CiteULike: jyuh's Schmidt

iTools: a framework for classification, categorization and integration of computational biology resources.

ID Dinov — 2008-06-05T16:59:30-00:00

PLoS ONE, Vol. 3, No. 5. (2008)

The advancement of the computational biology field hinges on progress in three fundamental directions--the development of new computational algorithms, the availability of informatics resource management infrastructures and the capability of tools to interoperate and synergize. There is an explosion in algorithms and tools for computational biology, which makes it difficult for biologists to find, compare and integrate such resources. We describe a new infrastructure, iTools, for managing the query, traversal and comparison of diverse computational biology resources. Specifically, iTools stores information about three types of resources--data, software tools and web-services. The iTools design, implementation and resource meta-data content reflect the broad research, computational, applied and scientific expertise available at the seven National Centers for Biomedical Computing. iTools provides a system for classification, categorization and integration of different computational biology resources across space-and-time scales, biomedical problems, computational infrastructures and mathematical foundations. A large number of resources are already iTools-accessible to the community and this infrastructure is rapidly growing. iTools includes human and machine interfaces to its resource meta-data repository. Investigators or computer programs may utilize these interfaces to search, compare, expand, revise and mine meta-data descriptions of existent computational biology resources. We propose two ways to browse and display the iTools dynamic collection of resources. The first one is based on an ontology of computational biology resources, and the second one is derived from hyperbolic projections of manifolds or complex structures onto planar discs. iTools is an open source project both in terms of the source code development as well as its meta-data content. iTools employs a decentralized, portable, scalable and lightweight framework for long-term resource management. We demonstrate several applications of iTools as a framework for integrated bioinformatics. iTools and the complete details about its specifications, usage and interfaces are available at the iTools web page http://iTools.ccb.ucla.edu.

Antibody Microarrays as an Experimental Platform for the Analysis of Signal Transduction Networks.

Ulrike Korf — 2008-07-04T23:23:35-00:00

Advances in biochemical engineering/biotechnology (5 June 2008)

A significant bottleneck for the time-resolved and quantitative description of signaling networks is the limited sample capacity and sensitivity of existing methods. Recently, antibody microarrays have emerged as a promising experimental platform for the quantitative and comprehensive determination of protein abundance and protein phosphorylation. This review summarizes the development of microarray applications involving antibody-based capture of target proteins with a focus on quantitative applications. Technical aspects regarding the production of antibody microarrays, identification of suitable detection and capture antibody pairs, signal detection methods, detection limit, and data analysis are discussed in detail.

Mechanisms of Disease: advanced glycation end-products and their receptor in inflammation and diabetes complications

Shi Yan — 2008-06-29T03:27:07-00:00

Nat Clin Pract End Met, Vol. 4, No. 5. (May 2008), pp. 285-293.

The obesity-survival paradox in hemodialysis patients: why do overweight hemodialysis patients live longer?

D Schmidt — 2008-06-28T12:37:09-00:00

Nutrition in clinical practice : official publication of the American Society for Parenteral and Enteral Nutrition, Vol. 22, No. 1. (February 2007), pp. 11-15.

Obesity is increasingly common in the United States, and it frequently coexists with diabetes and hypertension. Given that diabetes and hypertension are the 2 most common causes of end-stage renal disease, it is not surprising that obesity is also highly prevalent in the US hemodialysis population. However, unlike in the general population, obesity is associated with improved survival in hemodialysis patients. This phenomenon, the obesity-survival paradox, is neither universally accepted nor completely understood. In this article, we review the available data and provide potential reasons for the obesity-survival paradox in the dialysis population.

Sexual Function in Chronic Kidney Disease

Priya Anantharaman — 2008-05-17T09:51:56-00:00

Advances in Chronic Kidney Disease, Vol. 14, No. 2. (April 2007), pp. 119-125.

Endocrine abnormalities are common in patients with chronic kidney disease (CKD) and lead to sexual dysfunction, anemia, hyperparathyroidism, and altered mineral metabolism. Common clinical problems include disturbances in menstruation in women, erectile dysfunction in men, and decreased libido and infertility in both sexes. Organic factors tend to be prominent and are related to uremia and other comorbid illnesses. Psychological factors and depression may exacerbate the primary problem. Alterations in the hypothalamic-pituitary axis are seen early in CKD and tend to worsen after patients start dialysis. Hypogonadism plays a dominant role in male sexual function, whereas changes in hypothalamic-pituitary function predominate in female sexual dysfunction. In patients on dialysis, treatment strategies include optimizing dose of dialysis, correction of anemia with erythropoietin, and correction of hyperparathyroidism. Successful kidney transplantation may restore normal sexual function, especially in younger patients.

The standard protein mix database: a diverse data set to assist in the production of improved Peptide and protein identification software tools.

J Klimek — 2008-05-13T04:56:34-00:00

Journal of proteome research, Vol. 7, No. 1. (January 2008), pp. 96-103.

Tandem mass spectrometry (MS/MS) is frequently used in the identification of peptides and proteins. Typical proteomic experiments rely on algorithms such as SEQUEST and MASCOT to compare thousands of tandem mass spectra against the theoretical fragment ion spectra of peptides in a database. The probabilities that these spectrum-to-sequence assignments are correct can be determined by statistical software such as PeptideProphet or through estimations based on reverse or decoy databases. However, many of the software applications that assign probabilities for MS/MS spectra to sequence matches were developed using training data sets from 3D ion-trap mass spectrometers. Given the variety of types of mass spectrometers that have become commercially available over the last 5 years, we sought to generate a data set of reference data covering multiple instrumentation platforms to facilitate both the refinement of existing computational approaches and the development of novel software tools. We analyzed the proteolytic peptides in a mixture of tryptic digests of 18 proteins, named the "ISB standard protein mix", using 8 different mass spectrometers. These include linear and 3D ion traps, two quadrupole time-of-flight platforms (qq-TOF), and two MALDI-TOF-TOF platforms. The resulting data set, which has been named the Standard Protein Mix Database, consists of over 1.1 million spectra in 150+ replicate runs on the mass spectrometers. The data were inspected for quality of separation and searched using SEQUEST. All data, including the native raw instrument and mzXML formats and the PeptideProphet validated peptide assignments, are available at http://regis-web.systemsbiology.net/PublicDatasets/.

Identification of cross-linked peptides from large sequence databases

Oliver Rinner — 2008-03-28T16:35:47-00:00

Nature Methods, Vol. 5, No. 4. (09 March 2008), pp. 315-318.

Monovalent, reduced-size quantum dots for imaging receptors on living cells

Mark Howarth — 2008-04-29T17:53:28-00:00

Nat Meth, Vol. 5, No. 5. (May 2008), pp. 397-399.

Reactome: a knowledgebase of biological pathways.

G Joshi-Tope — 2006-02-28T11:50:48-00:00

Nucleic Acids Res, Vol. 33, No. Database issue. (1 January 2005)

Reactome, located at http://www.reactome.org is a curated, peer-reviewed resource of human biological processes. Given the genetic makeup of an organism, the complete set of possible reactions constitutes its reactome. The basic unit of the Reactome database is a reaction; reactions are then grouped into causal chains to form pathways. The Reactome data model allows us to represent many diverse processes in the human system, including the pathways of intermediary metabolism, regulatory pathways, and signal transduction, and high-level processes, such as the cell cycle. Reactome provides a qualitative framework, on which quantitative data can be superimposed. Tools have been developed to facilitate custom data entry and annotation by expert biologists, and to allow visualization and exploration of the finished dataset as an interactive process map. Although our primary curational domain is pathways from Homo sapiens, we regularly create electronic projections of human pathways onto other organisms via putative orthologs, thus making Reactome relevant to model organism research communities. The database is publicly available under open source terms, which allows both its content and its software infrastructure to be freely used and redistributed.

