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PubMed2006

Differentiation of HELLP patients from healthy pregnant women by proteome analysis--on the way towards a clinical marker set.

As the pathogenesis of the HELLP-syndrome is unknown, a complex consideration regarding the changes in the plasma as the main transport medium in the body is of great benefit because it is well available and can rapidly be investigated in the clinics. Besides that, the liver which is early affected in HELLP-syndrome produces the main part of plasma proteins. For the purpose of our study plasma protein abundances from patients with HELLP-syndrome and from control individuals were determined before and after delivery. In the differential analysis using two-dimensional gel electrophoresis, six areas with variable protein spot intensities were detected. The reference gel that we developed for HELLP plasma samples integrates the changes of plasma proteins when comparing HELLP patients to healthy women prior to and after delivery. A specific plasma protein profile for the HELLP-syndrome was generated involving protein areas that contain inter-alpha-trypsin inhibitor heavy chain H4, kininogen 1, fibrinogen gamma chain, transthyretin, haptoglobins, and serum amyloid A with statistically significant expression differences when compared to controls. The most striking difference between the majority of the gels from HELLP patients and the gels from non-HELLP samples were clearly overexpressed protein spots at about 11 kDa which were identified as serum amyloid A (SAA). This differential expression was validated and quantitatively assayed by ELISA measurements against human SAA in plasma. Our results show that significant differences in SAA expressions between healthy controls and HELLP patients were obtained, that could function as markers for the HELLP-syndrome. According to our data it is possible to draw a line of separation with no overlap between the HELLP group for which SAA plasma levels were found to be above 3.51 mg/L and the non-HELLP groups in which SAA plasma levels were below 3.51 mg/L. It now is possible to clinically elucidate if the differentially expressed proteins are suited for longitudinal studies concerning both, to function as markers or perhaps even as disease predictors that might become relevant for diagnostic tests.

Adult↗
PubMed2003

Trace element biology: the knowledge base and its application for the nutrition of individuals and populations.

Impressive strides are being made in the understanding of trace element metabolism and function. This is underscored by the many contributions in these proceedings. However, not so impressive are: i) the precise recognition of mild trace element deficiencies and how to establish their functional consequences, possibly confounded by concurrent trace element inadequacies, are difficult to assess, ii) approaches to the quantitative determination of requirements for trace elements remain unsatisfactory and archaic, in so many ways, iii) our understanding of the extent of the biological basis for the variation in requirements among apparently similar individuals is poor, and iv) much needs to be learned about the quantitative extent to which genetic, epigenetic and dietary factors interact to determine the nutritional phenotype. Some ideas are presented as to how we might embrace, in the context of a reconstructive approach, the exciting new knowledge and related techniques emerging during the postgenome era and develop new paradigms for assessing trace element needs and status, and for establishing effective nutrient intake under different conditions of complex genotype-environment interactions. Metabolites are functional cellular entities and I also urge a vigorous application of metabolomics and of metabolic profiling that is closely linked with genomics, proteomics, trace element kinetics and system analysis, as components of the new integrative paradigm. We need to understand the system and its strategy, not only the molecular details of its component parts and its individual controls. An interdisciplinary research and teaching enterprise will be necessary to best achieve this aim. All of this is related to our common goal to promote, through expanded biological knowledge and its effective application, the enhanced role of trace elements for human well-being.

Animals↗
PubMed2004

Biomarkers of congenital obstructive nephropathy: past, present and future.

PURPOSE: Congenital obstructive nephropathy constitutes one of the major causes of renal insufficiency in infants and children. This review addresses the need to define biomarkers that serve as surrogate end points for measuring the severity of obstruction, the evolution of renal maldevelopment and injury, and the response to medical or surgical intervention. MATERIALS AND METHODS: The literature from the last 10 years was reviewed for biomarkers of congenital obstructive nephropathy. Sources of biomarkers included urine, blood, amniotic fluid, tissue and imaging techniques. RESULTS: Previous markers of congenital obstructive nephropathy include sonographic renal pelvic diameter, quantitative diuretic renography, and markers of glomerular and tubular function. Attempts to correlate renal histological changes with differential renal function have been disappointing. Immunohistochemical analysis and laser capture microscopy should improve specificity. Most promising is the application of new insights into the cellular response of the developing kidney to urinary tract obstruction. These findings include components of the renin-angiotensin system, transforming growth factor-beta 1, monocyte chemoattractant protein-1 and epidermal growth factor. Microarray studies show unique patterns of gene expression by the neonatal rat kidney subjected to ureteral obstruction, and proteomics should provide even more sensitive biomarkers of obstructive nephropathy. CONCLUSIONS: We must define the cellular and molecular bases of renal maldevelopment, focusing on the link between functional and developmental pathophysiology. These findings will lead to biomarkers that will optimize our management of congenital obstructive nephropathy.

