environments (>2,500 m) provide scientists with a natural laboratory to study the physiological and genetic effects of low ambient oxygen tension on human populations. One approach to understanding how life at high altitude has affected human metabolism is to survey genome-wide datasets for signatures of natural selection. In this work, we report on a study to identify selection-nominated candidate genes involved in adaptation to hypoxia in one highland group, Andeans from the South American Altiplano. We analysed dense microarray genotype data using four test statistics that detect departures from neutrality. Using a candidate gene, single nucleotide polymorphism-based approach, we identified genes exhibiting preliminary evidence of recent genetic adaptation in this population. These included genes that are part of the hypoxia-inducible transcription factor (HIF) pathway, a biochemical pathway involved in oxygen homeostasis, as well as three other genomic regions previously not known to be associated with high-altitude phenotypes. In addition to identifying selection-nominated candidate genes, we also tested whether the HIF pathway shows evidence of natural selection. Our results indicate that the genes of this biochemical pathway as a group show no evidence of having evolved in response to hypoxia in Andeans. Results from particular HIF-targeted genes, however, suggest that genes in this pathway could play a role in Andean adaptation to high altitude, even if the pathway as a whole does not show higher relative rates of evolution. These data suggest a genetic role in high-altitude adaptation and provide a basis for genotype/phenotype association studies that are necessary to confirm the role of putative natural selection candidate genes and gene regions in adaptation to altitude.

2009-01-01

336

Constructing de novo biosynthetic pathways for chemical synthesis inside living cells†  

PubMed Central

Living organisms have evolved a vast array of catalytic functions that make them ideally suited for the production of medicinally and industrially relevant small-molecule targets. Indeed, native metabolic pathways in microbial hosts have long been exploited and optimized for the scalable production of both fine and commodity chemicals. Our increasing capacity for DNA sequencing and synthesis has revealed the molecular basis for the biosynthesis of a variety of complex and useful metabolites and enables the de novo construction of novel metabolic pathways for the production of new and exotic molecular targets in genetically tractable microbes. However, the development of commercially viable processes for these engineered pathways is currently limited by our ability to quickly identify or engineer enzymes with the correct reaction and substrate selectivity as well as the speed by which metabolic bottlenecks can be determined and corrected. Efforts in understanding the relationship between sequence, structure, and function in the basic biochemical sciences can advance these goals for synthetic biology applications while also serving as an experimental platform to elucidate the in vivo specificity and function of enzymes and to reconstitute complex biochemical traits for study in a living model organism. Furthermore, the continuing discovery of natural mechanisms for the regulation of metabolic pathways has revealed new principles for the design of high-flux pathways with minimized metabolic burden and has inspired the development of new tools and approaches to engineer synthetic pathways in microbial hosts for chemical production.

Weeks, Amy M.; Chang, Michelle C. Y.

2011-01-01

337

The Jasmonate Pathway  

NSDL National Science Digital Library

Plants are faced with many of the same problems as animalsâÂÂa need for regulation of metabolic processes and reproduction and for defense against enemies. Jasmonates in plants serve key roles in gene and metabolic regulation, defense, responses to trauma, reproduction, and possibly communication. Some remarkable features of plant responses, such as production of repellent volatiles as a defense against herbivorous insects, or the massive transcriptional reprogramming that occurs in response to wounding, are under the control of the jasmonate pathway. Details of the jasmonate signaling pathway are currently at the center of active research that is generating exciting results. The Jasmonate Biochemical Pathway at the STKE Connections Maps is designed to present and keep pace with these developments.

