A Versatile Structural Domain Analysis Server Using Profile Weight Matrices

Journal of Chemical Information and Computer Sciences. Mar-Apr, 2002  |  Pubmed ID: 11911710

The WEB tool "AnDom" assigns to a given protein sequence all experimentally determined structural domains contained within it, including multidomain and large proteins. The server uses profile specific matrices from custom generated multiple sequence alignments of all known SCOP domains (SCOP version 1.50). Prediction time is short allowing numerous applications for structural genomics including investigation of complex eucaryotic protein families. The WWW server is at http://www.bork.embl-heidelberg.de/AnDom, and profiles can be downloaded at ftp.bork.embl-heidelberg.de/pub/users/ schmidt/AnDom.

Comparative Genome Analysis and Pathway Reconstruction

Pharmacogenomics. Mar, 2002  |  Pubmed ID: 11972445

Pathway reconstruction builds on genome and biochemical data with the aim of reconstructing higher level interactions between identified enzymes in a specific genome, in particular the different enzyme pathways (species or individual/patient). Metabolite flow in a pathway is analyzed by different tools, such as elementary mode analysis. This reveals key enzymes and pharmacological targets in the enzyme network. An overview of bioinformatic tools and algorithms for these tasks, application examples and recent results from these techniques are presented. Target selection, drug development and optimization can all be sped up using these approaches.

Suspected Utility of Enzymes with Multiple Activities in the Small Genome Mycoplasma Species: the Replacement of the Missing "household" Nucleoside Diphosphate Kinase Gene and Activity by Glycolytic Kinases

Omics : a Journal of Integrative Biology. 2002  |  Pubmed ID: 12427276

The small genome Mollicutes whose DNAs are completely sequenced (Mycoplasma genitalium, Mycoplasma pneumoniae, Mycoplasma pulmonis, and Ureaplasma urealyticum [parvum]) lack a gene (ndk) for the presumably essential nucleoside diphosphate kinase (NDPK). We hypothesized that other activities might replace NDPK activity. We found in M. genitalium G37(T), Mycoplasma pneumoniae FH(T), Mycoplasma fermentans PG18(T), and Mycoplasma capricolum subsp. capricolum Kid(T) that their 6-phosphofructokinases (6-PFKs), phosphoglycerate kinases (PGKs), pyruvate kinases (PKs), and acetate kinases (AKs), besides reactant ADP/ATP, could use other ribo- and deoxyribo-purine and pyrimidine NDPs and NTPs. These activities could compensate for the absence of an orthologous ndk gene in the Mycoplasmataceae. They suggest a metabolically varied and consequential role for unrelated and perhaps unsuspected "replacement" or compensatory enzymes that may confound metabolic prediction. We partially purified and biochemically characterized the PKs, 6-PFKs, PGKs, and AKs from M. capricolum subsp. capricolum Kid(T) and M. fermentans PG18(T).

A Software Tool-box for Analysis of Regulatory RNA Elements

Nucleic Acids Research. Jul, 2003  |  Pubmed ID: 12824342

We describe an integrated tool-box to identify regulatory RNA elements. The RNA analyzer collects general and specific information on any submitted RNA sequence or batch of sequences in FASTA format. It determines and rapidly scans the different regions of an RNA (including 5' UTR, CDS, 3' UTR in mRNA) and screens for specific RNA signals (in each of these regions, e.g. polyA-site, AU rich region etc. in 3' UTR). It runs a fast folding RNA routine to provide an overview of the RNA fold. Furthermore it analyzes structure content, fold energy and stem loops. In addition, consensus templates are used to determine whether there are any functional structures present for translational control (template: IRE), structured RNA (template: tRNA consensus) or catalytic RNA (template: trans-splicing RNA), giving indications as to how well the structures found match to these templates. The tool box has been implemented as a WWW server at http://wb2x01.biozentrum.uni-wuerzburg.de/.

Metabolites: a Helping Hand for Pathway Evolution?

Trends in Biochemical Sciences. Jun, 2003  |  Pubmed ID: 12826406

The evolution of enzymes and pathways is under debate. Recent studies show that recruitment of single enzymes from different pathways could be the driving force for pathway evolution. Other mechanisms of evolution, such as pathway duplication, enzyme specialization, de novo invention of pathways or retro-evolution of pathways, appear to be less abundant. Twenty percent of enzyme superfamilies are quite variable, not only in changing reaction chemistry or metabolite type but in changing both at the same time. These variable superfamilies account for nearly half of all known reactions. The most frequently occurring metabolites provide a helping hand for such changes because they can be accommodated by many enzyme superfamilies. Thus, a picture is emerging in which new pathways are evolving from central metabolites by preference, thereby keeping the overall topology of the metabolic network.

Toward Computer-based Cleavage Site Prediction of Cysteine Endopeptidases

Biological Chemistry. Jun, 2003  |  Pubmed ID: 12887057

Identification of relevant substrates is essential for elucidation of in vivo functions of peptidases. The recent availability of the complete genome sequences of many eukaryotic organisms holds the promise of identifying specific peptidase substrates by systematic proteome analyses in combination with computer-based screening of genome databases. Currently available proteomics and bioinformatics tools are not sufficient for reliable endopeptidase substrate predictions. To address these shortcomings the bioinformatics tool 'PEPS' (Prediction of Endopeptidase Substrates) has been developed and is presented here. PEPS uses individual rule-based endopeptidase cleavage site scoring matrices (CSSM). The efficiency of PEPS in predicting putative caspase 3, cathepsin B and cathepsin L cleavage sites is demonstrated in comparison to established algorithms. Mortalin, a member of the heat shock protein family HSP70, was identified by PEPS as a putative cathepsin L substrate. Comparative proteome analyses of cathepsin L-deficient and wild-type mouse fibroblasts showed that mortalin is enriched in the absence of cathepsin L. These results indicate that CSSM/PEPS can correctly predict relevant peptidase substrates.

A Method for Classifying Metabolites in Topological Pathway Analyses Based on Minimization of Pathway Number

Bio Systems. Aug, 2003  |  Pubmed ID: 12941488

Metabolic pathway analysis based on the concept of elementary flux mode is a valuable tool for reconstruction of bacterial metabolisms and in predicting optimal conversion yields in biotechnology. However, pathway analysis of large and highly entangled metabolic networks meets the problem of combinatorial explosion of possible routes across the networks. Here we propose a method for coping with this problem by suitably classifying metabolites as external or internal. External metabolites are considered to have buffered concentrations while internal metabolites have to fulfil a balance condition at steady state. For many substances such as nutrients and excreted products, there are biochemical reasons to classify them as external. In addition, other substances (especially at central branching points) can operationally be considered external in order to avoid combinatorial explosion. We suggest to find such a classification of metabolites that minimizes the number of elementary flux modes (pathways). This is motivated by the objectives of finding such a description of the system that reduces as much as possible the amount of necessary data and of removing the ambiguity and arbitrariness in the classification of metabolites in an automated, systematic way. For networks of moderate size, the solution to this combinatorial minimization problem can be found by exhaustive search. To tackle also larger systems, a stochastic optimization program based on the Metropolis algorithm was developed. Both methods are applied, for illustration, to several reaction schemes including a larger network representing glutathione metabolism.

In Search of the Prototype of Nitric Oxide Synthase

FEBS Letters. Nov, 2003  |  Pubmed ID: 14596904

Recent identification of the prokaryotic genes related to the catalytic oxygenase domain of mammalian nitric oxide synthase (NOS) has led to speculations on the origins of the NO signaling network. NOS activity in eukaryotes relies on the concerted action of the oxygenase domain with an electron-donating reductase domain that is fused to it. A fused reductase domain is, however, absent in prokaryotes. Consequently, we searched bacterial genomes for homologs of the reductase domain and identified candidate genes. On the basis of genomic sequence and protein structural analysis, we here propose that sulfite reductase flavoprotein is a prototype of the mammalian NOS reductase domain and a complementing interaction partner of the bacterial NOS oxygenase protein.

An Open Source Protein Gel Documentation System for Proteome Analyses

Journal of Chemical Information and Computer Sciences. Jan-Feb, 2004  |  Pubmed ID: 14741024

Data organization and data mining represents one of the main challenges for modern high throughput technologies in pharmaceutical chemistry and medical chemistry. The presented open source documentation and analysis system provides an integrated solution (tutorial, setup protocol, sources, executables) aimed at substituting the traditionally used lab-book. The data management solution provided incorporates detailed information about the processing of the gels and the experimental conditions used and includes basic data analysis facilities which can be easily extended. The sample database and User-Interface are available free of charge under the GNU license from http://webber.physik.uni-freiburg.de/~fallerd/tutorial.htm.

NO-bound Myoglobin: Structural Diversity and Dynamics of the NO Ligand

Journal of the American Chemical Society. Feb, 2004  |  Pubmed ID: 14971914

We used femtosecond infrared polarization spectroscopy and density functional theory in a study on the key signaling molecule nitric oxide (NO) bound to myoglobin. Our results show that after photolysis, a substantial fraction of NO recombines within the first few picoseconds. We discovered that the diatomic ligand is severely tilted in the protein and present evidence that the Fe-NO moiety can sample a wide range of off-axis tilting and bending conformations.

A Novel Conserved Family of Nitric Oxide Synthase?

Trends in Biochemical Sciences. May, 2004  |  Pubmed ID: 15130557

Phylogeny of Firmicutes with Special Reference to Mycoplasma (Mollicutes) As Inferred from Phosphoglycerate Kinase Amino Acid Sequence Data

International Journal of Systematic and Evolutionary Microbiology. May, 2004  |  Pubmed ID: 15143038

The phylogenetic position of the Mollicutes has been re-examined by using phosphoglycerate kinase (Pgk) amino acid sequences. Hitherto unpublished sequences from Mycoplasma mycoides subsp. mycoides, Mycoplasma hyopneumoniae and Spiroplasma citri were included in the analysis. Phylogenetic trees based on Pgk data indicated a monophyletic origin for the Mollicutes within the Firmicutes, whereas Bacilli (Firmicutes) and Clostridia (Firmicutes) appeared to be paraphyletic. With two exceptions, i.e. Thermotoga (Thermotogae) and Fusobacterium (Fusobacteria), which clustered within the Firmicutes, comparative analyses show that at a low taxonomic level, the resolved phylogenetic relationships that were inferred from both the Pgk protein and 16S rRNA gene sequence data are congruent.

Pharmacogenomic Strategies Against Resistance Development in Microbial Infections

Pharmacogenomics. Jun, 2004  |  Pubmed ID: 15165173

There are several promising new strategies against resistance development in microbial infections. This paper discusses typical experimental and bioinformatical strategies to study the impact of infectious challenges on host-pathogen interaction, followed by several novel approaches and sources for new pharmaceutical strategies against resistance development. Genomics reveals promising new targets by providing a better understanding of cellular pathways, through the identification of new pathways, and by identifying new intervention areas, such as phospholipids, glycolipids, innate immunity, and antibiotic peptides. Additional antibiotic resources come from new genomes, including marine organisms, lytic phages and probiotic strategies. A system perspective regards all interactions between the host, pathogen and environment to develop new pharmacogenomic strategies against resistance development.

