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At least 235 records · Page 13Linked to original sources
PubMed2005

Peptide models XLV: conformational properties of N-formyl-L-methioninamide and its relevance to methionine in proteins.

The conformational space of the most biologically significant backbone folds of a suitable methionine peptide model was explored by density functional computational method. Using a medium [6-31G(d)] and a larger basis set [6-311++G(2d,2p)], the systematic exploration of low-energy backbone structures restricted for the "L-region" in the Ramachandran map of N-formyl-L-methioninamide results in conformers corresponding to the building units of an extended backbone structure (betaL), an inverse gamma-turn (gammaL), or a right-handed helical structure (alphaL). However, no poly-proline II type (epsilonL) fold was found, indicating that this conformer has no intrinsic stability, and highlighting the effect of molecular environment in stabilizing this backbone structure. This is in agreement with the abundance of the epsilonL-type backbone conformation of methionine found in proteins. Stability properties (DeltaE) and distinct backbone-side-chain interactions support the idea that specific intramolecular contacts are operative in the selection of the lowest energy conformers. Apart from the number of different folds, all stable conformers are within a 10 kcal x mol(-1) energy range, indicating the highly flexible behavior of methionine. This conformational feature can be important in supporting catalytic processes, facilitating protein folding and dimerization via metal ion binding. In both of the biological examples discussed (HIV-1 reverse transcriptase and PcoC copper-resistant protein), the conformational properties of Met residues were found to be of key importance. Spatial proximity to other types of residues or the same type of residue seems to be crucial for the structural integrity of a protein, whether Met is buried or exposed.

Amino Acids↗
PubMed2005

Effects of background fluorescence in fluorescence molecular tomography.

Recent advances in optical imaging systems and systemically administered fluorescent probes have significantly improved the ways by which we can visualize proteomics in vivo. A key component in the design of fluorescent probes is a favorable biodistribution, i.e., localization only in the targeted diseased tissue, in order to achieve high contrast and good detection characteristics. In practice, however, there is always some level of background fluorescence present that could result in distorted or obscured visualization and quantification of measured signals. In this study we observe the effects of background fluorescence in tomographic imaging. We demonstrate that increasing levels of background fluorescence result in artifacts when using a linear perturbation algorithm, along with a significant loss of image fidelity and quantification accuracy. To correct for effects of background fluorescence, we have applied cubic polynomial fits to bulk raw measurements obtained from spatially homogeneous and heterogeneous phantoms. We show that subtraction of the average fluorescence response from the raw data before reconstruction can improve image quality and quantification accuracy as shown in relatively homogeneous or heterogeneous phantoms. Subtraction methods thus appear to be a promising route for adaptively correcting nonspecific background fluorochrome distribution.

Algorithms↗
PubMedDate not supplied

The role of mass spectrometry in plant systems biology.

Large-scale analyses of proteins and metabolites are intimately bound to advancements in MS technologies. The aim of these non-targeted "omic" technologies is to extend our understanding beyond the analysis of only parts of the system. Here, metabolomics and proteomics emerged in parallel with the development of novel mass analyzers and hyphenated techniques such as gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF-MS) and multidimensional liquid chromatography coupled to mass spectrometry (LC-MS). The analysis of (i) proteins (ii) phosphoproteins, and (iii) metabolites is discussed in the context of plant physiology and environment and with a focus on novel method developments. Recently published studies measuring dynamic (quantitative) behavior at these levels are summarized; for these works, the completely sequenced plants Arabidopsis thaliana and Oryza sativa (rice) have been the primary models of choice. Particular emphasis is given to key physiological processes such as metabolism, development, stress, and defense. Moreover, attempts to combine spatial, tissue-specific resolution with systematic profiling are described. Finally, we summarize the initial steps to characterize the molecular plant phenotype as a corollary of environment and genotype.

Mass Spectrometry↗
PubMed2000

Strategies for the physiome project.