ProfCom: a web tool for profiling the complex functionality of gene groups identified from high-throughput data.

Alexey V Antonov — 2008-05-10T08:46:46-00:00

Nucleic acids research (6 May 2008)

ProfCom is a web-based tool for the functional interpretation of a gene list that was identified to be related by experiments. A trait which makes ProfCom a unique tool is an ability to profile enrichments of not only available Gene Ontology (GO) terms but also of 'complex functions'. A 'Complex function' is constructed as Boolean combination of available GO terms. The complex functions inferred by ProfCom are more specific in comparison to single terms and describe more accurately the functional role of genes. ProfCom provides a user friendly dialog-driven web page submission available for several model organisms and supports most available gene identifiers. In addition, the web service interface allows the submission of any kind of annotation data. ProfCom is freely available at http://webclu.bio.wzw.tum.de/profcom/.

The future is now - will the real disease gene please stand up?

ER Martin — 2008-05-03T09:56:38-00:00

Human heredity, Vol. 66, No. 2. (2008), pp. 127-135.

The transmission/disequilibrium test (TDT) [Spielman et al.: Am J Hum Genet 1993;52:506-516] has been postulated as the future of gene mapping for complex diseases, provided one is able to genotype a dense enough map of markers across the genome. Risch and Merikangas [Science 1996;273:1516-1517] suggested a million-marker screen in affected sibpair (ASP) families, demonstrating that the TDT is a more powerful test of linkage than traditional linkage tests based on allele-sharing when there is also association between marker and disease alleles. While the future of genotyping has arrived, successes in family-based association studies have been modest. This is often attributed to excessive false positives in candidate gene studies. This problem is only exacerbated by the increasing numbers of whole genome association (WGA) screens. When applied in ASPs, the TDT statistic, which assumes transmissions to siblings are independent, is not expected to have a constant variance in the presence of variable linkage. This results in generally more extreme statistics, hence will further aggravate the problem of having a large number of positive results to sort through. So an important question is how many positive TDT results will show up on a chromosome containing a disease gene due only to linkage, and will they obfuscate the true disease gene location. To answer this question we combined theory and computer simulations. These studies show that in ASPs the normal version of the TDT statistic has a mean of 0 and a variance of 1 in unlinked regions, but has a variance larger than 1 in linked regions. In contrast, the pedigree disequilibrium test (PDT) statistic adjusts for correlation between siblings due to linkage and maintains a constant variance of 1 at unassociated markers irrespective of linkage. The TDT statistic is generally larger than the PDT statistic across linked regions. This is true for unassociated as well as associated markers. To compare the two tests we ranked both statistics at the disease locus, or an associated marker, among statistics at all other markers. The TDT did better job than PDT placing the score of the associated marker near the top. Though, strictly speaking, the TDT in ASPs should be interpreted as a test of linkage and not a test of association, there is a good chance that if a marker stands out, the marker is associated as well as linked. In conclusion, our results suggest that TDT is an effective screening tool for WGA studies, especially in multiplex families.

Receptor for Advanced Glycation End Products. Fundamental Roles in the Inflammatory Response: Winding the Way to the Pathogenesis of Endothelial Dysfunction and Atherosclerosis

Ravichandran Ramasamy — 2008-04-24T08:35:11-00:00

Annals of the New York Academy of Sciences, Vol. 1126, No. 1. (2008), pp. 7-13.

The multiligand receptor for advanced glycation end products (RAGE) of the immunoglobulin superfamily is expressed on multiple cell types implicated in the immune-inflammatory response and in atherosclerosis. Multiple studies have elucidated that ligand-RAGE interaction on cells, such as monocytes, macrophages, and endothelial cells, mediates cellular migration and upregulation of proinflammatory and prothrombotic molecules. In addition, recent studies reveal definitive rules for RAGE in effective T lymphocyte priming in vivo. RAGE ligand AGEs may be formed in diverse settings; although AGEs are especially generated in hyperglycemia, their production in settings characterized by oxidative stress and inflammation suggests that these species, in part via RAGE, may contribute to the pathogenesis of atherosclerosis. In murine models of atherosclerosis, vascular inflammation is a key factor and one which is augmented, in parallel with even further increases in RAGE ligands, in diabetic macrovessels. The findings that antagonism and genetic disruption of RAGE in atherosclerosis-susceptible mice strikingly reduces vascular inflammation and atherosclerotic lesion area and complexity link RAGE intimately to these processes and suggest that RAGE is a logical target for therapeutic intervention in aberrant inflammatory mechanisms and in atherosclerosis.

Plasma creatinine determination in mice and rats: An enzymatic method compares favorably with a high-performance liquid chromatography assay

A Keppler — 2008-04-20T15:54:43-00:00

Kidney Int, Vol. 71, No. 1. (1 November 2006), pp. 74-78.

Receptor for AGE (RAGE): weaving tangled webs within the inflammatory response.

R Clynes — 2008-04-20T09:05:21-00:00

Current molecular medicine, Vol. 7, No. 8. (December 2007), pp. 743-751.

The family of RAGE ligands, including Advanced Glycation Endproducts (AGEs), S100/calgranulins, High Mobility Group Box-1 (HMGB1) and amyloid beta peptide (Abeta) and beta-sheet fibrils are highly enriched in immune and inflammatory foci. In parallel, upregulation of Receptor for AGE (RAGE) is noted in diverse forms of inflammation and autoimmunity, based on experiments examining human tissues as well as animal models. Indeed, prior to the demonstration that S100/calgranulins were signal transduction ligands of RAGE, these molecules were considered "biomarkers" of disease and disease activity in disorders such as colitis and arthritis. Premiere roles for RAGE in advancing cellular migration implicate this receptor in targeting immune cells to vulnerable foci. Once engaged, ligand-RAGE interaction in inflammatory and vascular cells amplifies upregulation of inflammatory cytokines, adhesion molecules and matrix metalloproteinases (MMPs). Discerning the primal versus chronic injury-provoking roles for this ligand-receptor interaction is a challenge in delineating the functions of the ligand/RAGE axis. As RAGE is expressed by many of the key cell types linked integrally to the immune response, we propose that the sites and time course of ligand-RAGE stimulation determine the phenotype produced by this axis. Ultimately, drawing the fine line between antagonism versus stimulation of the receptor in health and disease will depend on the full characterization of RAGE in repair versus injury.

Expression of HSP47, a collagen-specific chaperone, in normal and diseased human liver

Kyle Brown — 2005-04-04T17:32:19-00:00

Laboratory Investigation, Vol. aop, No. current. (04 April 2005)

Automated production of recombinant human proteins as resource for proteome research

Thorsten Kohl — 2008-01-31T12:17:03-00:00

Proteome Science, Vol. 6 (28 January 2008), 4.

An approach to handling and interpretation of ambiguous data in transcriptome and proteome comparisons.