Animals↗
PubMed2002

Two-dimensional gel electrophoresis: recent advances in sample preparation, detection and quantitation.

A strength of two-dimensional polyacrylamide gel electrophoresis (2D PAGE) is its ability to resolve and investigate the abundance of several thousand proteins in a single sample. This enables identification of the major proteins in a tissue or subcellular fraction by mass spectrometric methods. In addition, 2D PAGE can be used to compare quantities of proteins in related samples, such as those from altered environments or from mutant and wild type, thus allowing the response of classes of proteins to be determined. Those proteins showing a correlated difference in expression may participate in related processes, and this subsequently helps to define protein function. Although there are many limitations of the 2D gel technology that mean it will never be comprehensive in protein coverage, its use for the identification of relatively abundant proteins is now widespread. However, there are still surprisingly few examples of quantitative analysis of changes in protein abundance. In this review we highlight recent advances towards true quantitative analysis of 2D gels that will lead to better prediction of protein function. Despite the development of promising alternatives, 2D PAGE is likely to remain in extensive use for the foreseeable future, because the technology is now simple and readily available to many laboratories.

Animals↗
PubMed2003

Biomarkers, validation and pharmacokinetic-pharmacodynamic modelling.

Four elements are crucial to successful pharmacokinetic-pharmacodynamic (PK/PD) modelling and simulation for efficient and effective rational drug development: (i) mechanism-based biomarker selection and correlation to clinical endpoints; (ii) quantification of drug and/or metabolites in biological fluids under good laboratory practices (GLP); (iii) GLP-like biomarker method validation and measurements and; (iv) mechanism-based PK/PD modelling and validation. Biomarkers can provide great predictive value in early drug development if they reflect the mechanism of action for the intervention even if they do not become surrogate endpoints. PK/PD modelling and simulation can play a critical role in this process. Data from genomic and proteomics differentiating healthy versus disease states lead to biomarker discovery and identification. Multiple genes control complex diseases via hosts of gene products in biometabolic pathways and cell/organ signal transduction. Pilot exploratory studies should be conducted to identify pivotal biomarkers to be used for predictive clinical assessment of disease progression and the effect of drug intervention. Most biomarkers are endogenous macromolecules, which could be measured in biological fluids. Many exist in heterogeneous forms with varying activity and immunoreactivity, posting challenges for bioanalysis. Reliable and selective assays could be validated under a GLP-like environment for quantitative methods. While the need for consistent reference standards and quality control monitoring during sample analysis for biomarker assays are similar to that of drug molecules, many biomarkers have special requirements for sample collection that demand a well coordinated team management. Bioanalytical methods should be validated to meet study objectives at various drug development stages, and possess adequate performance to quantify biochemical responses specific to the target disease progression and drug intervention. Protocol design to produce sufficient data for PK/PD modelling would be more complex than that of PK. Knowledge of mechanism from discovery and preclinical studies are helpful for planning clinical study designs in cascade, sequential, crossover or replicate mode. The appropriate combination of biomarker identification and selection, bioanalytical methods development and validation for drugs and biomarkers, and mechanism-based PK/PD models for fitting data and predicting future clinical endpoints/outcomes provide powerful insights and guidance for effective and efficient rational drug development, toward safe and efficacious medicine for individual patients.

Biomarkers↗
PubMed2005

The complement of enzymatic sets in different species.

We present here a comprehensive analysis of the complement of enzymes in a large variety of species. As enzymes are a relatively conserved group there are several classification systems available that are common to all species and link a protein sequence to an enzymatic function. Enzymes are therefore an ideal functional group to study the relationship between sequence expansion, functional divergence and phenotypic changes. By using information retrieved from the well annotated SWISS-PROT database together with sequence information from a variety of fully sequenced genomes and information from the EC functional scheme we have aimed here to estimate the fraction of enzymes in genomes, to determine the extent of their functional redundancy in different domains of life and to identify functional innovations and lineage specific expansions in the metazoa lineage. We found that prokaryote and eukaryote species differ both in the fraction of enzymes in their genomes and in the pattern of expansion of their enzymatic sets. We observe an increase in functional redundancy accompanying an increase in species complexity. A quantitative assessment was performed in order to determine the degree of functional redundancy in different species. Finally, we report a massive expansion in the number of mammalian enzymes involved in signalling and degradation.

Animals↗
PubMed2002

Laser capture microdissection and two-dimensional polyacrylamide gel electrophoresis: evaluation of tissue preparation and sample limitations.