The physiome is the quantitative description of the functioning organism in normal and pathophysiological states. The human physiome can be regarded as the virtual human. It is built upon the morphome, the quantitative description of anatomical structure, chemical and biochemical composition, and material properties of an intact organism, including its genome, proteome, cell, tissue, and organ structures up to those of the whole intact being. The Physiome Project is a multicentric integrated program to design, develop, implement, test and document, archive and disseminate quantitative information, and integrative models of the functional behavior of molecules, organelles, cells, tissues, organs, and intact organisms from bacteria to man. A fundamental and major feature of the project is the databasing of experimental observations for retrieval and evaluation. Technologies allowing many groups to work together are being rapidly developed. Internet II will facilitate this immensely. When problems are huge and complex, a particular working group can be expert in only a small part of the overall project. The strategies to be worked out must therefore include how to pull models composed of many submodules together even when the expertise in each is scattered amongst diverse institutions. The technologies of bioinformatics will contribute greatly to this effort. Developing and implementing code for large-scale systems has many problems. Most of the submodules are complex, requiring consideration of spatial and temporal events and processes. Submodules have to be linked to one another in a way that preserves mass balance and gives an accurate representation of variables in nonlinear complex biochemical networks with many signaling and controlling pathways. Microcompartmentalization vitiates the use of simplified model structures. The stiffness of the systems of equations is computationally costly. Faster computation is needed when using models as thinking tools and for iterative data analysis. Perhaps the most serious problem is the current lack of definitive information on kinetics and dynamics of systems, due in part to the almost total lack of databased observations, but also because, though we are nearly drowning in new information being published each day, either the information required for the modeling cannot be found or has never been obtained. "Simple" things like tissue composition, material properties, and mechanical behavior of cells and tissues are not generally available. The development of comprehensive models of biological systems is a key to pharmaceutics and drug design, for the models will become gradually better predictors of the results of interventions, both genomic and pharmaceutic. Good models will be useful in predicting the side effects and long term effects of drugs and toxins, and when the models are really good, to predict where genomic intervention will be effective and where the multiple redundancies in our biological systems will render a proposed intervention useless. The Physiome Project will provide the integrating scientific basis for the Genes to Health initiative, and make physiological genomics a reality applicable to whole organisms, from bacteria to man.

Computer Simulation↗
PubMed2006

Optimizing UV laser focus profiles for improved MALDI performance.

Matrix assisted laser desorption/ionization (MALDI) applications, such as proteomics, genomics, clinical profiling and MALDI imaging, have created a growing demand for faster instrumentation. Since the commonly used nitrogen lasers have throughput and life span limitations, diode-pumped solid-state lasers are an alternative. Unfortunately this type of laser shows clear performance limitations in MALDI in terms of sensitivity, resolution and ease of use, for applications such as thin-layer sample preparations, acceptance of various matrices (e.g. DHB for glycopeptides) and MALDI imaging. While it is obvious that the MALDI process has some dependence on the characteristics of the laser used, it is unclear which features are the most critical in determining laser performance for MALDI. In this paper we show, for the first time, that a spatially structured laser beam profile in lieu of a Gaussian profile is of striking importance. This result enabled us to design diode-pumped Nd : YAG lasers that on various critical applications perform as well for MALDI as the nitrogen lasers and in some respects even better. The modulation of the beam profile appears to be a new parameter for optimizing the MALDI process. In addition, the results trigger new questions directing us to a better understanding of the MALDI process.

Algorithms↗
PubMed2006

Cell++--simulating biochemical pathways.

MOTIVATION: With the generation of a wealth of information, detailing cellular components, their functions and interactions, there is a growing need for the development of new computational tools capable of interpreting these data within spatial and dynamic contexts. Here, we introduce Cell++, a novel stochastic simulation environment with the capacity to study a wide variety of biochemical processes within a spatial context. RESULTS: Focusing on three case studies, we highlight the potential impact of spatial organization in the evolution and engineering of signaling and metabolic pathways. In addition to altering signaling and metabolic efficiency, simulations also demonstrated features consistent with the phenomenon of metabolic channeling. AVAILABILITY: Cell++ is licensed under the GNU general public license (GPL) and has been successfully implemented under Linux and IRIX operating systems. Source code together with a simple tutorial is available at http://www.compsysbio.org/CellSim/.

Biochemistry↗
PubMed2002

Continuous sample deposition from reversed-phase liquid chromatography to tracks on a matrix-assisted laser desorption/ionization precoated target for the analysis of protein digests.

Peptide mass fingerprinting by matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS) is one of the standard high-throughput methods for protein identification today. Traditionally this method has been based on spotting peptide mixtures onto MALDI targets. While this method works well for more abundant proteins, low-abundance proteins mixed with high-abundance proteins tend to go undetected due to ion suppression effects, instrumental dynamic range limitations and chemical noise interference. We present an alternative approach where liquid chromatography (LC) effluent is continuously collected as linear tracks on a MALDI target. In this manner the chromatographic separation is spatially preserved on the target, which enables generation of off-line LC-MS and LC-MS/MS data by MALDI. LC-MALDI sample collection provides improved sensitivity and dynamic range, spatial resolution of peptides along the sample track, and permits peptide mass mapping of low-abundance proteins in mixtures containing high-abundance proteins. In this work, standard and ribosomal protein digests are resolved and captured using LC-MALDI sample collection and analyzed by MALDI-TOF-MS.