Riboswitch Finder--a Tool for Identification of Riboswitch RNAs

Nucleic Acids Research. Jul, 2004  |  Pubmed ID: 15215370

We describe a dedicated RNA motif search program and web server to identify RNA riboswitches. The Riboswitch finder analyses a given sequence using the web interface, checks specific sequence elements and secondary structure, calculates and displays the energy folding of the RNA structure and runs a number of tests including this information to determine whether high-sensitivity riboswitch motifs (or variants) according to the Bacillus subtilis type are present in the given RNA sequence. Batch-mode determination (all sequences input at once and separated by FASTA format) is also possible. The program has been implemented and is available both as local software for in-house installation and as a web server at http://www.biozentrum.uni-wuerzburg.de/bioinformatik/Riboswitch/.

Accurate and Robust Phylogeny Estimation Based on Profile Distances: a Study of the Chlorophyceae (Chlorophyta)

BMC Evolutionary Biology. Jun, 2004  |  Pubmed ID: 15222898

In phylogenetic analysis we face the problem that several subclade topologies are known or easily inferred and well supported by bootstrap analysis, but basal branching patterns cannot be unambiguously estimated by the usual methods (maximum parsimony (MP), neighbor-joining (NJ), or maximum likelihood (ML)), nor are they well supported. We represent each subclade by a sequence profile and estimate evolutionary distances between profiles to obtain a matrix of distances between subclades.

Shared Components of Protein Complexes--versatile Building Blocks or Biochemical Artefacts?

BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology. Dec, 2004  |  Pubmed ID: 15551274

Protein complexes perform many important functions in the cell. Large-scale studies of protein-protein interactions have not only revealed new complexes but have also placed many proteins into multiple complexes. Whilst the advocates of hypothesis-free research touted the discovery of these shared components as new links between diverse cellular processes, critical commentators denounced many of the findings as artefacts, thus questioning the usefulness of large-scale approaches. Here, we survey proteins known to be shared between complexes, as established in the literature, and compare them to shared components found in high-throughput screens. We discuss the various challenges to the identification and functional interpretation of bona fide shared components, namely contaminants, variant and megacomplexes, and transient interactions, and suggest that many of the novel shared components found in high-throughput screens are neither the results of contamination nor central components, but appear to be primarily regulatory links in cellular processes.

Metabolic Interdependence of Obligate Intracellular Bacteria and Their Insect Hosts

Microbiology and Molecular Biology Reviews : MMBR. Dec, 2004  |  Pubmed ID: 15590782

Mutualistic associations of obligate intracellular bacteria and insects have attracted much interest in the past few years due to the evolutionary consequences for their genome structure. However, much less attention has been paid to the metabolic ramifications for these endosymbiotic microorganisms, which have to compete with but also to adapt to another metabolism--that of the host cell. This review attempts to provide insights into the complex physiological interactions and the evolution of metabolic pathways of several mutualistic bacteria of aphids, ants, and tsetse flies and their insect hosts.

L1Base: from Functional Annotation to Prediction of Active LINE-1 Elements

Nucleic Acids Research. Jan, 2005  |  Pubmed ID: 15608246

L1Base is a dedicated database containing putatively active LINE-1 (L1) insertions residing in human and rodent genomes that are as follows: (i) intact in the two open reading frames (ORFs), full-length L1s (FLI-L1s) and (ii) intact ORF2 but disrupted ORF1 (ORF2-L1s). In addition, due to their regulatory potential, the full-length (>6000 bp) non-intact L1s (FLnI-L1s) were also included in the database. Application of a novel annotation methodology, L1Xplorer, allowed in-depth annotation of functional sequence features important for L1 activity, such as transcription factor binding sites and amino acid residues. The L1Base is available online at http://l1base.molgen.mpg.de. In addition, the data stored in the database can be accessed from the Ensembl web browser via a DAS service (http://l1das.molgen.mpg.de:8080/das).

ProfDist: a Tool for the Construction of Large Phylogenetic Trees Based on Profile Distances

Bioinformatics (Oxford, England). May, 2005  |  Pubmed ID: 15677706

SUMMARY: ProfDist is a user-friendly software package using the profile-neighbor-joining method (PNJ) in inferring phylogenies based on profile distances on DNA or RNA sequences. It is a tool for reconstructing and visualizing large phylogenetic trees providing new and standard features with a special focus on time efficency, robustness and accuracy. AVAILABILITY: A Windows version of ProfDist comes with a graphical user interface and is freely available at http://profdist.bioapps.biozentrum.uni-wuerzburg.de

Current Efforts in the Analysis of RNAi and RNAi Target Genes

Briefings in Bioinformatics. Mar, 2005  |  Pubmed ID: 15826358

RNAi is RNA interference by short RNAs. It influences gene-expression by down-regulation of mRNAs, typically by complementarity to the 3' UTR (untranslated region) of the mRNA. microRNAs (miRNAs) are short RNAs acting as natural RNAi. miRNAs mediate down-regulation of many mRNAs from developmental genes and transcription factor genes. Natural examples for this additional level of post-transcriptional control are increasing. Suitable computer-based search strategies for new miRNA candidates include precursor folding as well as different compositional search strategies. Example programs for this are presented. New own and other data are provided for an overview on such strategies. A strategy feasible in plants for miRNA target identification is direct base pairing of miRNAs to potential mRNA target 3' UTRs. Correct identification in animals usually requires comparative genomics and conserved UTR regions pairing to conserved miRNA substructures. A number of example programs and target examples for these tasks are examined. Finally, strategies and programs for artificial gene silencing by designed RNAi are explained.

ITS-2 and 18S RRNA Gene Phylogeny of Aplysinidae (Verongida, Demospongiae)

Journal of Molecular Evolution. Mar, 2005  |  Pubmed ID: 15871043

18S ribosomal DNA and internal transcribed spacer 2 (ITS-2) full-length sequences, each of which was sequenced three times, were used to construct phylogenetic trees with alignments based on secondary structures, in order to elucidate genealogical relationships within the Aplysinidae (Verongida). The first poriferan ITS-2 secondary structures are reported. Altogether 11 Aplysina sponges and 3 additional sponges (Verongula gigantea, Aiolochroia crassa, Smenospongia aurea) from tropical and subtropical oceans were analyzed. Based on these molecular studies, S. aurea, which is currently affiliated with the Dictyoceratida, should be reclassified to the Verongida. Aplysina appears as monophyletic. A soft form of Aplysina lacunosa was separated from other Aplysina and stands at a basal position in both 18S and ITS-2 trees. Based on ITS-2 sequence information, the Aplysina sponges could be distinguished into a single Caribbean-Eastern Pacific cluster and a Mediterranean cluster. The species concept for Aplysina sponges as well as a phylogenetic history with a possibly Tethyan origin is discussed.

YANA - a Software Tool for Analyzing Flux Modes, Gene-expression and Enzyme Activities

BMC Bioinformatics. 2005  |  Pubmed ID: 15929789

A number of algorithms for steady state analysis of metabolic networks have been developed over the years. Of these, Elementary Mode Analysis (EMA) has proven especially useful. Despite its low user-friendliness, METATOOL as a reliable high-performance implementation of the algorithm has been the instrument of choice up to now. As reported here, the analysis of metabolic networks has been improved by an editor and analyzer of metabolic flux modes. Analysis routines for expression levels and the most central, well connected metabolites and their metabolic connections are of particular interest.

Metabolites and Pathway Flexibility

In Silico Biology. 2005  |  Pubmed ID: 15972010

Flexibility of metabolites and enzymes is investigated (i) on the level of the individual molecule, (ii) on the pathway level and (iii) combined effects on the systems and network level. Tools and results from our current research are summarized including data from our metabolite enzyme database. Including our latest census we find frequently used metabolites stimulate evolutionary flexibility in specific enzyme superfamilies. Furthermore, simultaneous changes of reactions and metabolites are observed in these flexible enzyme superfamilies. Both effects provide a strong source for resistance in parasites and pathogens. Specific adaptations scenarios and some counter strategies are discussed.

CBCAnalyzer: Inferring Phylogenies Based on Compensatory Base Changes in RNA Secondary Structures

In Silico Biology. 2005  |  Pubmed ID: 15996120

The CBCAnalyzer (CBC=compensatory base change) is a custom written software toolbox consisting of three parts, CTTransform, CBCDetect, and CBCTree. CTTransform reads several ct-file formats, and generates a so called "bracket-dot-bracket" format that typically is used as input for other tools such as RNAforester, RNAmovie or MARNA. The latter one creates a multiple alignment based on primary sequences and secondary structures that now can be used as input for CBCDetect. CBCDetect counts CBCs in all against all of the aligned sequences. This is important in detecting species that are discriminated by their sexual incompatibility. The count (distance) matrix obtained by CBCDetect is used as input for CBCTree that reconstructs a phylogram by using the algorithm of BIONJ. In this note we describe the features of the toolbox as well as application examples. The toolbox provides a graphical user interface. It is written in C++ and freely available at: http://cbcanalyzer.bioapps.biozentrum.uni-wuerzburg.de.

Homology Modeling Revealed More Than 20,000 RRNA Internal Transcribed Spacer 2 (ITS2) Secondary Structures

RNA (New York, N.Y.). Nov, 2005  |  Pubmed ID: 16244129

Structural genomics meets phylogenetics and vice versa: Knowing rRNA secondary structures is a prerequisite for constructing rRNA alignments for inferring phylogenies, and inferring phylogenies is a precondition to understand the evolution of such rRNA secondary structures. Here, both scientific worlds go together. The rRNA internal transcribed spacer 2 (ITS2) region is a widely used phylogenetic marker. Because of its high variability at the sequence level, correct alignments have to take into account structural information. In this study, we examine the extent of the conservation in structure. We present (1) the homology modeled secondary structure of more than 20,000 ITS2 covering about 14,000 species; (2) a computational approach for homology modeling of rRNA structures, which additionally can be applied to other RNA families; and (3) a database providing about 25,000 ITS2 sequences with their associated secondary structures, a refined ITS2 specific general time reversible (GTR) substitution model, and a scoring matrix, available at http://its2.bioapps.biozentrum.uni-wuerzburg.de.