Martin Irmler — 2008-02-22T03:02:28-00:00

Proteomics (18 February 2008)

A major challenge towards a comprehensive analysis of biological systems is the integration of data from different "omics" sources and their interpretation at a functional level. Here we address this issue by analysing transcriptomic and proteomic datasets from mouse brain tissue at embryonic days 9.5 and 13.5. We observe a high concordance between transcripts and their corresponding proteins when they were compared at the level of expression ratios between embryonic stages. Absolute expression values show marginal correlation. We show in examples, that poor concordance between protein and transcript expression is in part explained by the fact, that single genes give rise to multiple transcripts and protein variants. The integration of transcriptomic and proteomic data therefore requires proper handling of such ambiguities. A closer inspection of such cases in our datasets suggests, that comparing gene expression at exon level instead of gene level could improve the comparability. To address the biological relevance of differences in expression profiles, literature-data mining and analysis of gene ontology terms are widely used. We show here, that this can be complemented by the inspection of physical properties of genes, transcripts, and proteins.

Regulation of renal glucose transporters during severe inflammation.

C Schmidt — 2008-02-20T05:50:26-00:00

Am J Physiol Renal Physiol, Vol. 292, No. 2. (February 2007)

Severe sepsis is accompanied by acute renal failure (ARF) with renal tubular dysfunction and glucosuria. In this study, we aimed to determine the regulation of renal tubular glucose transporters during severe experimental inflammation. Male C57BL/6J mice were injected with LPS or proinflammatory cytokines, and renal perfusion, glomerular filtration rate (GFR), fractional glucose excretion, and expression of tubular glucose transporters were determined. We found a decreased plasma glucose concentration with impaired renal tissue perfusion and GFR and increased fractional glucose excretion associated with decreased expression of SGLT2, SGLT3, and GLUT2 after LPS injection. Similar alterations were observed after application of TNF-alpha, IL-1beta, IL-6, or IFN-gamma. To clarify the role of proinflammatory cytokines, we performed LPS injections in knockout mice with deficiencies for TNF-alpha, IL-1 receptor type 1, IFN-gamma, or IL-6 as well as LPS injections in glucocorticoid-treated wild-type mice. LPS-induced alterations of glucose transporters also were present in single-cytokine knockout mice. In contrast, glucocorticoid treatment clearly attenuated LPS-induced changes in renal glucose transporter expression and improved GFR and fractional glucose excretion. LPS-induced decrease of renal perfusion was not improved by glucocorticoids, indicating a minor role of ischemia in the development of septic renal dysfunction. Our results demonstrate modifications of tubular glucose transporters during severe inflammation that are probably mediated by proinflammatory cytokines and account for the development of ARF with increased fractional glucose excretion. In addition, our findings provide an explanation why single anti-cytokine strategies fail in the therapy of septic patients and contribute to an understanding of the beneficial effects of glucocorticoids on septic renal dysfunction.

Arguing for the motion: yes, RAGE is a receptor for advanced glycation endproducts.

R Ramasamy — 2008-01-21T14:24:22-00:00

Mol Nutr Food Res, Vol. 51, No. 9. (September 2007), pp. 1111-1115.

Advanced glycation endproducts (AGEs) are an heterogenous class of compounds formed by diverse stimuli, including hyperglycemia, oxidative stress, inflammation, renal failure, and innate aging. Recent evidence suggests that dietary sources of AGE may contribute to pathology. AGEs impart diverse effects in cells; evidence strongly suggests that crosslinking of proteins by AGEs may irrevocably alter basement membrane integrity and function. In addition, the ability of AGEs to bind to cells and activate signal transduction, thereby affecting broad properties in the cellular milieu, indicates that AGEs are not innocent bystanders in the diseases of AGEing. Here, we present evidence that receptor for AGE (RAGE) is a receptor for AGEs.

Hsp90--from signal transduction to cell transformation.

MA Brown — 2008-01-21T12:52:54-00:00

Biochem Biophys Res Commun, Vol. 363, No. 2. (16 November 2007), pp. 241-246.

The molecular chaperone, Hsp90, facilitates the maturation and/or activation of over 100 'client proteins' involved in signal transduction and transcriptional regulation. Largely an enigma among the families of heat shock proteins, Hsp90 is central to processes broadly ranging from cell cycle regulation to cellular transformation. Here, we review the contemporary body of knowledge regarding the biochemical mechanisms of Hsp90 and update the most current paradigms defining its involvement in both normal and pathological cell physiology.

Advanced glycation end products induce tubular epithelial-myofibroblast transition through the RAGE-ERK1/2 MAP kinase signaling pathway.

JH Li — 2008-01-20T08:08:44-00:00

Am J Pathol, Vol. 164, No. 4. (April 2004), pp. 1389-1397.

Advanced glycation end products (AGEs) have been shown to play a role in tubular epithelial-myofibroblast transdifferentiation (TEMT) in diabetic nephropathy, but the intracellular signaling pathway remains unknown. We report here that AGEs signal through the receptor for AGEs (RAGE) to induce TEMT, as determined by de novo expression of a mesenchymal marker (alpha-smooth muscle actin, alpha-SMA) and loss of epithelial marker (E-cadherin), directly through the MEK1-ERK1/2 MAP kinase pathway, which is TGF-beta independent. This is supported by the following findings: AGEs induced de novo alpha-SMA mRNA expression as early as 2 hours followed by a loss of E-cadherin before TGF-beta mRNA expression at 24 hours and occurred in the absence of TGF-beta and AGE-induced activation of ERK1/2 MAP kinase at 15 minutes and TEMT at 24 hours were completely blocked by a neutralizing RAGE antibody, a soluble RAGE receptor, an ERK1/2 MAP kinase inhibitor (PD98059), and DN-MEK1, but not by a neutralizing TGF-beta antibody. Thus, this study demonstrates that AGEs activate the RAGE-ERK1/2 MAP kinase pathway to mediate the early TEMT process. The findings from this study suggest that targeting the RAGE or the ERK MAP kinase pathway may provide new therapeutic strategies for diabetic nephropathy and shed new light on the pathogenesis of diabetic nephropathy.

Activation of tubular epithelial cells in diabetic nephropathy.

M Morcos — 2008-01-20T08:09:05-00:00

Diabetes, Vol. 51, No. 12. (December 2002), pp. 3532-3544.