Robin Liechti (University of Lausanne;Gene Expression Laboratory); Edward Farmer (University of Lausanne;Gene Expression Laboratory)

2002-05-31

338

Biochemical and genetic aspects of mevalonate kinase and its deficiency  

Microsoft Academic Search

Mevalonate kinase (MK) is an essential enzyme in the mevalonate pathway which produces numerous cellular isoprenoids. The enzyme has been characterized both at the biochemical and the molecular level in a variety of organisms. Despite the fact that mevalonate kinase is not the rate-limiting enzyme in isoprenoid biosynthesis, its activity is subject to feedback regulation by the branch-point intermediates geranyldiphosphate,

Sander M Houten; Ronald J. A Wanders; Hans R Waterham

2000-01-01

339

Genomic encyclopedia of sugar utilization pathways in the Shewanella genus  

PubMed Central

Background Carbohydrates are a primary source of carbon and energy for many bacteria. Accurate projection of known carbohydrate catabolic pathways across diverse bacteria with complete genomes constitutes a substantial challenge due to frequent variations in components of these pathways. To address a practically and fundamentally important challenge of reconstruction of carbohydrate utilization machinery in any microorganism directly from its genomic sequence, we combined a subsystems-based comparative genomic approach with experimental validation of selected bioinformatic predictions by a combination of biochemical, genetic and physiological experiments. Results We applied this integrated approach to systematically map carbohydrate utilization pathways in 19 genomes from the Shewanella genus. The obtained genomic encyclopedia of sugar utilization includes ~170 protein families (mostly metabolic enzymes, transporters and transcriptional regulators) spanning 17 distinct pathways with a mosaic distribution across Shewanella species providing insights into their ecophysiology and adaptive evolution. Phenotypic assays revealed a remarkable consistency between predicted and observed phenotype, an ability to utilize an individual sugar as a sole source of carbon and energy, over the entire matrix of tested strains and sugars. Comparison of the reconstructed catabolic pathways with E. coli identified multiple differences that are manifested at various levels, from the presence or absence of certain sugar catabolic pathways, nonorthologous gene replacements and alternative biochemical routes to a different organization of transcription regulatory networks. Conclusions The reconstructed sugar catabolome in Shewanella spp includes 62 novel isofunctional families of enzymes, transporters, and regulators. In addition to improving our knowledge of genomics and functional organization of carbohydrate utilization in Shewanella, this study led to a substantial expansion of our current version of the Genomic Encyclopedia of Carbohydrate Utilization. A systematic and iterative application of this approach to multiple taxonomic groups of bacteria will further enhance it, creating a knowledge base adequate for the efficient analysis of any newly sequenced genome as well as of the emerging metagenomic data.

2010-01-01

340

Genetic and Biochemical Characterization of Human AP Endonuclease 1 Mutants Deficient in Nucleotide Incision Repair Activity  

PubMed Central

Background Human apurinic/apyrimidinic endonuclease 1 (APE1) is a key DNA repair enzyme involved in both base excision repair (BER) and nucleotide incision repair (NIR) pathways. In the BER pathway, APE1 cleaves DNA at AP sites and 3?-blocking moieties generated by DNA glycosylases. In the NIR pathway, APE1 incises DNA 5? to a number of oxidatively damaged bases. At present, physiological relevance of the NIR pathway is fairly well established in E. coli, but has yet to be elucidated in human cells. Methodology/Principal Finding We identified amino acid residues in the APE1 protein that affect its function in either the BER or NIR pathway. Biochemical characterization of APE1 carrying single K98A, R185A, D308A and double K98A/R185A amino acid substitutions revealed that all mutants exhibited greatly reduced NIR and 3??5? exonuclease activities, but were capable of performing BER functions to some extent. Expression of the APE1 mutants deficient in the NIR and exonuclease activities reduced the sensitivity of AP endonuclease-deficient E. coli xth nfo strain to an alkylating agent, methylmethanesulfonate, suggesting that our APE1 mutants are able to repair AP sites. Finally, the human NIR pathway was fully reconstituted in vitro using the purified APE1, human flap endonuclease 1, DNA polymerase ? and DNA ligase I proteins, thus establishing the minimal set of proteins required for a functional NIR pathway in human cells. Conclusion/Significance Taken together, these data further substantiate the role of NIR as a distinct and separable function of APE1 that is essential for processing of potentially lethal oxidative DNA lesions.

Gelin, Aurore; Redrejo-Rodriguez, Modesto; Laval, Jacques; Fedorova, Olga S.; Saparbaev, Murat; Ishchenko, Alexander A.