B-Raf and C-Raf Signaling Investigated in a Simplified Model of the Mitogenic Kinase Cascade

Biological Chemistry. Nov, 2005  |  Pubmed ID: 16307482

Signaling pathways based on the reversible phosphorylation of proteins control most aspects of cellular life in higher organisms. Extracellular stimuli can induce growth, differentiation, survival and the stress response through a number of highly conserved signaling pathways. We discuss how the intensity and duration of signals may have dramatic consequences on the way cells respond to stimuli. Picking the central Ras-Raf-MEK-ERK signal cascade, we developed a mathematical model of how stimuli induce different signal patterns and thereby different cellular responses, depending on cell type and the ratio between B-Raf and C-Raf. Based on biochemical data for activation and dephosphorylation, as well as the differential equations of our model, we suggest a different signaling pattern and response result for B-Raf (strong activation, sustained signal) and C-Raf (steep activation, transient signal). We further support the significance of such differential modulatory signaling by showing different Raf isoform expression in various cell lines and experimental testing of the predicted kinase activities in B-Raf, C-Raf and mutated versions.

Understanding Platelets. Lessons from Proteomics, Genomics and Promises from Network Analysis

Thrombosis and Haemostasis. Nov, 2005  |  Pubmed ID: 16363231

New large-scale analysis techniques such as bioinformatics, mass spectrometry and SAGE data analysis will allow a new framework for understanding platelets. This review analyses some important options and tasks for these tools and examines an outline of the new, refined picture of the platelet outlined by these new techniques. Looking at the platelet-specific building blocks of genome, (active) transcriptome and proteome (notably secretome and phospho-proteome), we summarize current bioinformatical and biochemical approaches, tasks as well as their limitations. Understanding the surprisingly complex platelet regarding compartmentalization, key cascades, and pathways including clinical implications will remain an exciting and hopefully fruitful challenge for the future.

Analysis of and Function Predictions for Previously Conserved Hypothetical or Putative Proteins in Blochmannia Floridanus

BMC Microbiology. 2006  |  Pubmed ID: 16401340

There is an increasing interest to better understand endosymbiont capabilities in insects both from an ecological point of view and for pest control. Blochmannia floridanus provides important nutrients for its host, the ant Camponotus, while the bacterium in return is provided with a niche to proliferate. Blochmannia floridanus proteins and metabolites are difficult to study due to its endosymbiontic life style; however, its complete genome sequence became recently available.

13C Isotopologue Perturbation Studies of Listeria Monocytogenes Carbon Metabolism and Its Modulation by the Virulence Regulator PrfA

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2006  |  Pubmed ID: 16461909

The carbon metabolism of Listeria monocytogenes (Lm) EGD and the two isogenic mutant strains LmDeltaprfA and LmDeltaprfApPRFA* (showing no or enhanced expression, respectively, of the virulence factor PrfA) was determined by 13C isotopologue perturbation. After growth of the bacteria in a defined medium containing a mixture of [U-13C6]glucose and glucose with natural 13C abundance (1:25, wt/wt), 14 amino acids were isolated and analyzed by NMR spectroscopy. Multiply 13C-labeled isotopologues were determined quantitatively by signal deconvolution. The 13C enrichments and isotopologue patterns allowed the reconstruction of most amino acid biosynthesis pathways and illustrated that overproduced PrfA may strongly influence the synthesis of some amino acids, notably that of the branched amino acids (Val, Ile, and Leu). Retrobiosynthetic analysis of the isotopologue compositions showed that degradation of glucose occurs to a large extent via the pentose phosphate pathway and that the citrate cycle is incomplete because of the absence of 2-oxoglutarate dehydrogenase activity. The reconstructed labeling pattern of oxaloacetate indicated its formation by carboxylation of pyruvate. This metabolic reaction seems to have a strong impact on the growth requirement in defined minimal medium. Bioinformatical steady-state network analyses and flux distribution predictions confirmed the experimental data and predicted metabolite fluxes through the enzymes of the pathways under study.

RIP Death Domain Structural Interactions Implicated in TNF-mediated Proliferation and Survival

Proteins. May, 2006  |  Pubmed ID: 16470584

Death domain (DD)-containing proteins are involved in both apoptosis and survival/proliferation signaling induced by activated death receptors. Here, a phylogenetic and structural analysis was performed to highlight differences in DD domains and their key regulatory interaction sites. The phylogenetic analysis shows that receptor DDs are more conserved than DDs in adaptors. Adaptor DDs can be subdivided into those that activate or inhibit apoptosis. Modeling of six homotypic DD interactions involved in the TNF signaling pathway implicates that the DD of RIP (Receptor interacting protein kinase 1) is capable of interacting with the DD of TRADD (TNFR1-associated death domain protein) in two different, exclusive ways: one that subsequently recruits CRADD (apoptosis/inflammation) and another that recruits NFkappaB (survival/proliferation).

Analysis of SAGE Data in Human Platelets: Features of the Transcriptome in an Anucleate Cell

Thrombosis and Haemostasis. Apr, 2006  |  Pubmed ID: 16601835

A comprehensive SAGE (serial analysis of gene expression) library of purified human platelets was established. Twenty-five thousand (25,000) tags were sequenced, and after removal of mitochondrial tags, 12,609 (51%) non-mitochondrial-derived tags remained, corresponding to 2,300 different transcripts with expression levels of up to 30,000 tags per million. This new, highly purified SAGE library of platelets is enriched in specific transcripts. The complexity in terms of tag distribution is similar to cells that are still able to replenish their mRNA pool by transcription. We show that our SAGE data are consistent with recently published microarray data but show further details of the platelet transcriptome, including (i) longer UTR regions and more stable folding in the enriched mRNAs, (ii) biologically interesting new candidate mRNAs that show regulatory elements, including elements for RNA stabilization or for translational control, and (iii) significant enrichment of specific, highly transcribed mRNAs compared to a battery of SAGE libraries from other tissues. Among several regulatory mRNA elements known to be involved in mRNA localization and translational control, CPE elements are in particular enriched in the platelet transcriptome. mRNAs previously reported to be translationally regulated were found to be present in the library and were validated by real-time PCR. Furthermore, specific molecular functions such as signal transduction activity were found to be significantly enriched in the platelet transcriptome. These findings emphasize the richness and diversity of the platelet transcriptome.

Genome-wide Mapping of Foamy Virus Vector Integrations into a Human Cell Line

The Journal of General Virology. May, 2006  |  Pubmed ID: 16603537

Integration-site selection by retroviruses and retroviral vectors has gained increased scientific interest. Foamy viruses (FVs) constitute a unique subfamily (Spumavirinae) of the family Retroviridae, for which the integration pattern into the human genome has not yet been determined. To accomplish this, 293 cells were transduced with FV vectors and the integration sites into the cellular genome were determined by a high-throughput method based on inverse PCR. For comparison, a limited number of murine leukemia virus (MLV) and human immunodeficiency virus (HIV) integration sites were analysed in parallel. Altogether, 628 FV, 87 HIV and 141 MLV distinct integration sites were mapped to the human genome. The sequences were analysed for RefSeq genes, promoter regions, CpG islands and insertions into cellular oncogenes. Compared with the integration-site preferences of HIV, which strongly favours active genes, and MLV, which favours integration near transcription-start regions, our results indicate that FV integration has neither of these preferences. However, once integration has occurred into a transcribed region of the genome, FVs tend to target promoter-close regions, albeit with less preference than MLV. Furthermore, our study revealed a palindromic consensus sequence for integration, which was centred on the virus-specific, four-base-duplicated target site. In summary, it is shown that the integration pattern of FVs appears to be unique compared with those of other retroviral genera.

Lysosomal, Cytoskeletal, and Metabolic Alterations in Cardiomyopathy of Cathepsin L Knockout Mice

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Jun, 2006  |  Pubmed ID: 16636100

Although lysosomal proteases are expressed in the heart at considerable levels, their specific functions in this organ remain elusive. Mice deficient for the lysosomal cysteine protease cathepsin L (CTSL) develop a late onset dilated cardiomyopathy (DCM) that is characterized by cardiac chamber dilation, fibrosis, and impaired cardiac contraction at 12 month of age. Investigation of the pathogenic sequence of DCM in ctsl-/- mice revealed numerous dysmorphic lysosome-like structures in heart muscle as early as 3 days after birth, whereas skeletal muscle was not affected. Labeling of the acidic cell compartment of neonatal cardiomyocytes and detection of lysosomal markers after subcellular fractionation confirmed increased lysosome content in CTSL deficient myocardium; however, specific storage materials were not detected. The myocardium of ctsl+/+ and ctsl-/- mice revealed no differences in incidence of cell death, proliferation, and capillary density during DCM progression. However, an observed increase in mRNA expression of natriuretic peptides in young adult mice indicates the activation of the adaptive "fetal" gene program, while proteome analysis revealed decreased levels of the sarcomere-associated proteins alpha-tropomyosin, desmin, and calsarcin 1, as well as considerable changes of metabolic enzymes. Bioinformatic pathway analysis suggested a switch to anaerobic catabolism and impairment of mitochondrial respiration. This interpretation was supported by a 50% reduction in resting state oxygen consumption and impaired respiration capacity in ctsl-/- myocardial homogenates. In summary, the data indicate an essential role of CTSL in maintaining the structure of the endosomal/lysosomal compartment in cardiomyocytes. Lysosomal impairment in ctsl-/- hearts results in metabolic and sarcomeric alterations that promote DCM development.

Iron Regulation and the Cell Cycle: Identification of an Iron-responsive Element in the 3'-untranslated Region of Human Cell Division Cycle 14A MRNA by a Refined Microarray-based Screening Strategy

The Journal of Biological Chemistry. Aug, 2006  |  Pubmed ID: 16760464

Iron regulatory proteins (IRPs) 1 and 2 post-transcriptionally control mammalian iron homeostasis by binding to iron-responsive elements (IREs), conserved RNA stem-loop structures located in the 5'- or 3'-untranslated regions of genes involved in iron metabolism (e.g. FTH1, FTL, and TFRC). To identify novel IRE-containing mRNAs, we integrated biochemical, biocomputational, and microarray-based experimental approaches. IRP/IRE messenger ribonucleoproteins were immunoselected, and their mRNA composition was analyzed using an IronChip microarray enriched for genes predicted computationally to contain IRE-like motifs. Among different candidates, this report focuses on a novel IRE located in the 3'-untranslated region of the cell division cycle 14A mRNA. We show that this IRE motif efficiently binds both IRP1 and IRP2. Differential splicing of cell division cycle 14A produces IRE- and non-IRE-containing mRNA isoforms. Interestingly, only the expression of the IRE-containing mRNA isoforms is selectively increased by cellular iron deficiency. This work describes a new experimental strategy to explore the IRE/IRP regulatory network and uncovers a previously unrecognized regulatory link between iron metabolism and the cell cycle.