Previous studies have shown that renal function in type 2 diabetes correlates better with tubular changes than with glomerular pathology. Since advanced glycation end products (AGEs; AGE-albumin) and in particular carboxymethyllysine (CML) are known to play a central role in diabetic nephropathy, we studied the activation of nuclear factor kappaB (NF-kappaB) in tubular epithelial cells in vivo and in vitro by AGE-albumin and CML. Urine samples from healthy control subjects (n = 50) and type 2 diabetic patients (n = 100) were collected and tested for excretion of CML and the presence of proximal tubular epithelial cells (pTECs). CML excretion was significantly higher in diabetic patients than in healthy control subjects (P < 0.0001) and correlated with the degree of albuminuria (r = 0.7, P < 0.0001), while there was no correlation between CML excretion and HbA(1c) (r = 0.03, P = 0.76). Urine sediments from 20 of 100 patients contained pTECs, evidenced by cytokeratin 18 positivity, while healthy control subjects (n = 50) showed none (P < 0.0001). Activated NF-kappaB could be detected in the nuclear region of excreted pTECs in 8 of 20 patients with pTECs in the urine sediment (40%). Five of eight NF-kappaBp65 antigen-positive cells stained positive for interleukin-6 (IL-6) antigen (62%), while only one of the NF-kappaB-negative cells showed IL-6 positivity. pTECs in the urine sediment correlated positively with albuminuria (r = 0.57, P < 0.0001) and CML excretion (r = 0.55, P < 0.0001). Immunohistochemistry in diabetic rat kidneys and a human diabetic kidney confirmed strong expression of NF-kappaB in tubular cells. To further prove an AGE/CML-induced NF-kappaB activation in pTECs, NF-kappaB activation was studied in cultured human pTECs by electrophoretic mobility shift assays (EMSAs) and Western blot. Stimulation of NF-kappaB binding activity was dose dependent and was one-half maximal at 250 nmol/l AGE-albumin or CML and time dependent at a maximum of activation after 4 days. Functional relevance of the observed NF-kappaB activation was demonstrated in pTECs transfected with a NF-kappaB-driven luciferase reporter plasmid and was associated with an increased release of IL-6 into the supernatant. The AGE- and CML-dependent activation of NF-kappaBp65 and NF-kappaB-dependent IL-6 expression could be inhibited using the soluble form of the receptor for AGEs (RAGE) (soluble RAGE [sRAGE]), RAGE-specific antibody, or the antioxidant thioctic acid. In addition transcriptional activity and IL-6 release from transfected cells could be inhibited by overexpression of the NF-kappaB-specific inhibitor kappaBalpha. The findings that excreted pTECs demonstrate activated NF-kappaB and IL-6 antigen and that AGE-albumin and CML lead to a perpetuated activation of NF-kappaB in vitro infer that a perpetuated increase in proinflammtory gene products, such as IL-6, plays a role in damaging the renal tubule.

CORUM: the comprehensive resource of mammalian protein complexes.

A Ruepp — 2008-01-18T03:50:41-00:00

Nucleic Acids Res, Vol. 36, No. Database issue. (January 2008)

Protein complexes are key molecular entities that integrate multiple gene products to perform cellular functions. The CORUM (http://mips.gsf.de/genre/proj/corum/index.html) database is a collection of experimentally verified mammalian protein complexes. Information is manually derived by critical reading of the scientific literature from expert annotators. Information about protein complexes includes protein complex names, subunits, literature references as well as the function of the complexes. For functional annotation, we use the FunCat catalogue that enables to organize the protein complex space into biologically meaningful subsets. The database contains more than 1750 protein complexes that are built from 2400 different genes, thus representing 12% of the protein-coding genes in human. A web-based system is available to query, view and download the data. CORUM provides a comprehensive dataset of protein complexes for discoveries in systems biology, analyses of protein networks and protein complex-associated diseases. Comparable to the MIPS reference dataset of protein complexes from yeast, CORUM intends to serve as a reference for mammalian protein complexes.

A biochemical approach to identifying microRNA targets

Fedor Karginov — 2007-11-30T08:58:31-00:00

Proceedings of the National Academy of Sciences (27 November 2007), 0709971104.

Identifying the downstream targets of microRNAs (miRNAs) is essential to understanding cellular regulatory networks. We devised a direct biochemical method for miRNA target discovery that combined RNA-induced silencing complex (RISC) purification with microarray analysis of bound mRNAs. Because targets of miR-124a have been analyzed, we chose it as our model. We honed our approach both by examining the determinants of stable binding between RISC and synthetic target RNAs in vitro and by determining the dependency of both repression and RISC coimmunoprecipitation on miR-124a seed sites in two of its well characterized targets in vivo. Examining the complete spectrum of miR-124 targets in 293 cells yielded both a set that were down-regulated at the mRNA level, as previously observed, and a set whose mRNA levels were unaffected by miR-124a. Reporter assays validated both classes, extending the spectrum of mRNA targets that can be experimentally linked to the miRNA pathway. 10.1073/pnas.0709971104

S100B-RAGE-mediated augmentation of angiotensin II-induced activation of JAK2 in vascular smooth muscle cells is dependent on PLD2.

SS Shaw — 2008-01-16T02:41:50-00:00

Diabetes, Vol. 52, No. 9. (September 2003), pp. 2381-2388.

Angiotensin II (Ang II), a vasoactive peptide that is also considered a growth factor, has been implicated in both normal and diabetic cellular proliferation. We recently found that activation of janus kinase 2 (JAK2) is essential for the Ang II-induced proliferation of vascular smooth muscle cells (VSMCs) and that high glucose augments Ang II-induced proliferation of VSMCs by increasing signal transduction through activation of JAK2. Here, we demonstrate that S100B, a ligand for the receptor of advanced glycation end products (RAGEs), augmented both Ang II-induced tyrosine phosphorylation of JAK2 and cell proliferation in VSMCs in a receptor-dependent manner. We also found that S100B-RAGE interaction triggered intracellular generation of reactive oxygen species (ROS), VSMC proliferation, and JAK2 tyrosine phosphorylation via activation of phospholipase D (PLD)2. These results provide direct evidence for linkages between PLD2, ROS production, and S100B-RAGE-induced enhancement of Ang II-induced cell proliferation and activation of JAK2 in VSMCs.

Receptor for advanced glycation end products (RAGE) in a dash to the rescue: inflammatory signals gone awry in the primal response to stress.

K Herold — 2008-01-14T12:11:51-00:00

J Leukoc Biol, Vol. 82, No. 2. (August 2007), pp. 204-212.

The multiligand receptor for advanced glycation end products (RAGE) of the Ig superfamily transduces the biological impact of discrete families of ligands, including advanced glycation end products, certain members of the S100/calgranulin family, high mobility group box-1, Mac-1 (alpha(M)beta(2), CD11b/CD18), and amyloid-beta peptide and beta-sheet fibrils. Although structurally dissimilar, at least at the monomeric level, recent evidence suggests that oligomeric forms of these RAGE ligands may be especially apt to activate the receptor and up-regulate a program of inflammatory and tissue injury-provoking genes. The challenge in probing the biology of RAGE and its impact in acute responses to stress and the potential development of chronic disease is to draw the line between mechanisms that evoke repair versus those that sustain inflammation and tissue damage. In this review, we suggest the concept that the ligands of RAGE comprise a primal program in the acute response to stress. When up-regulated in environments laden with oxidative stress, inflammation, innate aging, or high glucose, as examples, the function of these ligand families may be transformed from ones linked to rapid repair to those that drive chronic disease. Identification of the threshold beyond which ligands of RAGE mediate repair versus injury is a central component in delineating optimal strategies to target RAGE in the clinic.

Vascular and inflammatory stresses mediate atherosclerosis via RAGE and its ligands in apoE mice.

E Harja — 2008-01-12T15:40:49-00:00

J Clin Invest, Vol. 118, No. 1. (January 2008), pp. 183-194.

Endothelial dysfunction is a key triggering event in atherosclerosis. Following the entry of lipoproteins into the vessel wall, their rapid modification results in the generation of advanced glycation endproduct epitopes and subsequent infiltration of inflammatory cells. These inflammatory cells release receptor for advanced glycation endproduct (RAGE) ligands, specifically S100/calgranulins and high-mobility group box 1, which sustain vascular injury. Here, we demonstrate critical roles for RAGE and its ligands in vascular inflammation, endothelial dysfunction, and atherosclerotic plaque development in a mouse model of atherosclerosis, apoE(-/-) mice. Experiments in primary aortic endothelial cells isolated from mice and in cultured human aortic endothelial cells revealed the central role of JNK signaling in transducing the impact of RAGE ligands on inflammation. These data highlight unifying mechanisms whereby endothelial RAGE and its ligands mediate vascular and inflammatory stresses that culminate in atherosclerosis in the vulnerable vessel wall.