2010-01-01

341

A Biochemical Double Slit  

NASA Astrophysics Data System (ADS)

Radical-ion-pair reactions, fundamental in photosynthesis and at the basis of the avian magnetic compass mechanism, have been recently shown to offer a rich playground for applying methods and concepts from quantum measurement/quantum information science. We will demonstrate that radical-ion-pair reactions are almost the exact analog of the optical double slit experiment, i.e. Nature has already engineered biochemical reactions performing the act of quantum interference. We will further elaborate on the non-trivial quantum effects pertaining in these reactions and the recent debate on their fundamental theoretical description that these effects have sparked.

Kominis, Iannis

2011-03-01

342

The Genetic and Biochemical Basis of FANCD2 Monoubiquitination  

PubMed Central

Summary Fanconi anaemia (FA) is a cancer predisposition syndrome characterized by cellular sensitivity to DNA interstrand crosslinkers. The molecular defect in FA is an impaired DNA repair pathway. The critical event in activating this pathway is monoubiquitination of FANCD2. In vivo, a multisubunit FA core complex catalyzes this step, but its mechanism is unclear. Here, we report purification of a native avian FA core complex and biochemical reconstitution of FANCD2 monoubiquitination. This demonstrates that the catalytic FANCL E3 ligase subunit must be embedded within the complex for maximal activity and site specificity. We genetically and biochemically define a minimal subcomplex comprising just three proteins (FANCB, FANCL, and FAAP100) that functions as the monoubiquitination module. Residual FANCD2 monoubiquitination activity is retained in cells defective for other FA core complex subunits. This work describes the in vitro reconstitution and characterization of this multisubunit monoubiquitin E3 ligase, providing key insight into the conserved FA DNA repair pathway.

Rajendra, Eeson; Oestergaard, Vibe H.; Langevin, Frederic; Wang, Meng; Dornan, Gillian L.; Patel, Ketan J.; Passmore, Lori A.

2014-01-01

343

The Genetic and Biochemical Basis of FANCD2 Monoubiquitination.  

PubMed

Fanconi anaemia (FA) is a cancer predisposition syndrome characterized by cellular sensitivity to DNA interstrand crosslinkers. The molecular defect in FA is an impaired DNA repair pathway. The critical event in activating this pathway is monoubiquitination of FANCD2. In vivo, a multisubunit FA core complex catalyzes this step, but its mechanism is unclear. Here, we report purification of a native avian FA core complex and biochemical reconstitution of FANCD2 monoubiquitination. This demonstrates that the catalytic FANCL E3 ligase subunit must be embedded within the complex for maximal activity and site specificity. We genetically and biochemically define a minimal subcomplex comprising just three proteins (FANCB, FANCL, and FAAP100) that functions as the monoubiquitination module. Residual FANCD2 monoubiquitination activity is retained in cells defective for other FA core complex subunits. This work describes the in vitro reconstitution and characterization of this multisubunit monoubiquitin E3 ligase, providing key insight into the conserved FA DNA repair pathway. PMID:24905007

Rajendra, Eeson; Oestergaard, Vibe H; Langevin, Frédéric; Wang, Meng; Dornan, Gillian L; Patel, Ketan J; Passmore, Lori A

2014-06-01

344

Misleading biochemical laboratory test results  

PubMed Central

This article reviews the general and specific factors that interfere with the performance of common biochemical laboratory tests and the interpretation of their results. The clinical status of the patient, drug interactions, and in-vivo and in-vitro biochemical interactions and changes may alter the results obtained from biochemical analysis of blood constituents. Failure to recognize invalid laboratory test results may lead to injudicious and dangerous management of patients.

Nanji, Amin A.

1984-01-01

345

Potential drug targets in Mycobacterium tuberculosis through metabolic pathway analysis  

Microsoft Academic Search

The emergence of multidrug resistant varieties of Mycobacterium tuberculosis has led to a search for novel drug targets. We have performed an insilico comparative analysis of metabolic pathways of the host Homo sapiens and the pathogen M. tuberculosis. Enzymes from the biochemical pathways of M. tuberculosis from the KEGG metabolic pathway database were compared with proteins from the hostH. sapiens,

Sharmila Anishetty; Mrudula Pulimi; Pennathur Gautam