The Internal Transcribed Spacer 2 Database--a Web Server for (not Only) Low Level Phylogenetic Analyses

Nucleic Acids Research. Jul, 2006  |  Pubmed ID: 16845103

The internal transcribed spacer 2 (ITS2) is a phylogenetic marker which has been of broad use in generic and infrageneric level classifications, as its sequence evolves comparably fast. Only recently, it became clear, that the ITS2 might be useful even for higher level systematic analyses. As the secondary structure is highly conserved within all eukaryotes it serves as a valuable template for the construction of highly reliable sequence-structure alignments, which build a fundament for subsequent analyses. Thus, any phylogenetic study using ITS2 has to consider both sequence and structure. We have integrated a homology based RNA structure prediction algorithm into a web server, which allows the detection and secondary structure prediction for ITS2 in any given sequence. Furthermore, the resource contains more than 25,000 pre-calculated secondary structures for the currently known ITS2 sequences. These can be taxonomically searched and browsed. Thus, our resource could become a starting point for ITS2-based phylogenetic analyses and is therefore complementary to databases of other phylogenetic markers, which focus on higher level analyses. The current version of the ITS2 database can be accessed via http://its2.bioapps.biozentrum.uni-wuerzburg.de.

Whole-genome Sequence of Listeria Welshimeri Reveals Common Steps in Genome Reduction with Listeria Innocua As Compared to Listeria Monocytogenes

Journal of Bacteriology. Nov, 2006  |  Pubmed ID: 16936040

We present the complete genome sequence of Listeria welshimeri, a nonpathogenic member of the genus Listeria. Listeria welshimeri harbors a circular chromosome of 2,814,130 bp with 2,780 open reading frames. Comparative genomic analysis of chromosomal regions between L. welshimeri, Listeria innocua, and Listeria monocytogenes shows strong overall conservation of synteny, with the exception of the translocation of an F(o)F(1) ATP synthase. The smaller size of the L. welshimeri genome is the result of deletions in all of the genes involved in virulence and of "fitness" genes required for intracellular survival, transcription factors, and LPXTG- and LRR-containing proteins as well as 55 genes involved in carbohydrate transport and metabolism. In total, 482 genes are absent from L. welshimeri relative to L. monocytogenes. Of these, 249 deletions are commonly absent in both L. welshimeri and L. innocua, suggesting similar genome evolutionary paths from an ancestor. We also identified 311 genes specific to L. welshimeri that are absent in the other two species, indicating gene expansion in L. welshimeri, including horizontal gene transfer. The species L. welshimeri appears to have been derived from early evolutionary events and an ancestor more compact than L. monocytogenes that led to the emergence of nonpathogenic Listeria spp.

A Java Applet for Exploring the New Higher Level Classification of Eukaryotes with Emphasis on the Taxonomy of Protists

The Journal of Eukaryotic Microbiology. Sep-Oct, 2006  |  Pubmed ID: 16968448

We have converted the hierarchically organized new higher level classification of eukaryotes with emphasis on the taxonomy of protists proposed by Adl et al. into an interactive and dynamic Java applet. The current version of the applet can be accessed via http://phylogenetics.bioapps.biozentrum.uni-wuerzburg.de/etv. We use the layout from a Degree-of-Interest tree (DOITree) that effectively displays all the taxonomic information as well as the phylogenetic relationships described in the original article by Adl et al. The tree was made using the Prefuse Toolkit for interactive information visualization. All browsers capable of using Java applets will be able to view the tree. The applet is freely available for scientists, teachers, and students.

Modelling Interaction Sites in Protein Domains with Interaction Profile Hidden Markov Models

Bioinformatics (Oxford, England). Dec, 2006  |  Pubmed ID: 17000753

Due to the growing number of completely sequenced genomes, functional annotation of proteins becomes a more and more important issue. Here, we describe a method for the prediction of sites within protein domains, which are part of protein-ligand interactions. As recently demonstrated, these sites are not trivial to detect because of a varying degree of conservation of their location and type within a domain family.

InGeno--an Integrated Genome and Ortholog Viewer for Improved Genome to Genome Comparisons

BMC Bioinformatics. 2006  |  Pubmed ID: 17054788

Systematic genome comparisons are an important tool to reveal gene functions, pathogenic features, metabolic pathways and genome evolution in the era of post-genomics. Furthermore, such comparisons provide important clues for vaccines and drug development. Existing genome comparison software often lacks accurate information on orthologs, the function of similar genes identified and genome-wide reports and lists on specific functions. All these features and further analyses are provided here in the context of a modular software tool "inGeno" written in Java with Biojava subroutines.

XML Schemas for Common Bioinformatic Data Types and Their Application in Workflow Systems

BMC Bioinformatics. 2006  |  Pubmed ID: 17087823

Today, there is a growing need in bioinformatics to combine available software tools into chains, thus building complex applications from existing single-task tools. To create such workflows, the tools involved have to be able to work with each other's data--therefore, a common set of well-defined data formats is needed. Unfortunately, current bioinformatic tools use a great variety of heterogeneous formats.

4SALE--a Tool for Synchronous RNA Sequence and Secondary Structure Alignment and Editing

BMC Bioinformatics. 2006  |  Pubmed ID: 17101042

In sequence analysis the multiple alignment builds the fundament of all proceeding analyses. Errors in an alignment could strongly influence all succeeding analyses and therefore could lead to wrong predictions. Hand-crafted and hand-improved alignments are necessary and meanwhile good common practice. For RNA sequences often the primary sequence as well as a secondary structure consensus is well known, e.g., the cloverleaf structure of the t-RNA. Recently, some alignment editors are proposed that are able to include and model both kinds of information. However, with the advent of a large amount of reliable RNA sequences together with their solved secondary structures (available from e.g. the ITS2 Database), we are faced with the problem to handle sequences and their associated secondary structures synchronously.

An Integrated View of Gene Expression and Solute Profiles of Arabidopsis Tumors: a Genome-wide Approach

The Plant Cell. Dec, 2006  |  Pubmed ID: 17172353

Transformation of plant cells with T-DNA of virulent agrobacteria is one of the most extreme triggers of developmental changes in higher plants. For rapid growth and development of resulting tumors, specific changes in the gene expression profile and metabolic adaptations are required. Increased transport and metabolic fluxes are critical preconditions for growth and tumor development. A functional genomics approach, using the Affymetrix whole genome microarray (approximately 22,800 genes), was applied to measure changes in gene expression. The solute pattern of Arabidopsis thaliana tumors and uninfected plant tissues was compared with the respective gene expression profile. Increased levels of anions, sugars, and amino acids were correlated with changes in the gene expression of specific enzymes and solute transporters. The expression profile of genes pivotal for energy metabolism, such as those involved in photosynthesis, mitochondrial electron transport, and fermentation, suggested that tumors produce C and N compounds heterotrophically and gain energy mainly anaerobically. Thus, understanding of gene-to-metabolite networks in plant tumors promotes the identification of mechanisms that control tumor development.

Use of Pathway Analysis and Genome Context Methods for Functional Genomics of Mycoplasma Pneumoniae Nucleotide Metabolism

Gene. Jul, 2007  |  Pubmed ID: 17467928

Elementary modes analysis allows one to reveal whether a set of known enzymes is sufficient to sustain functionality of the cell. Moreover, it is helpful in detecting missing reactions and predicting which enzymes could fill these gaps. Here, we perform a comprehensive elementary modes analysis and a genomic context analysis of Mycoplasma pneumoniae nucleotide metabolism, and search for new enzyme activities. The purine and pyrimidine networks are reconstructed by assembling enzymes annotated in the genome or found experimentally. We show that these reaction sets are sufficient for enabling synthesis of DNA and RNA in M. pneumoniae. Special focus is on the key modes for growth. Moreover, we make an educated guess on the nutritional requirements of this micro-organism. For the case that M. pneumoniae does not require adenine as a substrate, we suggest adenylosuccinate synthetase (EC 6.3.4.4), adenylosuccinate lyase (EC 4.3.2.2) and GMP reductase (EC 1.7.1.7) to be operative. GMP reductase activity is putatively assigned to the NRDI_MYCPN gene on the basis of the genomic context analysis. For the pyrimidine network, we suggest CTP synthase (EC 6.3.4.2) to be active. Further experiments on the nutritional requirements are needed to make a decision. Pyrimidine metabolism appears to be more appropriate as a drug target than purine metabolism since it shows lower plasticity.

Distinguishing Species

RNA (New York, N.Y.). Sep, 2007  |  Pubmed ID: 17652131

Given two organisms, how can one distinguish whether they belong to the same species or not? This might be straightforward for two divergent organisms, but can be extremely difficult and laborious for closely related ones. A molecular marker giving a clear distinction would therefore be of immense benefit. The internal transcribed spacer 2 (ITS2) has been widely used for low-level phylogenetic analyses. Case studies revealed that a compensatory base change (CBC) in the helix II or helix III ITS2 secondary structure between two organisms correlated with sexual incompatibility. We analyzed more than 1300 closely related species to test whether this correlation is generally applicable. In 93%, where a CBC was found between organisms classified within the same genus, they belong to different species. Thus, a CBC in an ITS2 sequence-structure alignment is a sufficient condition to distinguish even closely related species.

Deep Metazoan Phylogeny

In Silico Biology. 2007  |  Pubmed ID: 17688440

We reconstructed a robust phylogenetic tree of the Metazoa, consisting of almost 1,500 taxa, by profile neighbor joining (PNJ), an automated computational method that inherits the efficiency of the neighbor joining algorithm. This tree supports the one proposed in the latest review on metazoan phylogeny. Our main goal is not to discuss aspects of the phylogeny itself, but rather to point out that PNJ can be a valuable tool when the basal branching pattern of a large phylogenetic tree must be estimated, whereas traditional methods would be computationally impractical.

Integrated Network Reconstruction, Visualization and Analysis Using YANAsquare

BMC Bioinformatics. 2007  |  Pubmed ID: 17725829

Modeling of metabolic networks includes tasks such as network assembly, network overview, calculation of metabolic fluxes and testing the robustness of the network.

Exonization of Active Mouse L1s: a Driver of Transcriptome Evolution?

BMC Genomics. 2007  |  Pubmed ID: 17963496

Long interspersed nuclear elements (LINE-1s, L1s) have been recently implicated in the regulation of mammalian transcriptomes.