Hypoxia-inducible Factor-1 Mediates Neuronal Expression of the Receptor for Advanced Glycation End Products following Hypoxia/Ischemia

Paola Pichiule — 2008-01-12T02:09:02-00:00

J. Biol. Chem., Vol. 282, No. 50. (14 December 2007), pp. 36330-36340.

Activation of the receptor for advanced glycation endproducts (RAGE) by its multiple ligands can trigger diverse signaling pathways with injurious or pro-survival consequences. In this study, we show that Rage mRNA and protein levels were stimulated in the mouse brain after experimental stroke and systemic hypoxia. In both cases, RAGE expression was primarily associated with neurons. Activation of RAGE-dependent pathway(s) post-ischemia appears to have a neuroprotective role because mice genetically deficient for RAGE exhibited increased infarct size 24 h after injury. Up-regulation of RAGE expression was also observed in primary neurons subjected to hypoxia or oxygen-glucose deprivation, an in vitro model of ischemia. Treatment of neurons with low concentrations of S100B decreased neuronal death after oxygen-glucose deprivation, and this effect was abolished by a neutralizing antibody against RAGE. Conversely, high concentrations of exogenous S100B had a cytotoxic effect that seems to be RAGE-independent. As an important novel finding, we demonstrate that hypoxic stimulation of RAGE expression is mediated by the transcription factor hypoxia-inducible factor-1. This conclusion is supported by the finding that HIF-1alpha down-regulation by Cre-mediated excision drastically decreased RAGE induction by hypoxia or desferrioxamine. In addition, we showed that the mouse RAGE promoter region contains at least one functional HIF-1 binding site, located upstream of the proposed transcription start site. A luciferase reporter construct containing this RAGE promoter fragment was activated by hypoxia, and mutation at the potential HIF-1 binding site decreased hypoxia-dependent promoter activation. Specific binding of HIF-1 to this putative HRE in hypoxic cells was detected by chromatin immunoprecipitation assay. 10.1074/jbc.M706407200

Identification, classification, and expression of RAGE gene splice variants.

Barry I Hudson — 2007-12-31T05:15:32-00:00

FASEB J (18 December 2007)

The receptor for advanced glycation end-products (RAGE) is a single-transmembrane, multiligand receptor of the immunoglobulin superfamily. RAGE up-regulation is implicated in numerous pathological states including vascular disease, diabetes, cancer, and neurodegeneration. The understanding of the regulation of RAGE is important in both disease pathogenesis and normal homeostasis. Here, we demonstrate the characterization and identification of human RAGE splice variants by analysis of RAGE cDNA from tissue and cells. We identified a vast range of splice forms that lead to changes in the protein coding region of RAGE, which we have classified according to the Human Gene Nomenclature Committee (HGNC). These resulted in protein changes in the ligand-binding domain of RAGE or the removal of the transmembrane domain and cytosolic tail. Analysis of splice variants for premature termination codons reveals approximately 50% of identified variants are targeted to the nonsense-mediated mRNA decay pathway. Expression analysis revealed the RAGE_v1 variant to be the primary secreted soluble isoform of RAGE. Taken together, identification of functional splice variants of RAGE underscores the biological diversity of the RAGE gene and will aid in the understanding of the gene in the normal and pathological state. -Hudson, B. I., Carter, A. M., Harja, E., Kalea, A. Z., Arriero, M., Yang, H., Grant, P. J., Schmidt, A. M. Identification, classification, and expression of RAGE gene splice variants.

Blockade of receptor for advanced glycation endproducts: a new target for therapeutic intervention in diabetic complications and inflammatory disorders.

BI Hudson — 2007-12-01T16:12:12-00:00

Arch Biochem Biophys, Vol. 419, No. 1. (1 November 2003), pp. 80-88.

The glycation and oxidation of proteins/lipids leads to the generation of a new class of biologically active moieties, the advanced glycation endproducts (AGEs). Recent studies have elucidated that carboxymethyllysine (CML) adducts of proteins/lipids are a highly prevalent AGE in vivo. CML-modified adducts are signal transduction ligands of the receptor for AGE (RAGE), a member of the immunoglobulin superfamily. Importantly, CML-modified adducts accumulate in diverse settings. In addition to enhanced formation in settings of high glucose, these adducts form in inflammatory milieu. Studies performed both in vitro and in vivo have suggested that the proinflammatory/tissue destructive consequences of RAGE activation in the diabetic/inflamed environment may be markedly attenuated by blockade of the ligand-RAGE axis. Here, we will summarize the known consequences of RAGE activation in the tissues and highlight novel areas for therapeutic intervention in these disease states.

Reactome: a knowledgebase of biological pathways and processes

Imre Vastrik — 2007-03-17T17:43:21-00:00

Genome Biology, Vol. 8 (16 March 2007), R39.

Iron homeostasis.

NC Andrews — 2007-11-11T01:47:53-00:00

Annu Rev Physiol, Vol. 69 (2007), pp. 69-85.

Iron is needed by all mammalian cells but is toxic in excess. Specialized transport mechanisms conduct iron across cellular membranes. These are regulated to ensure homeostasis both systemically in living organisms and within individual cells. Over the past decade, major advances have been made in identifying and characterizing the proteins involved in the transport, handling, and homeostatic regulation of iron. Molecular understanding of these processes has provided important insights into the pathophysiology of human iron disorders.

Toxicity of green tea extracts and their constituents in rat hepatocytes in primary culture.

M Schmidt — 2007-11-10T09:32:00-00:00

Food Chem Toxicol, Vol. 43, No. 2. (February 2005), pp. 307-314.

Recent reports on sporadic cases of liver disorders (acute hepatitis, icterus, hepatocellular necrosis) after ingestion of dietary supplements based on hydro-alcoholic extracts from green tea leaves led to restrictions of the marketing of such products in certain countries of the EU. Since green tea is considered to exert a number of beneficial health effects, and, therefore, green tea products are widely used as dietary supplements, we were interested in the possible mechanism of hepatotoxicity of green tea extracts and in the components involved in such effects. Seven hours after seeding on collagen, rat hepatocytes in primary culture were treated with various hydro-alcoholic green tea extracts (two different native 80% ethanolic dry extracts and an 80% ethanolic dry extract cleared from lipophilic compounds). Cells were washed, and reduction of resazurin, used as a viability parameter monitoring intact mitochondrial function, was determined. It was found that all seven green tea extracts examined enhanced resazurin reduction significantly at a concentration range of 100-500 microg/ml medium, while a significant decrease was observed at 1-3mg/ml medium. Decreased levels were concomitant with abundant necrosis as observed by microscopic inspection of the cultures and with increased leakage of lactate dehydrogenase activity from the cells. In a separate series of experiments, the green tea constituents (-)-epicatechin, (-)-epigallocatechin-3-gallate, caffeine and theanine were tested at concentrations reflecting their levels in a typical green tea extract. Synthetic (+)-epigallocatechin (200 microM) was used for comparison. Cytotoxicity was found with (-)-epigallocatechin-3-gallate only. The concomitant addition of 0.25 mM ascorbate/0.05 mM alpha-tocopherol had no influence on cytotoxicity. In conclusion, our results suggest that high concentrations of green tea extract can exert acute toxicity in rat liver cells. (-)-Epigallocatechin-3-gallate seems to be a key constituent responsible for this effect. The relatively low bioavailability of catechins reported after oral exposure to green tea argues, however, against a causal role of these constituents in the reported liver disorders.