Amino Acid Sequence↗
PubMed2007

Direct identification of proteins from T47D cells and murine brain tissue by matrix-assisted laser desorption/ionization post-source decay/collision-induced dissociation.

The purpose of this study is to determine the feasibility of the direct matrix-assisted laser desorption/ionization (MALDI) identification of proteins in fixed T47D breast cancer cells and murine brain tissues. The ability to identify proteins from cells and tissue may lead to biomarkers that effectively predict the onset of defined disease states, and their dynamic behavior could be an important hint for drug target discoveries. Direct tissue application of trypsin allows protein identification in cells and tissues, while maintaining spatial integrity and intracellular organization. Using a chemical printer, matrix was co-registered on trypsinized human T47D breast cancer cells and cryo-preserved sections of murine brain tissue, followed by MALDI post-source decay (PSD) or MALDI collision-induced dissociation (CID), respectively. Mass-to-charge (m/z) data from the cells and brain tissues were processed using Mascot software interrogation of the National Center for Biotechnology Information (NCBI) database. Histone H2B was identified from cultured T47D human breast cancer cells. Tubulin beta2 was identified from mouse brain cortex following an induced stroke. These results suggest that MALDI PSD/CID, combined with bioinformatics, can be used for the direct identification of proteins from cells and tissues. Refinements in preparation techniques may improve this approach to provide a tool for quantitative proteomics and clinical analysis.

Animals↗
PubMed2005

Identification of differentially expressed genes between fetal and adult mouse kidney: candidate gene in kidney development.

BACKGROUND: The kidney development involves a wide variety of developmental processes requiring a lot of genes expressed in a sequential manner. The aim of the present study is to identify new genes involved in these processes. METHODS: To obtain a view of the mouse embryonic kidney transcriptome we used the SADE method, which allows large-scale quantitative gene expression measurements. RESULTS: 7,689 tags were sequenced from our library. Among the 4,507 unique transcripts yielded, 64% correspond to known genes, 22% ESTs, 12% unidentified genes. 472 genes were differentially expressed as compared to published adult kidney library. Among these, we identified several candidate genes and focused on a particular one: thymosin beta4 (Tbeta4), an actin-sequestering protein more highly expressed in fetal kidney. First we studied the in vivo expression patterns of Tbeta4 transcript during kidney development. Tbeta4 increases throughout the kidney development and remains high during active nephrogenesis. Moreover, the spatial distribution of Tbeta4 mRNA was analysed and reveals that during active nephrogenesis (i.e., 18 dpc) Tbeta4 is localised in differentiating glomeruli. In adult kidney, Tbeta4 remains expressed in podocytes and collecting ducts. CONCLUSION: Our results provide the first demonstration of Tbeta4 production in vivo by embryonic kidney and further show that Tbeta4 is implicated in kidney organogenesis.

Aging↗
PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗
PubMed2006

Shootin1: A protein involved in the organization of an asymmetric signal for neuronal polarization.

Neurons have the remarkable ability to polarize even in symmetrical in vitro environments. Although recent studies have shown that asymmetric intracellular signals can induce neuronal polarization, it remains unclear how these polarized signals are organized without asymmetric cues. We describe a novel protein, named shootin1, that became up-regulated during polarization of hippocampal neurons and began fluctuating accumulation among multiple neurites. Eventually, shootin1 accumulated asymmetrically in a single neurite, which led to axon induction for polarization. Disturbing the asymmetric organization of shootin1 by excess shootin1 disrupted polarization, whereas repressing shootin1 expression inhibited polarization. Overexpression and RNA interference data suggest that shootin1 is required for spatially localized phosphoinositide-3-kinase activity. Shootin1 was transported anterogradely to the growth cones and diffused back to the soma; inhibiting this transport prevented its asymmetric accumulation in neurons. We propose that shootin1 is involved in the generation of internal asymmetric signals required for neuronal polarization.

Amino Acid Sequence↗
PubMed2004

POINT: a database for the prediction of protein-protein interactions based on the orthologous interactome.