Interactions of Methylene Blue with Human Disulfide Reductases and Their Orthologues from Plasmodium Falciparum

Antimicrobial Agents and Chemotherapy. Jan, 2008  |  Pubmed ID: 17967916

Methylene blue (MB) has experienced a renaissance mainly as a component of drug combinations against Plasmodium falciparum malaria. Here, we report biochemically relevant pharmacological data on MB such as rate constants for the uncatalyzed reaction of MB at pH 7.4 with cellular reductants like NAD(P)H (k = 4 M(-1) s(-1)), thioredoxins (k = 8.5 to 26 M(-1) s(-1)), dihydrolipoamide (k = 53 M(-1) s(-1)), and slowly reacting glutathione. As the disulfide reductases are prominent targets of MB, optical tests for enzymes reducing MB at the expense of NAD(P)H under aerobic conditions were developed. The product leucomethylene blue (leucoMB) is auto-oxidized back to MB at pH 7 but can be stabilized by enzymes at pH 5.0, which makes this colorless compound an interesting drug candidate. MB was found to be an inhibitor and/or a redox-cycling substrate of mammalian and P. falciparum disulfide reductases, with the kcat values ranging from 0.03 s(-1) to 10 s(-1) at 25 degrees C. Kinetic spectroscopy of mutagenized glutathione reductase indicates that MB reduction is conducted by enzyme-bound reduced flavin rather than by the active-site dithiol Cys58/Cys63. The enzyme-catalyzed reduction of MB and subsequent auto-oxidation of the product leucoMB mean that MB is a redox-cycling agent which produces H2O2 at the expense of O2 and of NAD(P)H in each cycle, turning the antioxidant disulfide reductases into pro-oxidant enzymes. This explains the terms subversive substrate or turncoat inhibitor for MB. The results are discussed in cell-pathological and clinical contexts.

Explorative Data Analysis of MCL Reveals Gene Expression Networks Implicated in Survival and Prognosis Supported by Explorative CGH Analysis

BMC Cancer. 2008  |  Pubmed ID: 18416826

Mantle cell lymphoma (MCL) is an incurable B cell lymphoma and accounts for 6% of all non-Hodgkin's lymphomas. On the genetic level, MCL is characterized by the hallmark translocation t(11;14) that is present in most cases with few exceptions. Both gene expression and comparative genomic hybridization (CGH) data vary considerably between patients with implications for their prognosis.

Platelet Protein Interactions: Map, Signaling Components, and Phosphorylation Groundstate

Arteriosclerosis, Thrombosis, and Vascular Biology. Jul, 2008  |  Pubmed ID: 18451328

Assembly of a comprehensive proteome and transcriptome database of human platelets, derivation of a model of the platelet-specific interactome, and generation of a functional interaction map of platelet phosphorylations and kinases.

The Linear Chromosome of the Plant-pathogenic Mycoplasma 'Candidatus Phytoplasma Mali'

BMC Genomics. 2008  |  Pubmed ID: 18582369

Phytoplasmas are insect-transmitted, uncultivable bacterial plant pathogens that cause diseases in hundreds of economically important plants. They represent a monophyletic group within the class Mollicutes (trivial name mycoplasmas) and are characterized by a small genome with a low GC content, and the lack of a firm cell wall. All mycoplasmas, including strains of 'Candidatus (Ca.) Phytoplasma asteris' and 'Ca. P. australiense', examined so far have circular chromosomes, as is the case for almost all walled bacteria.

Identifying Functional Modules in Protein-protein Interaction Networks: an Integrated Exact Approach

Bioinformatics (Oxford, England). Jul, 2008  |  Pubmed ID: 18586718

With the exponential growth of expression and protein-protein interaction (PPI) data, the frontier of research in systems biology shifts more and more to the integrated analysis of these large datasets. Of particular interest is the identification of functional modules in PPI networks, sharing common cellular function beyond the scope of classical pathways, by means of detecting differentially expressed regions in PPI networks. This requires on the one hand an adequate scoring of the nodes in the network to be identified and on the other hand the availability of an effective algorithm to find the maximally scoring network regions. Various heuristic approaches have been proposed in the literature.

ITS2 Data Corroborate a Monophyletic Chlorophycean DO-group (Sphaeropleales)

BMC Evolutionary Biology. 2008  |  Pubmed ID: 18655698

Within Chlorophyceae the ITS2 secondary structure shows an unbranched helix I, except for the 'Hydrodictyon' and the 'Scenedesmus' clade having a ramified first helix. The latter two are classified within the Sphaeropleales, characterised by directly opposed basal bodies in their flagellar apparatuses (DO-group). Previous studies could not resolve the taxonomic position of the 'Sphaeroplea' clade within the Chlorophyceae without ambiguity and two pivotal questions remain open: (1) Is the DO-group monophyletic and (2) is a branched helix I an apomorphic feature of the DO-group? In the present study we analysed the secondary structure of three newly obtained ITS2 sequences classified within the 'Sphaeroplea' clade and resolved sphaeroplealean relationships by applying different phylogenetic approaches based on a combined sequence-structure alignment.

ProfDistS: (profile-) Distance Based Phylogeny on Sequence--structure Alignments

Bioinformatics (Oxford, England). Oct, 2008  |  Pubmed ID: 18723521

MOTIVATION: The Profile Neighbor Joining (PNJ) algorithm as implemented in the software ProfDist is computationally efficient in reconstructing very large trees. Besides the huge amount of sequence data the structure is important in RNA alignment analysis and phylogenetic reconstruction. RESULTS: For this ProfDistS provides a phylogenetic workflow that uses individual RNA secondary structures in reconstructing phylogenies based on sequence-structure alignments-using PNJ with manual or iterative and automatic profile definition. Moreover, ProfDistS can deal also with protein sequences.

Synchronous Visual Analysis and Editing of RNA Sequence and Secondary Structure Alignments Using 4SALE

BMC Research Notes. 2008  |  Pubmed ID: 18854023

The function of a noncoding RNA sequence is mainly determined by its secondary structure and therefore a family of noncoding RNA sequences is much more conserved on the structural level than on the sequence level. Understanding the function of noncoding RNA sequence families requires two things: a hand-crafted or hand-improved alignment and detailed analyses of the secondary structures. There are several tools available that help performing these tasks, but all of them are specialized and focus on only one aspect, editing the alignment or plotting the secondary structure. The problem is both these tasks need to be performed simultaneously.

New Trends in Pharmacogenomic Strategies Against Resistance Development in Microbial Infections

Pharmacogenomics. Nov, 2008  |  Pubmed ID: 19018725

This review summarizes some of the new trends in the fight against drug resistant bacteria. We review Gram-positive (e.g., S.aureus) and Gram-negative (e.g., Pseudomonas aeruginosa, Helicobacter pylori) bacteria, the current antibiotic resistance situation, as well as resistance spread and some recently discovered resistance mechanisms, such as those based on integrons and complex transposons. We then summarize several current routes to identify new drugs such as cationic antimicrobial peptides, novel acyldepsipeptides, RNA aptamers and lipopeptides. New drug strategies to treat resistant pathogens include eliciting growth in dormant bacteria, or a new way to attack efflux systems. Typical approaches from pharmacogenomics combined with systems biology and bioinformatics support these routes (simulations, metagenomics and metabolic network modeling), as well as the patient treatment (e.g., haplotyping and immune response).

Modelling Phagosomal Lipid Networks That Regulate Actin Assembly

BMC Systems Biology. 2008  |  Pubmed ID: 19061496

When purified phagosomes are incubated in the presence of actin under appropriate conditions, microfilaments start growing from the membrane in a process that is affected by ATP and the lipid composition of the membrane. Isolated phagosomes are metabolically active organelles that contain enzymes and metabolites necessary for lipid interconversion. Hence, addition of ATP, lipids, and actin to the system alter the steady-state composition of the phagosomal membrane at the same time that the actin nucleation is initiated. Our aim was to model all these processes in parallel.

Use of Functional Highly Purified Human Platelets for the Identification of New Proteins of the IPP Signaling Pathway

Thrombosis Research. 2008  |  Pubmed ID: 17931695

Identification of the full content of platelet proteins and their mRNAs would be helpful for further studies of human platelet function. For this purpose, proteomic as well as transcriptomic methods (SAGE and qRT-PCR) can be utilized, but the purity of the platelet samples studied is crucial. Here we report the development of a new, effective, and efficient technique for purification of human platelets from washed apheresis platelet concentrates and whole blood.

The ITS2 Database II: Homology Modelling RNA Structure for Molecular Systematics

Nucleic Acids Research. Jan, 2008  |  Pubmed ID: 17933769

An increasing number of phylogenetic analyses are based on the internal transcribed spacer 2 (ITS2). They mainly use the fast evolving sequence for low-level analyses. When considering the highly conserved structure, the same marker could also be used for higher level phylogenies. Furthermore, structural features of the ITS2 allow distinguishing different species from each other. Despite its importance, the correct structure is only rarely found by standard RNA folding algorithms. To overcome this hindrance for a wider application of the ITS2, we have developed a homology modelling approach to predict the structure of RNA and present the results of modelling the ITS2 in the ITS2 Database. Here, we describe the database and the underlying algorithms which allowed us to predict the structure for 86 784 sequences, which is more than 55% of all GenBank entries concerning the ITS2. These are not equally distributed over all genera. There is a substantial amount of genera where the structure of nearly all sequences is predicted whereas for others no structure at all was found despite high sequence coverage. These genera might have evolved an ITS2 structure diverging from the standard one. The current version of the ITS2 Database can be accessed via http://its2.bioapps.biozentrum.uni-wuerzburg.de.

Unsupervised Meta-analysis on Diverse Gene Expression Datasets Allows Insight into Gene Function and Regulation

Bioinformatics and Biology Insights. 2008  |  Pubmed ID: 19812781

Over the past years, microarray databases have increased rapidly in size. While they offer a wealth of data, it remains challenging to integrate data arising from different studies. Here we propose an unsupervised approach of a large-scale meta-analysis on Arabidopsis thaliana whole genome expression datasets to gain additional insights into the function and regulation of genes. Applying kernel principal component analysis and hierarchical clustering, we found three major groups of experimental contrasts sharing a common biological trait. Genes associated to two of these clusters are known to play an important role in indole-3-acetic acid (IAA) mediated plant growth and development or pathogen defense. Novel functions could be assigned to genes including a cluster of serine/threonine kinases that carry two uncharacterized domains (DUF26) in their receptor part implicated in host defense. With the approach shown here, hidden interrelations between genes regulated under different conditions can be unraveled.

Tardigrade Workbench: Comparing Stress-related Proteins, Sequence-similar and Functional Protein Clusters As Well As RNA Elements in Tardigrades

BMC Genomics. 2009  |  Pubmed ID: 19821996

Tardigrades represent an animal phylum with extraordinary resistance to environmental stress.

Molecular Mechanisms of Tolerance in Tardigrades: New Perspectives for Preservation and Stabilization of Biological Material

Biotechnology Advances. Jul-Aug, 2009  |  Pubmed ID: 19472511

Certain organisms found across a range of taxa, including bacteria, yeasts, plants and many invertebrates such as nematodes and tardigrades are able to survive almost complete loss of body water. The dry organisms may remain in this state, which is known as anhydrobiosis, for decades without apparent damage. When water again becomes available, they rapidly rehydrate and resume active life. Research in anhydrobiosis has focused mainly on sugar metabolism and stress proteins. Despite the discovery of various molecules which are involved in desiccation and water stress, knowledge of the regulatory network governing the stability of the cellular architecture and the metabolic machinery during dehydration is still fragmentary and not well understood. A combination of transcriptional, proteomic and metabolic approaches with bioinformatics tools can provide a better understanding of gene regulation that underlie the biological functions and physiology related to anhydrobiosis. The development of this concept will raise exciting possibilities and techniques for the preservation and stabilization of biological materials in the dry state.