Reproductive factors and familial predisposition for breast cancer by age 50 years. A case-control-family study for assessing main effects and possible gene-environment interaction.

H Becher — 2007-11-07T02:11:39-00:00

Int J Epidemiol, Vol. 32, No. 1. (February 2003), pp. 38-48.

BACKGROUND: The effect of environmental/lifestyle factors on breast cancer risk may be modified by genetic predisposition. METHODS: In a population-based case-control-family study performed in Germany including 706 cases by age 50 years, 1381 population, and 252 sister controls, we investigated main effects for environmental/lifestyle factors and genetic susceptibility and gene-environment interaction (G x E). Different surrogate measures for genetic predisposition using pedigree information were used: first-degree family history of breast or ovarian cancer; and gene carrier probability using a genetic model based on rare dominant genes. Possible G x E interaction was studied by (1) logistic regression using cases and population controls including an interaction term; (2) comparing results using sister controls and population controls; (3) case-only analysis with logistic regression and (4) a mixture logistic model. RESULTS: Familial predisposition showed the strongest main effect and the estimated gene carrier probability gave the best fit. High parity and longer duration of breastfeeding reduced breast cancer risk significantly, a history of abortions increased risk and age at menarche showed no significant effect. We found significant G x E interaction between parity and genetic susceptibility using different surrogate measures. In women most likely to have a high genetic susceptibility, high parity was less protective. Later age at menarche was protective in women with a positive family history. No evidence for G x E interaction was found for breastfeeding and abortion. CONCLUSIONS: These findings corroborate results from other studies and provide further evidence that the magnitude of protection from parity is reduced in women most likely to have a genetic risk in spite of the limitations of using surrogate genetic measures.

The full-ORF clone resource of the German cDNA Consortium

Stephanie Bechtel — 2007-11-01T21:37:11-00:00

BMC Genomics, Vol. 8 (31 October 2007), 399.

Utilization of extended donor criteria in liver transplantation: a comprehensive review of the literature.

Arash Nickkholgh — 2007-10-29T03:54:10-00:00

Nephrol Dial Transplant, Vol. 22, No. suppl_8. (September 2007)

Organ shortage has driven many transplant programs to extend their criteria to accept donors. The goal of the present work is to further characterize the most important extended donor criteria (EDC) in liver transplantation and to identify factors that impact outcomes for this type of grafts through a comprehensive review of the most recent findings and current opinions. Age, steatosis, positive viral hepatitis serology, intensive care unit stay, and history of malignancy in donor have been the matter of substantial debate in recent years and are therefore discussed in further detail here. Cold and warm ischemic times have also been discussed separately as they have been identified as important independent risk factors for mortality. The use of grafts with EDC provides an immediate expansion of the donor pool. However, in order to optimize effective utilization of EDC, attempts should be made to carefully match the most appropriate graft-recipient pair.

Partial liver transplantation-living donor liver transplantation and split liver transplantation.

Sascha A Müller — 2007-10-29T03:53:49-00:00

Nephrol Dial Transplant, Vol. 22, No. suppl_8. (September 2007)

In the last two decades, liver transplantation (LTx) has become the treatment of choice for several liver diseases including hepatocellular carcinoma in selected cases. Improvements in surgical and anesthesiological procedures have increased patient survival after LTx, resulting in excellent 1-year survival rates. The rate-limiting factor to further increase the number of LTx is the extreme shortage of suitable organs with the consequence that pediatric and adult patients are dying on the waiting list. At present, mortality reported for pediatric and adult patients on the waiting list is 10 to 20%. Living-donor liver transplantation and split liver transplantation are measurements to reduce the severe lack of cadaveric grafts by expanding the donor pool. Major centers around the world now routinely perform partial LTx in infants and adults with survival success equivalent to that after full-size liver transplantation.

Fulminant hepatic failure: etiology and indications for liver transplantation.

Daniel Gotthardt — 2007-10-21T05:22:56-00:00

Nephrol Dial Transplant, Vol. 22, No. suppl_8. (September 2007)

Fulminant hepatic failure is characterized by the development of severe liver injury with impaired synthetic capacity and encephalopathy in patients with previous normal liver or at least well compensated liver disease. The etiology of fulminant hepatic failure refers to a wide variety of causes, of which toxin-induced or viral hepatitis are most common. In spite of specific therapeutic options in distinctive etiologies, orthotopic liver transplantation is the only therapy proven to improve patient survival in the majority of patients. The outcome is determined by the complications like severe coagulopathy, infections, renal impairment or increased intracranial pressure. The decision for transplantation depends on the possibility of spontaneous hepatic recovery, which may be estimated by several factors. The most important variables for predicting the need of transplantation in fulminant hepatic failure are the degree of encephalopathy, patients age and the underlying cause of liver failure.

Renin-angiotensin system and cardiovascular risk

Roland Schmieder — 2007-10-15T10:05:43-00:00

The Lancet, Vol. 369, No. 9568., pp. 1208-1219.

Summary The renin-angiotensin system is a major regulatory system of cardiovascular and renal function. Basic research has revealed exciting new aspects, which could lead to novel or modified therapeutic approaches. Renin-angiotensin system blockade exerts potent antiatherosclerotic effects, which are mediated by their antihypertensive, anti-inflammatory, antiproliferative, and oxidative stress lowering properties. Inhibitors of the system--ie, angiotensin converting enzyme inhibitors and angiotensin receptor blockers, are now first-line treatments for hypertensive target organ damage and progressive renal disease. Their effects are greater than expected by their ability to lower blood pressure alone. Angiotensin receptor blockers reduce the frequency of atrial fibrillation and stroke. Renin-angiotensin system blockade delays or avoids the onset of type 2 diabetes and prevents cardiovascular and renal events in diabetic patients. Thus, blockade of this system will remain a cornerstone of our strategies to reduce cardiovascular risk.

Infrared-based protein detection arrays for quantitative proteomics.

C Loebke — 2007-10-12T02:50:49-00:00

Proteomics, Vol. 7, No. 4. (February 2007), pp. 558-564.

The advancement of efficient technologies to comply with the needs of systems biology and drug discovery has so far not received adequate attention. A substantial bottleneck for the time-resolved quantitative description of signaling networks is the limited throughput and the inadequate sensitivity of currently established methods. Here, we present an improved protein microarray-based approach towards the sensitive detection of proteins in the fg-range which is based on signal detection in the near-infrared range. The high sensitivity of the assay permits the specific quantification of proteins derived from as little as only 20,000 cells with an error rate of only 5%. The capacity is limited to the analysis of up to 500 different samples per microarray. Protein abundance is determined qualitatively, and quantitatively, if recombinant protein is available. This novel approach was called IPAQ (infrared-based protein arrays with quantitative readout). IPAQ offers a highly sensitive experimental approach superior to the established standard protein quantification technologies, and is suitable for quantitative proteomics. Employing the IPAQ approach, a detailed analysis of activated signaling networks in biopsy samples and of crosstalk between signaling modules as required in drug discovery strategies can easily be performed.

The mitogen-activated protein kinase p38 regulates activator protein 1 by direct phosphorylation of c-Jun.