One possible path towards understanding the biological function of a target protein is through the discovery of how it interfaces within protein-protein interaction networks. The goal of this study was to create a virtual protein-protein interaction model using the concepts of orthologous conservation (or interologs) to elucidate the interacting networks of a particular target protein. POINT (the prediction of interactome database) is a functional database for the prediction of the human protein-protein interactome based on available orthologous interactome datasets. POINT integrates several publicly accessible databases, with emphasis placed on the extraction of a large quantity of mouse, fruit fly, worm and yeast protein-protein interactions datasets from the Database of Interacting Proteins (DIP), followed by conversion of them into a predicted human interactome. In addition, protein-protein interactions require both temporal synchronicity and precise spatial proximity. POINT therefore also incorporates correlated mRNA expression clusters obtained from cell cycle microarray databases and subcellular localization from Gene Ontology to further pinpoint the likelihood of biological relevance of each predicted interacting sets of protein partners.

Animals↗
PubMed2005

Electrospray interfacing of polymer microfluidics to MALDI-MS.

The off-line coupling of polymer microfluidics to MALDI-MS is presented using electrospray deposition. Using polycarbonate microfluidic chips with integrated hydrophobic membrane electrospray tips, peptides and proteins are deposited onto a stainless steel target followed by MALDI-MS analysis. Microchip electrospray deposition is found to yield excellent spatial control and homogeneity of deposited peptide spots, and significantly improved MALDI-MS spectral reproducibility compared to traditional target preparation methods. A detection limit of 3.5 fmol is demonstrated for angiotensin. Furthermore, multiple electrospray tips on a single chip provide the ability to simultaneously elute parallel sample streams onto a MALDI target for high-throughput multiplexed analysis. Using a three-element electrospray tip array with 150 microm spacing, the simultaneous deposition of bradykinin, fibrinopeptide, and angiotensin is achieved with no cross talk between deposited samples. In addition, in-line proteolytic digestion of intact proteins is successfully achieved during the electrospray process by binding trypsin within the electrospray membrane, eliminating the need for on-probe digestion prior to MALDI-MS. The technology offers promise for a range of microfluidic platforms designed for high-throughput multiplexed proteomic analyses in which simultaneous on-chip separations require an effective interface to MS.

Microfluidic Analytical Techniques↗
PubMed2003

From measurements of metabolites to metabolomics: an 'on the fly' perspective illustrated by recent studies of carbon-nitrogen interactions.

Techniques for surveying metabolite levels provide a powerful tool for basic plant research and biotechnology. They offer the possibility of specialised measurements of specific classes of metabolites, profiling of a very broad range of low molecular weight compounds, and resolving the spatial and/or temporal localisation of selected key metabolites. Recent applications in the field of carbon-nitrogen interactions provide a framework to discuss how metabolite datasets are being utilised in the post-genomic era to characterise system responses, link transcript data to phenotypic responses, analyse underlying regulation mechanisms, and implement an in-context analysis of gene function. A major future challenge concerns the integration of the information gained from metabolite profiling into an accessible body of knowledge.

Carbon↗
PubMed2004

Computational physiology and the Physiome Project.

Bioengineering analyses of physiological systems use the computational solution of physical conservation laws on anatomically detailed geometric models to understand the physiological function of intact organs in terms of the properties and behaviour of the cells and tissues within the organ. By linking behaviour in a quantitative, mathematically defined sense across multiple scales of biological organization--from proteins to cells, tissues, organs and organ systems--these methods have the potential to link patient-specific knowledge at the two ends of these spatial scales. A genetic profile linked to cardiac ion channel mutations, for example, can be interpreted in relation to body surface ECG measurements via a mathematical model of the heart and torso, which includes the spatial distribution of cardiac ion channels throughout the myocardium and the individual kinetics for each of the approximately 50 types of ion channel, exchanger or pump known to be present in the heart. Similarly, linking molecular defects such as mutations of chloride ion channels in lung epithelial cells to the integrated function of the intact lung requires models that include the detailed anatomy of the lungs, the physics of air flow, blood flow and gas exchange, together with the large deformation mechanics of breathing. Organizing this large body of knowledge into a coherent framework for modelling requires the development of ontologies, markup languages for encoding models, and web-accessible distributed databases. In this article we review the state of the field at all the relevant levels, and the tools that are being developed to tackle such complexity. Integrative physiology is central to the interpretation of genomic and proteomic data, and is becoming a highly quantitative, computer-intensive discipline.

Biophysical Phenomena↗
PubMed2005

Analysis of leukocyte membrane protein interactions using protein microarrays.