Bioinformatical Approaches to Detect and Analyze Protein Interactions

Methods in Molecular Biology (Clifton, N.J.). 2009  |  Pubmed ID: 19544036

Protein-protein interactions are the building blocks of cellular networks and at the heart of cellular regulation. However, their experimental identification is still a challenge. This chapter is concerned with the determination of protein-protein interactions by bioinformatical methods. These often can operate just on sequence information. Further required information is derived from public knowledge in literature databanks and biochemical databases as well as from the sequences themselves and iterative sequence comparisons. Further tools include domain analysis, structure prediction, and genome context methods. The results are predicted binary interactions and complete interaction networks.

Detecting Species-site Dependencies in Large Multiple Sequence Alignments

Nucleic Acids Research. Oct, 2009  |  Pubmed ID: 19661281

Multiple sequence alignments (MSAs) are one of the most important sources of information in sequence analysis. Many methods have been proposed to detect, extract and visualize their most significant properties. To the same extent that site-specific methods like sequence logos successfully visualize site conservations and sequence-based methods like clustering approaches detect relationships between sequences, both types of methods fail at revealing informational elements of MSAs at the level of sequence-site interactions, i.e. finding clusters of sequences and sites responsible for their clustering, which together account for a high fraction of the overall information of the MSA. To fill this gap, we present here a method that combines the Fisher score-based embedding of sequences from a profile hidden Markov model (pHMM) with correspondence analysis. This method is capable of detecting and visualizing group-specific or conflicting signals in an MSA and allows for a detailed explorative investigation of alignments of any size tractable by pHMMs. Applications of our methods are exemplified on an alignment of the Neisseria surface antigen LP2086, where it is used to detect sites of recombinatory horizontal gene transfer and on the vitamin K epoxide reductase family to distinguish between evolutionary and functional signals.

CA/C1 Peptidases of the Malaria Parasites Plasmodium Falciparum and P. Berghei and Their Mammalian Hosts--a Bioinformatical Analysis

Biological Chemistry. Nov, 2009  |  Pubmed ID: 19663681

In genome-wide screens we studied CA/C1 peptidases of malaria-causing plasmodia and their hosts (man and mouse). For Plasmodium falciparum and P. berghei, several new CA/C1 peptidase genes encoding proteases of the L- and B-family with specific promoter modules were identified. In addition, two new human CA/C1 peptidase loci and one new mouse gene locus were found; otherwise, the sets of CA/C1 peptidase genes in man and mouse seem to be complete now. In each species studied there is a multitude of CA/C1 peptidases with lysosomal localization signals and partial functional overlap according to similar but subfamily-specific structures. Individual target structures in plasmodia include residues specifically different in CA/C1 peptidase subsite 2. This is of medical interest considering CA/C1 peptidase inhibition for chemotherapy in malaria, malignancies and other diseases. Promoter structures and mRNA regulation differ widely among CA/C1 peptidase subfamilies and between mammals and plasmodia. We characterized promoter modules conserved in mouse and man for the CA/C1 peptidase families B and L (with the L-like subfamily, F-like subfamily and mouse-specific J-like subfamily). RNA motif searches revealed conserved regulatory elements such as GAIT elements; plasmodial CA/C1 peptidase mRNA elements include ARE elements and mammalian mRNAs contain 15-lox DICE elements.

The Highly Attenuated Oncolytic Recombinant Vaccinia Virus GLV-1h68: Comparative Genomic Features and the Contribution of F14.5L Inactivation

Molecular Genetics and Genomics : MGG. Oct, 2009  |  Pubmed ID: 19701652

As a new anticancer treatment option, vaccinia virus (VACV) has shown remarkable antitumor activities (oncolysis) in preclinical studies, but potential infection of other organs remains a safety concern. We present here genome comparisons between the de novo sequence of GLV-1h68, a recombinant VACV, and other VACVs. The identified differences in open reading frames (ORFs) include genes encoding host-range selection, virulence and immune modulation proteins, e.g., ankyrin-like proteins, serine proteinase inhibitor SPI-2/CrmA, tumor necrosis factor (TNF) receptor homolog CrmC, semaphorin-like and interleukin-1 receptor homolog proteins. Phylogenetic analyses indicate that GLV-1h68 is closest to Lister strains but has lost several ORFs present in its parental LIVP strain, including genes encoding CrmE and a viral Golgi anti-apoptotic protein, v-GAAP. The reduced pathogenicity of GLV-1h68 is confirmed in male mice bearing C6 rat glioma and in immunocompetent mice bearing B16-F10 murine melanoma. The contribution of foreign gene expression cassettes in the F14.5L, J2R and A56R loci is analyzed, in particular the contribution of F14.5L inactivation to the reduced virulence is demonstrated by comparing the virulence of GLV-1h68 with its F14.5L-null and revertant viruses. GLV-1h68 is a promising engineered VACV variant for anticancer therapy with tumor-specific replication, reduced pathogenicity and benign tissue tropism.

Modeling System States in Liver Cells: Survival, Apoptosis and Their Modifications in Response to Viral Infection

BMC Systems Biology. 2009  |  Pubmed ID: 19772631

The decision pro- or contra apoptosis is complex, involves a number of different inputs, and is central for the homeostasis of an individual cell as well as for the maintenance and regeneration of the complete organism.

Modelling Cross-hybridization on Phylogenetic DNA Microarrays Increases the Detection Power of Closely Related Species

Molecular Ecology Resources. Jan, 2009  |  Pubmed ID: 21564570

DNA microarrays are a popular technique for the detection of microorganisms. Several approaches using specific oligomers targeting one or a few marker genes for each species have been proposed. Data analysis is usually limited to call a species present when its oligomer exceeds a certain intensity threshold. While this strategy works reasonably well for distantly related species, it does not work well for very closely related species: Cross-hybridization of nontarget DNA prevents a simple identification based on signal intensity. The majority of species of the same genus has a sequence similarity of over 90%. For biodiversity studies down to the species level, it is therefore important to increase the detection power of closely related species. We propose a simple, cost-effective and robust approach for biodiversity studies using DNA microarray technology and demonstrate it on scenedesmacean green algae. The internal transcribed spacer 2 (ITS2) rDNA sequence was chosen as marker because it is suitable to distinguish all eukaryotic species even though parts of it are virtually identical in closely related species. We show that by modelling hybridization behaviour with a matrix algebra approach, we are able to identify closely related species that cannot be distinguished with a threshold on signal intensity. Thus this proof-of-concept study shows that by adding a simple and robust data analysis step to the evaluation of DNA microarrays, species detection can be significantly improved for closely related species with a high sequence similarity.

5.8S-28S RRNA Interaction and HMM-based ITS2 Annotation

Gene. Feb, 2009  |  Pubmed ID: 19026726

The internal transcribed spacer 2 (ITS2) of the nuclear ribosomal repeat unit is one of the most commonly applied phylogenetic markers. It is a fast evolving locus, which makes it appropriate for studies at low taxonomic levels, whereas its secondary structure is well conserved, and tree reconstructions are possible at higher taxonomic levels. However, annotation of start and end positions of the ITS2 differs markedly between studies. This is a severe shortcoming, as prediction of a correct secondary structure by standard ab initio folding programs requires accurate identification of the marker in question. Furthermore, the correct structure is essential for multiple sequence alignments based on individual structural features. The present study describes a new tool for the delimitation and identification of the ITS2. It is based on hidden Markov models (HMMs) and verifies annotations by comparison to a conserved structural motif in the 5.8S/28S rRNA regions. Our method was able to identify and delimit the ITS2 in more than 30000 entries lacking start and end annotations in GenBank. Furthermore, 45000 ITS2 sequences with a questionable annotation were re-annotated. Approximately 30000 entries from the ITS2-DB, that uses a homology-based method for structure prediction, were re-annotated. We show that the method is able to correctly annotate an ITS2 as small as 58 nt from Giardia lamblia and an ITS2 as large as 1160 nt from humans. Thus, our method should be a valuable guide during the first and crucial step in any ITS2-based phylogenetic analysis: the delineation of the correct sequence. Sequences can be submitted to the following website for HMM-based ITS2 delineation: http://its2.bioapps.biozentrum.uni-wuerzburg.de.

JANE: Efficient Mapping of Prokaryotic ESTs and Variable Length Sequence Reads on Related Template Genomes

BMC Bioinformatics. 2009  |  Pubmed ID: 19943962

ESTs or variable sequence reads can be available in prokaryotic studies well before a complete genome is known. Use cases include (i) transcriptome studies or (ii) single cell sequencing of bacteria. Without suitable software their further analysis and mapping would have to await finalization of the corresponding genome.

Including RNA Secondary Structures Improves Accuracy and Robustness in Reconstruction of Phylogenetic Trees

Biology Direct. 2010  |  Pubmed ID: 20078867

In several studies, secondary structures of ribosomal genes have been used to improve the quality of phylogenetic reconstructions. An extensive evaluation of the benefits of secondary structure, however, is lacking.

Characterization of a Novel Interaction Between Vasodilator-stimulated Phosphoprotein and Abelson Interactor 1 in Human Platelets: a Concerted Computational and Experimental Approach

Arteriosclerosis, Thrombosis, and Vascular Biology. Apr, 2010  |  Pubmed ID: 20110575

The goal of this study was systematic profiling of vasodilator-stimulated phosphoprotein (VASP)-Ena/VASP homology 1 (EVH1) interactors in human platelets using a combined in silico and in vitro approach.

BioNet: an R-Package for the Functional Analysis of Biological Networks

Bioinformatics (Oxford, England). Apr, 2010  |  Pubmed ID: 20189939

Increasing quantity and quality of data in transcriptomics and interactomics create the need for integrative approaches to network analysis. Here, we present a comprehensive R-package for the analysis of biological networks including an exact and a heuristic approach to identify functional modules.

The IronChip Evaluation Package: a Package of Perl Modules for Robust Analysis of Custom Microarrays

BMC Bioinformatics. 2010  |  Pubmed ID: 20193060

Gene expression studies greatly contribute to our understanding of complex relationships in gene regulatory networks. However, the complexity of array design, production and manipulations are limiting factors, affecting data quality. The use of customized DNA microarrays improves overall data quality in many situations, however, only if for these specifically designed microarrays analysis tools are available.

Proteomic Analysis of Tardigrades: Towards a Better Understanding of Molecular Mechanisms by Anhydrobiotic Organisms

PloS One. 2010  |  Pubmed ID: 20224743

Tardigrades are small, multicellular invertebrates which are able to survive times of unfavourable environmental conditions using their well-known capability to undergo cryptobiosis at any stage of their life cycle. Milnesium tardigradum has become a powerful model system for the analysis of cryptobiosis. While some genetic information is already available for Milnesium tardigradum the proteome is still to be discovered.