Matjaz Humar — 2007-09-21T08:34:28-00:00

Int J Biochem Cell Biol (1 July 2007)

The involvement of p38 in fundamental physiological processes and the fact that deregulation often leads to disease indicates the potential impact of p38 dependent mechanisms. Here we demonstrate a new pathway that includes the induction of the mitogen activated protein kinase p38 by protein kinase C and results in a specific phosphorylation of c-Jun in T-lymphocytes. P38 directly phosphorylates c-Jun within its transactivation domain at serine 63 and serine 73 and thus posttranscriptionally affects the presence of DNA-bound phosphorylated c-Jun, a prerequisite for activator protein 1 dependent gene transcription. Moreover, DNA-binding activity of c-Fos, FosB, and JunB were also dependent on the p38 protein kinase activity, whereas JunD, Fra-1 and Fra-2 were not affected. Although we show that stress induced mitogen activated protein kinases share c-Jun as a substrate for phosphorylation, p38 mediated effects could not be rescued by the c-Jun N-terminal kinases. This demonstrates that the protein kinase p38 plays a unique and non-redundant role in posttranslational c-Jun regulation. The induction of a p38 dependent c-Jun phosphorylation was comparable in both CD4(+) and CD8(+) T-cells, proposing a ubiquitous pathway that is not linked to T-cell subtype and effector function. In contrast, ATF-2 was predominantly phosphorylated in CD8(+) T-cells. Different cell lines show p38-dependent c-Jun phosphorylation upon phorbol ester induction but there is evidence that the simian virus 40 large T-antigen may interfere with this pathway.

Activation of the receptor for advanced glycation end products triggers a p21(ras)-dependent mitogen-activated protein kinase pathway regulated by oxidant stress.

HM Lander — 2007-09-18T12:30:05-00:00

J Biol Chem, Vol. 272, No. 28. (11 July 1997), pp. 17810-17814.

Advanced glycation end products (AGEs) exert their cellular effects on cells by interacting with specific cellular receptors, the best characterized of which is the receptor for AGE (RAGE). The transductional processes by which RAGE ligation transmits signals to the nuclei of cells is unknown and was investigated. AGE-albumin, a prototypic ligand, activated p21(ras) in rat pulmonary artery smooth muscle cells that express RAGE, whereas nonglycated albumin was without effect. MAP kinase activity was enhanced at concentrations of AGE-albumin, which activated p21(ras) and NF-kappaB. Depletion of intracellular glutathione rendered cells more sensitive to AGE-mediated activation of this signaling pathway. In contrast, signaling was blocked by preventing p21(ras) from associating with the plasma membrane or mutating Cys118 on p21(ras) to Ser. Signaling was receptor-dependent, because it was prevented by blocking access to RAGE with either anti-RAGE IgG or by excess soluble RAGE. These data suggest that RAGE-mediated induction of cellular oxidant stress triggers a cascade of intracellular signals involving p21(ras) and MAP kinase, culminating in transcription factor activation. The molecular mechanism that triggers this pathway likely involves oxidant modification and activation of p21(ras).

Down-regulation of c-Fos/c-Jun AP-1 dimer activity by sumoylation.

G Bossis — 2007-09-17T10:30:06-00:00

Mol Cell Biol, Vol. 25, No. 16. (August 2005), pp. 6964-6979.

The inducible transcriptional complex AP-1, composed of c-Fos and c-Jun proteins, is crucial for cell adaptation to many environmental changes. While its mechanisms of activation have been extensively studied, how its activity is restrained is poorly understood. We report here that lysine 265 of c-Fos is conjugated by the peptidic posttranslational modifiers SUMO-1, SUMO-2, and SUMO-3 and that c-Jun can be sumoylated on lysine 257 as well as on the previously described lysine 229. Sumoylation of c-Fos preferentially occurs in the context of c-Jun/c-Fos heterodimers. Using nonsumoylatable mutants of c-Fos and c-Jun as well as a chimeric protein mimicking sumoylated c-Fos, we show that sumoylation entails lower AP-1 transactivation activity. Interestingly, single sumoylation at any of the three acceptor sites of the c-Fos/c-Jun dimer is sufficient to substantially reduce transcription activation. The lower activity of sumoylated c-Fos is not due to inhibition of protein entry into the nucleus, accelerated turnover, and intrinsic inability to dimerize or to bind to DNA. Instead, cell fractionation experiments suggest that decreased transcriptional activity of sumoylated c-Fos is associated with specific intranuclear distribution. Interestingly, the phosphorylation of threonine 232 observed upon expression of oncogenically activated Ha-Ras is known to superactivate c-Fos transcriptional activity. We show here that it also inhibits c-Fos sumoylation, revealing a functional antagonism between two posttranslational modifications, each occurring within a different moiety of a bipartite transactivation domain of c-Fos. Finally we report that the sumoylation of c-Fos is a dynamic process that can be reversed via multiple mechanisms. This supports the idea that this modification does not constitute a final inactivation step that necessarily precedes protein degradation.

Effect of bosentan on NF-kappaB, inflammation, and tissue factor in angiotensin II-induced end-organ damage.

DN Muller — 2007-09-17T09:54:42-00:00

Hypertension, Vol. 36, No. 2. (August 2000), pp. 282-290.

Reports on the effectiveness of endothelin receptor blockers in angiotensin (Ang) II-induced end-organ damage are conflicting, and the mechanisms involved are uncertain. We tested the hypothesis that endothelin (ET)(A/B) receptor blockade with bosentan (100 mg/kg by gavage after age 4 weeks) ameliorates cardiac and renal damage by decreasing inflammation in rats harboring both human renin and angiotensinogen genes (dTGR). Furthermore, we elucidated the effect of bosentan on tissue factor (TF), which is a key regulator of the extrinsic coagulation cascade. We compared bosentan with hydralazine (80 mg/L in the drinking water for 3 weeks) as a blood pressure control. Untreated dTGR featured hypertension, focal necrosis in heart and kidney, and a 45% mortality rate (9 of 20) at age 7 weeks. Compared with Sprague-Dawley controls, both systolic blood pressure and 24-hour albuminuria were increased in untreated dTGR (203+/-8 versus 111+/-2 mm Hg and 67.1+/-8.6 versus 0.3+/-0.06 mg/d at week 7, respectively). Bosentan and hydralazine both reduced blood pressure and cardiac hypertrophy. Mortality rate was markedly reduced by bosentan (1/15) and partially by hydralazine (4/15). However, only bosentan decreased albuminuria and renal injury. Untreated and hydralazine-treated dTGR showed increased nuclear factor (NF)-kappaB and AP-1 expression in the kidney and heart; the p65 NF-kappaB subunit was increased in the endothelium, vascular smooth muscles cells, infiltrating cells, glomeruli, and tubules. In the heart and kidney, ET(A/B) receptor blockade inhibited NF-kappaB and AP-1 activation compared with hydralazine treatment. Macrophage infiltration, ICAM-1 expression, and the integrin expression on infiltrating cells were markedly reduced. Renal vasculopathy was accompanied by increased tissue factor expression on macrophages and vessels of untreated and hydralazine-treated dTGR, which was markedly reduced by bosentan. Thus, ET(A/B) receptor blockade inhibits NF-kappaB and AP-1 activation and the NF-kappaB- and/or AP-1-regulated genes ICAM-1, VCAM-1, and TF, independent of blood pressure-related effects. We conclude that Ang II-induced NF-kappaB and AP-1 activation and subsequent inflammation and coagulation involve at least in part the ET(A/B) receptors.