BACKGROUND: Protein microarrays represent an emerging class of proteomic tools to investigate multiple protein-protein interactions in parallel. A sufficient proportion of immobilized proteins must maintain an active conformation and an orientation that allows for the sensitive and specific detection of antibody and ligand binding. In order to establish protein array technology for the characterization of the weak interactions between leukocyte membrane proteins, we selected the human leukocyte membrane protein CD200 (OX2) and its cell surface receptor (hCD200R) as a model system. As antibody-antigen reactions are generally of higher affinity than receptor-ligand binding, we first analyzed the reactivity of monoclonal antibodies (mAb) to normal and mutant forms of immobilized CD200R. RESULTS: Fluorescently labelled mAb DX147, DX136 and OX108 were specifically reactive with immobilized recombinant hCD200R extracellular region, over a range of 0.1-40 microg ml(-1) corresponding to a limit of sensitivity of 0.01-0.05 femtomol per spot. Orientating hCD200R using capture antibodies, showed that DX147 reacts with an epitope spatially distinct from the more closely related DX136 and OX108 epitopes. A panel of soluble recombinant proteins with mutations in hCD200R domain 1 produced by transiently transfected cells, was arrayed directly without purification and screened for binding to the three mAb. Several showed decreased binding to the blocking mAb DX136 and OX108, suggesting close proximity of these epitopes to the CD200 binding site. Binding of hCD200 to directly immobilized rat, mouse, and hCD200R was achieved with multimeric ligands, in the form of biotinylated-hCD200 coupled to FITC-labelled avidin coated beads. CONCLUSION: We have achieved sensitive, specific and reproducible detection of immobilized CD200R with different antibodies and mapped antigenic epitopes for two mAb in the vicinity of the ligand binding site using protein microarrays. We also detected CD200 binding to its receptor, a low affinity interaction, using beads presenting multivalent ligands. Our results demonstrate the quantitative aspects of protein arrays and their potential use in detecting simultaneously multiple protein-protein interactions and in particular the weak interactions found between leukocyte membrane proteins.

Animals↗
PubMed2006

The molecular scanner in microscope mode.

The combination of microscope mode matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) with protein identification methodology: the molecular scanner, was explored. The molecular scanner approach provides improvement of sensitivity of detection and identification of high-mass proteins in microscope mode IMS. The methodology was tested on protein distributions obtained after separation by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). High-quality, high-spatial-resolution ion images were recorded on a TRIFT-II ion microscope after gold coating of the MALDI sample preparation on the poly(vinylidenedifluoride) capture membranes. The sensitivity of the combined method is estimated to be 5 pmol. The minimum amount of sample consumed, needed for identification, was estimated to be better than 100 fmol. Software tools were developed to analyze the spectral data and to generate broad mass range and single molecular component microscope mode ion images and single mass-to-charge ratio microprobe mode images.

Animals↗
PubMed2000

Molecular characterization of a bacteriophage (Chp2) from Chlamydia psittaci.

Comparisons of the proteome of abortifacient Chlamydia psittaci isolates from sheep by two-dimensional gel electrophoresis identified a novel abundant protein with a molecular mass of 61.4 kDa and an isoelectric point of 6.41. C-terminal sequence analysis of this protein yielded a short peptide sequence that had an identical match to the viral coat protein (VP1) of the avian chlamydiaphage Chp1. Electron microscope studies revealed the presence of a 25-nm-diameter bacteriophage (Chp2) with no apparent spike structures. Thin sections of chlamydia-infected cells showed that Chp2 particles were located to membranous structures surrounding reticulate bodies (RBs), suggesting that Chp2 is cytopathic for ovine C. psittaci RBs. Chp2 double-stranded circular replicative-form DNA was purified and used as a template for DNA sequence analysis. The Chp2 genome is 4,567 bp and encodes up to eight open reading frames (ORFs); it is similar in overall organization to the Chp1 genome. Seven of the ORFs (1 to 5, 7, and 8) have sequence homologies with Chp1. However, ORF 6 has a different spatial location and no cognate partner within the Chp1 genome. Chlamydiaphages have three viral structural proteins, VP1, VP2, and VP3, encoded by ORFs 1 to 3, respectively. Amino acid residues in the phiX174 procapsid known to mediate interactions between the viral coat protein and internal scaffolding proteins are conserved in the Chp2 VP1 and VP3 proteins. We suggest that VP3 performs a scaffolding-like function but has evolved into a structural protein.

Abortion, Veterinary↗