Transcriptome Survey of the Anhydrobiotic Tardigrade Milnesium Tardigradum in Comparison with Hypsibius Dujardini and Richtersius Coronifer

BMC Genomics. 2010  |  Pubmed ID: 20226016

The phenomenon of desiccation tolerance, also called anhydrobiosis, involves the ability of an organism to survive the loss of almost all cellular water without sustaining irreversible damage. Although there are several physiological, morphological and ecological studies on tardigrades, only limited DNA sequence information is available. Therefore, we explored the transcriptome in the active and anhydrobiotic state of the tardigrade Milnesium tardigradum which has extraordinary tolerance to desiccation and freezing. In this study, we present the first overview of the transcriptome of M. tardigradum and its response to desiccation and discuss potential parallels to stress responses in other organisms.

Carbon Metabolism of Intracellular Bacterial Pathogens and Possible Links to Virulence

Nature Reviews. Microbiology. Jun, 2010  |  Pubmed ID: 20453875

New technologies such as high-throughput methods and 13C-isotopologue-profiling analysis are beginning to provide us with insight into the in vivo metabolism of microorganisms, especially in the host cell compartments that are colonized by intracellular bacterial pathogens. In this Review, we discuss the recent progress made in determining the major carbon sources and metabolic pathways used by model intracellular bacterial pathogens that replicate either in the cytosol or in vacuoles of infected host cells. Furthermore, we highlight the possible links between intracellular carbon metabolism and the expression of virulence genes.

Developmental Profiling by Mass Spectrometry of Phosphocholine Containing Phospholipids in the Rat Nervous System Reveals Temporo-spatial Gradients

Journal of Neurochemistry. Aug, 2010  |  Pubmed ID: 20524967

Phospholipids are important components of the nervous system, in particular of neuronal and glial membranes. Ontogenesis of the nervous system is associated with fundamental alterations in lipid patterns. Here, matrix-assisted-laser-desorption/ionization time-of-flight mass spectrometry and electro-spray-ionization mass spectrometry were combined to analyze phosphatidylcholines and sphingomyelins, allowing an assessment of individual molecular species. Analysis in eight different regions of the nervous system during development of the Wistar rat, from embryonic day 14 to adulthood, produced informative patterns of developmental and regional changes in lipid contents. Phospholipids containing long chain fatty acyl residues exhibited a characteristic patterning, with dramatic increases in the caudal parts of the nervous system 2 weeks after birth. In contrast, relative contents of short chain phosphatidylcholines were low in the perinatal CNS, decreasing even further during development. The relative amounts of sphingomyelins carrying the fatty acid residues 18:0, 22:0, 24:0, and 24:1 increased developmentally in the caudal nervous system. The rostro-caudal gradient of long chain lipid accumulation is matched by expression gradients of myelin structural and regulatory genes, as evident from bioinformatic analysis. These observations characterize the accumulation of individual lipid classes in the nervous system as a highly regulated process, with structurally related lipids showing a similar temporo-spatial distribution and developmental patterning.

Protecs, a Comprehensive and Powerful Storage and Analysis System for OMICS Data, Applied for Profiling the Anaerobiosis Response of Staphylococcus Aureus COL

Proteomics. Aug, 2010  |  Pubmed ID: 20662099

Broad functional genomic studies call for comprehensive and powerful data repositories for storage of genome sequences, experimental information, protein identification data, protein properties and expression values. The better such data repositories can integrate and display complex data in a clear and structured way the more biologically meaningful conclusions or novel hypotheses can be derived from extensive omics data sets. This work presents the web accessible database system Protecs and how it was used to support analysis of 50 samples drawn from four Staphylococcus aureus cultivations under anaerobiosis. Protecs incorporates findings from visualization science, e.g. micro charts and heat maps in the user interface. Its integrated tools for expression data analysis in combination with TIGR Multi Experiment Viewer were used to highlight similar gene expression profiles in single or multiple experiments based on the continuously updated S. aureus master gel. Raw data analysis results are available online at www.protecs.uni-greifswald.de. Our meta-study revealed that S. aureus responds in different anaerobiotic experimental setups (growth without oxygen; growth without oxygen but with supplemental pyruvate and uracil; growth without oxygen but with NO(3)(-); growth without oxygen but with NO(3)(-) and without functional nreABC genes) with a general anaerobiosis response. Among others, this response is characterized by an induction of fermentation enzymes (PflB, Ldh1, SACOL0135, SACOL0660) as well as the response regulator SrrA. Interestingly, especially genes with a high codon adaptation index highly overlap with anaerobically induced genes.

Pharmacogenomic Strategies Against Microbial Resistance: from Bright to Bleak to Innovative

Pharmacogenomics. Sep, 2010  |  Pubmed ID: 20860457

The last decade saw an alarming increase in antibiotic resistance in infections, with more than 13 million deaths per year from infections. Counter strategies include hygiene, antibiotic restriction and new antibiotics such as quinupristin, linezolid, tigecycline, daptomycin and dalbavancin. Presently, pharmacogenomics with basic research is revealing new antimicrobial peptides and is applying old drugs in new ways to break resistance. New approaches with host-directed drug targeting emerge to circumvent resistance. A future systems perspective from large-scale molecular techniques and bioinformatic modeling allows pharmacogenomics to reveal new intervention angles. This includes the fight against resistance and its transmission, improved vaccines, disarmament of microbes and antibiotic options from novel molecular targets (lipids, RNA and carbohydrates). Such a system perspective is also essential for improved diagnostics and individualized medicine. However, an increase in public awareness and closer cooperation of industry and basic research are essential to turn research into powerful new drugs that will enable us to treat new arising infections in the future.

Deciphering the Intracellular Metabolism of Listeria Monocytogenes by Mutant Screening and Modelling

BMC Genomics. 2010  |  Pubmed ID: 20955543

The human pathogen Listeria monocytogenes resides and proliferates within the cytoplasm of epithelial cells. While the virulence factors essentially contributing to this step of the infection cycle are well characterized, the set of listerial genes contributing to intracellular replication remains to be defined on a genome-wide level.

High-throughput Microarray Technology in Diagnostics of Enterobacteria Based on Genome-wide Probe Selection and Regression Analysis

BMC Genomics. 2010  |  Pubmed ID: 20964857

The Enterobacteriaceae comprise a large number of clinically relevant species with several individual subspecies. Overlapping virulence-associated gene pools and the high overall genome plasticity often interferes with correct enterobacterial strain typing and risk assessment. Array technology offers a fast, reproducible and standardisable means for bacterial typing and thus provides many advantages for bacterial diagnostics, risk assessment and surveillance. The development of highly discriminative broad-range microbial diagnostic microarrays remains a challenge, because of marked genome plasticity of many bacterial pathogens.

Lead Expansion and Virtual Screening of Indinavir Derivate HIV-1 Protease Inhibitors Using Pharmacophoric - Shape Similarity Scoring Function

Bioinformation. 2010  |  Pubmed ID: 20978602

Indinavir (Crivaxan®) is a potent inhibitor of the HIV (human immunodeficiency virus) protease. This enzyme has an important role in viral replication and is considered to be very attractive target for new antiretroviral drugs. However, it becomes less effective due to highly resistant new viral strains of HIV, which have multiple mutations in their proteases. For this reason, we used a lead expansion method to create a new set of compounds with a new mode of action to protease binding site. 1300 compounds chemically diverse from the initial hit were generated and screened to determine their ability to interact with protease and establish their QSAR properties. Further computational analyses revealed one unique compound with different protease binding ability from the initial hit and its role for possible new class of protease inhibitors is discussed in this report.

Stress Response in Tardigrades: Differential Gene Expression of Molecular Chaperones

Cell Stress & Chaperones. Jul, 2010  |  Pubmed ID: 19943197

Semi-terrestrial tardigrades exhibit a remarkable tolerance to desiccation by entering a state called anhydrobiosis. In this state, they show a strong resistance against several kinds of physical extremes. Because of the probable importance of stress proteins during the phases of dehydration and rehydration, the relative abundance of transcripts coding for two alpha-crystallin heat-shock proteins (Mt-sHsp17.2 and Mt-sHsp19.5), as well for the heat-shock proteins Mt-sHsp10, Mt-Hsp60, Mt-Hsp70 and Mt-Hsp90, were analysed in active and anhydrobiotic tardigrades of the species Milnesium tardigradum. They were also analysed in the transitional stage (I) of dehydration, the transitional stage (II) of rehydration and in heat-shocked specimens. A variable pattern of expression was detected, with most candidates being downregulated. Gene transcripts of one Mt-hsp70 isoform in the transitional stage I and Mt-hsp90 in the anhydrobiotic stage were significantly upregulated. A high gene expression (778.6-fold) was found for the small alpha-crystallin heat-shock protein gene Mt-sHsp17.2 after heat shock. We discuss the limited role of the stress-gene expression in the transitional stages between the active and anhydrobiotic tardigrades and other mechanisms which allow tardigrades to survive desiccation.

Two Nucleus-localized CDK-like Kinases with Crucial Roles for Malaria Parasite Erythrocytic Replication Are Involved in Phosphorylation of Splicing Factor

Journal of Cellular Biochemistry. May, 2011  |  Pubmed ID: 21312235

The kinome of the human malaria parasite Plasmodium falciparum comprises representatives of most eukaryotic protein kinase groups, including kinases which regulate proliferation and differentiation processes. Despite extensive research on most plasmodial enzymes, little information is available regarding the four identified members of the cyclin-dependent kinase-like kinase (CLK) family. In other eukaryotes, CLKs regulate mRNA splicing through phosphorylation of Serine/Arginine-rich proteins. Here, we investigate two of the PfCLKs, the Lammer kinase homolog PfCLK-1, and PfCLK-2. Both PfCLKs show homology with the yeast Serine/Arginine protein kinase Sky1p and are transcribed throughout the asexual blood stages and in gametocytes. PfCLK-1/Lammer possesses two nuclear localization signal sites and PfCLK-2 possesses one of these signal sites upstream of the C-terminal catalytic domains. Indirect immunofluorescence, Western blot, and electron microscopy data confirm that the kinases are primarily localized in the parasite nucleus, and PfCLK-2 is further present in the cytoplasm. The two kinases are important for completion of the asexual replication cycle of P. falciparum, as demonstrated by reverse genetics approaches. In vitro kinase assays show substrate phosphorylation by the PfCLKs, including the Sky1p substrate, splicing factor Npl3p, and the plasmodial alternative splicing factor PfASF-1. Mass spectrometric analysis of co-immunoprecipitated proteins indicates assembly of the two PfCLKs with proteins with predicted nuclease, phosphatase, or helicase functions. Our data indicate a crucial role of PfCLKs for malaria blood stage parasites, presumably by participating in gene regulation through the post-transcriptional modification of mRNA.