Expression of advanced glycation end products and their cellular receptor RAGE in diabetic nephropathy and nondiabetic renal disease.

N Tanji — 2007-09-13T09:26:45-00:00

J Am Soc Nephrol, Vol. 11, No. 9. (September 2000), pp. 1656-1666.

Advanced glycation end products (AGE) contribute to diabetic tissue injury by two major mechanisms, i.e., the alteration of extracellular matrix architecture through nonenzymatic glycation, with formation of protein crosslinks, and the modulation of cellular functions through interactions with specific cell surface receptors, the best characterized of which is the receptor for AGE (RAGE). Recent evidence suggests that the AGE-RAGE interaction may also be promoted by inflammatory processes and oxidative cellular injury. To characterize the distributions of AGE and RAGE in diabetic kidneys and to determine their specificity for diabetic nephropathy, an immunohistochemical analysis of renal biopsies from patients with diabetic nephropathy (n = 26), hypertensive nephrosclerosis (n = 7), idiopathic focal segmental glomerulosclerosis (n = 11), focal sclerosis secondary to obesity (n = 7), and lupus nephritis (n = 11) and from normal control subjects (n = 2) was performed, using affinity-purified antibodies raised to RAGE and two subclasses of AGE, i.e., N(epsilon)-(carboxymethyl)-lysine (CML) and pentosidine (PENT). AGE were detected equally in diffuse and nodular diabetic nephropathy. CML was the major AGE detected in diabetic mesangium (96%), glomerular basement membranes (GBM) (42%), tubular basement membranes (85%), and vessel walls (96%). In diabetic nephropathy, PENT was preferentially located in interstitial collagen (90%) and was less consistently observed in vessel walls (54%), mesangium (77%), GBM (4%), and tubular basement membranes (31%). RAGE was expressed on normal podocytes and was upregulated in diabetic nephropathy. The restriction of RAGE mRNA expression to glomeruli was confirmed by reverse transcription-PCR analysis of microdissected renal tissue compartments. The extent of mesangial and GBM immunoreactivity for CML, but not PENT, was correlated with the severity of diabetic glomerulosclerosis, as assessed pathologically. CML and PENT were also identified in areas of glomerulosclerosis and arteriosclerosis in idiopathic and secondary focal segmental glomerulosclerosis, hypertensive nephrosclerosis, and lupus nephritis. In active lupus nephritis, CML and PENT were detected in the proliferative glomerular tufts and crescents. In conclusion, CML is a major AGE in renal basement membranes in diabetic nephropathy, and its accumulation involves upregulation of RAGE on podocytes. AGE are also accumulated in acute inflammatory glomerulonephritis secondary to systemic lupus erythematosus, possibly via enzymatic oxidation of glomerular matrix proteins.

Current methods for phosphoprotein isolation and enrichment.

SR Schmidt — 2007-09-12T05:20:09-00:00

J Chromatogr B Analyt Technol Biomed Life Sci, Vol. 849, No. 1-2. (15 April 2007), pp. 154-162.

The phosphorylation of proteins is a central paradigm of signal transduction. The substitution of neutral hydroxyl groups of serine, threonine and tyrosine with a negatively charged phosphate group alters the physicochemical and immunogenic properties of the protein, which then can be used to isolate these isoforms. In the last decades several different techniques were applied, attempting to selectively enrich protein populations with this post-translational modification. This review aims to give an overview on the arsenal of available methods to extract phosphoproteins focusing on chromatographic approaches.

The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins.

TG Schmidt — 2007-09-09T08:08:21-00:00

Nat Protoc, Vol. 2, No. 6. (2007), pp. 1528-1535.

The Strep-tag II is an eight-residue minimal peptide sequence (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) that exhibits intrinsic affinity toward streptavidin and can be fused to recombinant proteins in various fashions. We describe a protocol that enables quick and mild purification of corresponding Strep-tag II fusion proteins--including their complexes with interacting partners--both from bacterial and eukaryotic cell lysates using affinity chromatography on a matrix carrying an engineered streptavidin (Strep-Tactin), which can be accomplished within 1 h. A high-affinity monoclonal antibody (StrepMAB-Immo) permits stable immobilization of Strep-tag II fusion proteins to solid surfaces, for example, for surface plasmon resonance analysis. Selective and sensitive detection on western blots is achieved with Strep-Tactin/enzyme conjugates or another monoclonal antibody (StrepMAB-Classic). Thus, the Strep-tag II, which is short, biologically inert, proteolytically stable and does not interfere with membrane translocation or protein folding, offers a versatile tool both for the rapid isolation of a functional gene product and for its detection or molecular interaction analysis.

NF-kappaB and AP-1 connection: mechanism of NF-kappaB-dependent regulation of AP-1 activity.

S Fujioka — 2007-09-05T07:38:56-00:00

Mol Cell Biol, Vol. 24, No. 17. (September 2004), pp. 7806-7819.

Nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1) transcription factors regulate many important biological and pathological processes. Activation of NF-kappaB is regulated by the inducible phosphorylation of NF-kappaB inhibitor IkappaB by IkappaB kinase. In contrast, Fos, a key component of AP-1, is primarily transcriptionally regulated by serum responsive factors (SRFs) and ternary complex factors (TCFs). Despite these different regulatory mechanisms, there is an intriguing possibility that NF-kappaB and AP-1 may modulate each other, thus expanding the scope of these two rapidly inducible transcription factors. To determine whether NF-kappaB activity is involved in the regulation of fos expression in response to various stimuli, we analyzed activity of AP-1 and expression of fos, fosB, fra-1, fra-2, jun, junB, and junD, as well as AP-1 downstream target gene VEGF, using MDAPanc-28 and MDAPanc-28/IkappaBalphaM pancreatic tumor cells and wild-type, IKK1-/-, and IKK2-/- murine embryonic fibroblast cells. Our results show that elk-1, a member of TCFs, is one of the NF-kappaB downstream target genes. Inhibition of NF-kappaB activity greatly decreased expression of elk-1. Consequently, the reduced level of activated Elk-1 protein by extracellular signal-regulated kinase impeded constitutive, serum-, and superoxide-inducible c-fos expression. Thus, our study revealed a distinct and essential role of NF-kappaB in participating in the regulation of elk-1, c-fos, and VEGF expression.

SuperHirn - a novel tool for high resolution LC-MS-based peptide/protein profiling.

Lukas N Mueller — 2007-09-05T05:59:08-00:00

Proteomics (28 August 2007)

Label-free quantification of high mass resolution LC-MS data has emerged as a promising technology for proteome analysis. Computational methods are required for the accurate extraction of peptide signals from LC-MS data and the tracking of these features across the measurements of different samples. We present here an open source software tool, SuperHirn, that comprises a set of modules to process LC-MS data acquired on a high resolution mass spectrometer. The program includes newly developed functionalities to analyze LC-MS data such as feature extraction and quantification, LC-MS similarity analysis, LC-MS alignment of multiple datasets, and intensity normalization. These program routines extract profiles of measured features and comprise tools for clustering and classification analysis of the profiles. SuperHirn was applied in an MS1-based profiling approach to a benchmark LC-MS dataset of complex protein mixtures with defined concentration changes. We show that the program automatically detects profiling trends in an unsupervised manner and is able to associate proteins to their correct theoretical dilution profile.