A Metabolomics and Proteomics Study of the Adaptation of Staphylococcus Aureus to Glucose Starvation

Molecular BioSystems. Apr, 2011  |  Pubmed ID: 21327190

As a versatile pathogen Staphylococcus aureus can cause various disease patterns, which are influenced by strain specific virulence factor repertoires but also by S. aureus physiological adaptation capacity. Here, we present metabolomic descriptions of S. aureus central metabolic pathways and demonstrate the potential for combined metabolomics- and proteomics-based approaches for the basic research of this important pathogen. This study provides a time-resolved picture of more than 500 proteins and 94 metabolites during the transition from exponential growth to glucose starvation. Under glucose excess, cells exhibited higher levels of proteins involved in glycolysis and protein-synthesis, whereas entry into the stationary phase triggered an increase of enzymes of TCC and gluconeogenesis. These alterations in levels of metabolic enzymes were paralleled by more pronounced changes in the concentrations of associated metabolites, in particular, intermediates of the glycolysis and several amino acids.

Modeling Antibiotic and Cytotoxic Effects of the Dimeric Isoquinoline IQ-143 on Metabolism and Its Regulation in Staphylococcus Aureus, Staphylococcus Epidermidis and Human Cells

Genome Biology. 2011  |  Pubmed ID: 21418624

Xenobiotics represent an environmental stress and as such are a source for antibiotics, including the isoquinoline (IQ) compound IQ-143. Here, we demonstrate the utility of complementary analysis of both host and pathogen datasets in assessing bacterial adaptation to IQ-143, a synthetic analog of the novel type N,C-coupled naphthyl-isoquinoline alkaloid ancisheynine.

Immune Response of the Ant Camponotus Floridanus Against Pathogens and Its Obligate Mutualistic Endosymbiont

Insect Biochemistry and Molecular Biology. Aug, 2011  |  Pubmed ID: 21440063

Numerous insect species harbor mutualistic endosymbionts that play a role in nutrient cycling or confer other fitness benefits to their hosts. Insect hosts face the problem of having to maintain such mutualistic bacteria while staging an immune response towards pathogens upon infection. In addition, hosts may regulate the number of endosymbionts present in their tissues via the innate immune system. Camponotus floridanus ants harbor the obligate endosymbiont Blochmannia floridanus in specialized midgut cells and ovaries. We identified genes transcriptionally induced in response to septic injury by suppression subtractive hybridization (SSH). Among these were genes involved in pathogen recognition (e.g. GNBP), signal transduction (e.g. MAPK-kinase), antimicrobial activity (e.g. defensin and hymenoptaecin), or general stress response (e.g. heat shock protein). A quantitative analysis of immune-gene expression revealed different expression kinetics of individual factors and also characteristic expression profiles after injection of gram-negative and gram-positive bacteria. Likewise, B. floridanus injected into the hemocoel elicited a comparable immune response of its host C. floridanus. Thus, the host immune system may contribute to controlling the endosymbiont population.

Staphylococcus Aureus Physiological Growth Limitations: Insights from Flux Calculations Built on Proteomics and External Metabolite Data

Proteomics. May, 2011  |  Pubmed ID: 21472852

Comparing proteomics and metabolomics allows insights into Staphylococcus aureus physiological growth. We update genome and proteome information and deliver strain-specific metabolic models for three S. aureus strains (COL, N315, and Newman). We find a number of differences in metabolism and enzymes. Growth experiments (glucose or combined with oxygen limitation) were conducted to measure external metabolites. Fluxes of the central metabolism were calculated from these data with low error. In exponential phase, glycolysis is active and amino acids are used for growth. In later phases, dehydroquinate synthetase is suppressed and acetate metabolism starts. There are strain-specific differences for these phases. A time series of 2-D gel protein expression data on COL strain delivered a second data set (glucose limitation) on which fluxes were calculated. The comparison with the metabolite-predicted fluxes shows, in general, good correlation. Outliers point to different regulated enzymes for S. aureus COL under these limitations. In exponential growth, there is lower activity for some enzymes in upper glycolysis and pentose phosphate pathway and stronger activity for some in lower glycolysis. In transition phase, aspartate kinase is expressed to meet amino acid requirements and in later phases there is high expression of glyceraldehyde-3-phosphate dehydrogenase and lysine synthetase. Central metabolite fluxes and protein expression of their enzymes correlate in S. aureus.

Integration of Boolean Models Exemplified on Hepatocyte Signal Transduction

Briefings in Bioinformatics. Oct, 2011  |  Pubmed ID: 22016404

The number of mathematical models for biological pathways is rapidly growing. In particular, Boolean modelling proved to be suited to describe large cellular signalling networks. Systems biology is at the threshold to holistic understanding of comprehensive networks. In order to reach this goal, connection and integration of existing models of parts of cellular networks into more comprehensive network models is necessary. We discuss model combination approaches for Boolean models. Boolean modelling is qualitative rather than quantitative and does not require detailed kinetic information. We show that these models are useful precursors for large-scale quantitative models and that they are comparatively easy to combine. We propose modelling standards for Boolean models as a prerequisite for smooth model integration. Using these standards, we demonstrate the coupling of two logical models on two different examples concerning cellular interactions in the liver. In the first example, we show the integration of two Boolean models of two cell types in order to describe their interaction. In the second example, we demonstrate the combination of two models describing different parts of the network of a single cell type. Combination of partial models into comprehensive network models will take systems biology to the next level of understanding. The combination of logical models facilitated by modelling standards is a valuable example for the next step towards this goal.

Time-resolved in Silico Modeling of Fine-tuned CAMP Signaling in Platelets: Feedback Loops, Titrated Phosphorylations and Pharmacological Modulation

BMC Systems Biology. 2011  |  Pubmed ID: 22034949

Hemostasis is a critical and active function of the blood mediated by platelets. Therefore, the prevention of pathological platelet aggregation is of great importance as well as of pharmaceutical and medical interest. Endogenous platelet inhibition is predominantly based on cyclic nucleotides (cAMP, cGMP) elevation and subsequent cyclic nucleotide-dependent protein kinase (PKA, PKG) activation. In turn, platelet phosphodiesterases (PDEs) and protein phosphatases counterbalance their activity. This main inhibitory pathway in human platelets is crucial for countervailing unwanted platelet activation. Consequently, the regulators of cyclic nucleotide signaling are of particular interest to pharmacology and therapeutics of atherothrombosis. Modeling of pharmacodynamics allows understanding this intricate signaling and supports the precise description of these pivotal targets for pharmacological modulation.

Single-cell Genomics Reveals the Lifestyle of Poribacteria, a Candidate Phylum Symbiotically Associated with Marine Sponges

The ISME Journal. Jan, 2011  |  Pubmed ID: 20613790

In this study, we present a single-cell genomics approach for the functional characterization of the candidate phylum Poribacteria, members of which are nearly exclusively found in marine sponges. The microbial consortia of the Mediterranean sponge Aplysina aerophoba were singularized by fluorescence-activated cell sorting, and individual microbial cells were subjected to phi29 polymerase-mediated 'whole-genome amplification'. Pyrosequencing of a single amplified genome (SAG) derived from a member of the Poribacteria resulted in nearly 1.6 Mb of genomic information distributed among 554 contigs analyzed in this study. Approximately two-third of the poribacterial genome was sequenced. Our findings shed light on the functional properties and lifestyle of a possibly ancient bacterial symbiont of marine sponges. The Poribacteria are mixotrophic bacteria with autotrophic CO(2)-fixation capacities through the Wood-Ljungdahl pathway. The cell wall is of Gram-negative origin. The Poribacteria produce at least two polyketide synthases (PKSs), one of which is the sponge-specific Sup-type PKS. Several putative symbiosis factors such as adhesins (bacterial Ig-like domains, lamininin G domain proteins), adhesin-related proteins (ankyrin, fibronectin type III) and tetratrico peptide repeat domain-encoding proteins were identified, which might be involved in mediating sponge-microbe interactions. The discovery of genes coding for 24-isopropyl steroids implies that certain fossil biomarkers used to date the origins of metazoan life on earth may possibly be of poribacterial origin. Single-cell genomic approaches, such as those shown herein, contribute to a better understanding of beneficial microbial consortia, of which most members are, because of the lack of cultivation, inaccessible by conventional techniques.

PlateletWeb: a Systems Biologic Analysis of Signaling Networks in Human Platelets

Blood. Jan, 2012  |  Pubmed ID: 22123846

Understanding the cellular mechanisms of platelet activation and their pharmacologic modulation is of major interest for basic and clinical research. Here we introduce a comprehensive human platelet repository (PlateletWeb) for systems biologic analysis of platelets in the functional context of integrated networks. Functional, drug, and pathway associations provide a first systemic insight into various aspects of platelet functionality and pharmacologic regulation. Detailed manual curation of recent platelet proteome and transcriptome studies yielded more than 5000 platelet proteins. Integration of protein-protein interactions with kinase-substrate relationships unraveled the platelet signaling network involving more than 70% of all platelet proteins. Analysis of the platelet kinome in the context of the kinase phylogenetic background revealed an over-representation of tyrosine kinase substrates. The extraction and graphical visualization of specific subnetworks allow identification of all major signaling modules involved in activation and inhibition. An in-depth analysis of DOK1 signaling identifies putative signal modulators of the integrin network. Through integration of various information sources and high curation standards, the PlateletWeb knowledge base offers the systems biologic background for the investigation of signal transduction in human platelets (http://plateletweb.bioapps.biozentrum.uni-wuerzburg.de).

Toward a Systemic Understanding of Listeria Monocytogenes Metabolism During Infection

Frontiers in Microbiology. 2012  |  Pubmed ID: 22347216

Listeria monocytogenes is a foodborne human pathogen that can cause invasive infection in susceptible animals and humans. For proliferation within hosts, this facultative intracellular pathogen uses a reservoir of specific metabolic pathways, transporter, and enzymatic functions whose expression requires the coordinated activity of a complex regulatory network. The highly adapted metabolism of L. monocytogenes strongly depends on the nutrient composition of various milieus encountered during infection. Transcriptomic and proteomic studies revealed the spatial-temporal dynamic of gene expression of this pathogen during replication within cultured cells or in vivo. Metabolic clues are the utilization of unusual C(2)- and C(3)-bodies, the metabolism of pyruvate, thiamine availability, the uptake of peptides, the acquisition or biosynthesis of certain amino acids, and the degradation of glucose-phosphate via the pentose phosphate pathway. These examples illustrate the interference of in vivo conditions with energy, carbon, and nitrogen metabolism, thus affecting listerial growth. The exploitation, analysis, and modeling of the available data sets served as a first attempt to a systemic understanding of listerial metabolism during infection. L. monocytogenes might serve as a model organism for systems biology of a Gram-positive, facultative intracellular bacterium.