Laser capture microdissection (LCM) is now well established as a tool for facilitating the enrichment of cells of interest from tissue sections, overcoming the problem of tissue heterogeneity. LCM has been used extensively in combination with analysis at the DNA and RNA levels, but only a small number of studies have employed LCM with subsequent protein analysis, albeit with promising results. This study focuses on the potential of LCM in combination with two-dimensional polyacrylamide gel electrophoresis. The effects of tissue section preparation and sample type were evaluated to fully determine the suitability of using LCM in global protein profiling. The effects of several histochemical stains (hematoxylin and eosin, methyl green and toluidine blue) and immunolabeling on subsequent two-dimensional polyacrylamide gel electrophoresis were investigated. Quantitative analysis was performed to establish the extent of changes in the relative intensity of protein species and their reproducibility. All staining protocols tested were found to be compatible with protein analysis although there was variation in protein recovery and the quality of the protein profiles obtained. LCM of renal and cervix samples indicated that protein yield after dissection was acceptable, although the extent of enrichment and dissection time was tissue-dependent, which may preclude the use of this approach with some tissue types. These results indicate that LCM has potential as a tool in proteomic research.

Electrophoresis, Gel, Two-Dimensional↗
PubMedDate not supplied

Properties of 13C-substituted arginine in stable isotope labeling by amino acids in cell culture (SILAC).

We have recently described a method, stable isotope labeling by amino acids in cell culture (SILAC) for the accurate quantitation of relative protein abundances. Cells were metabolically labeled with deuterated leucine, leading to complete incorporation within about five cell doublings. Here, we investigate fully substituted 13C-labeled arginine in the SILAC method. After tryptic digestion, there is a single label at the C-terminal position in half of the peptides. Labeled and unlabeled peptides coelute in liquid chromatography-mass spectrometric analysis, eliminating quantitation error due to unequal sampling of ion profiles. Tandem mass spectrum interpretation and database identification are aided by the predictable shift of the y-ions in the labeled form. The quantitation of mixtures of total cell lysates in known ratios resolved on a one-dimensional SDS-PAGE gel produced consistent and reproducible results with relative standard deviations better than five percent under optimal conditions.

Amino Acids↗
PubMed2002

Metabolic control analysis in drug discovery and disease.

Metabolic control analysis (MCA) provides a quantitative description of substrate flux in response to changes in system parameters of complex enzyme systems. Medical applications of the approach include the following: understanding the threshold effect in the manifestation of metabolic diseases; investigating the gene dose effect of aneuploidy in inducing phenotypic transformation in cancer; correlating the contributions of individual genes and phenotypic characteristics in metabolic disease (e.g., diabetes); identifying candidate enzymes in pathways suitable as targets for cancer therapy; and elucidating the function of "silent" genes by identifying metabolic features shared with genes of known pathways. MCA complements current studies of genomics and proteomics, providing a link between biochemistry and functional genomics that relates the expression of genes and gene products to cellular biochemical and physiological events. Thus, it is an important tool for the study of genotype-phenotype correlations. It allows genes to be ranked according to their importance in controlling and regulating cellular metabolic networks. We can expect that MCA will have an increasing impact on the choice of targets for intervention in drug discovery.

Aneuploidy↗
PubMed2006

Overexpression of stathmin in oral squamous-cell carcinoma: correlation with tumour progression and poor prognosis.

Stathmin is an intracellular phosphoprotein that is overexpressed in a number of human malignancies. Our previous study using proteomic profiling showed that significant upregulation of stathmin occurs in oral squamous-cell carcinoma (OSCC)-derived cell lines. In the current study, to determine the potential involvement of stathmin in OSCC, we evaluated the state of stathmin protein and mRNA expression in OSCC-derived cell lines and human primary OSCCs. A significant increase in stathmin expression was observed in all OSCC-derived cell lines examined compared to human normal oral keratinocytes. In immunohistochemistry, 65% of the OSCCs were positive for stathmin, and no immunoreaction was observed in corresponding normal tissues. Real-time quantitative reverse transcriptase-polymerase chain reaction data were consistent with the protein expression status. Moreover, stathmin expression status was correlated with the TNM stage grading. Furthermore, we found a statistical correlation between the protein expression status and disease-free survival (P=0.029). These results suggest that expression of stathmin could contribute to cancer progression/prognosis, and that stathmin may have potential as a biomarker and a therapeutic target for OSCC.

Aged↗
PubMed2004

Identification of membrane proteins differentially expressed in human papillomavirus type 16 E5-transfected human keratinocytes by nanoelectrospray ionization mass spectrometry.

Membrane proteins differentially expressed in human papillomavirus type 16 (HPV-16) E5-transfected HaCaT cells have been identified. Membrane proteins were isolated and separated by two-dimensional gel electrophoresis. Spots showing quantitative differences between E5-transfected and control cells were extracted and the proteins were identified by nanoelectrospray ionization mass spectrometry. A total of 24 spots was analysed. Among the proteins showing differential expression, a decreased amount of calnexin and increased expression of hsp70, proteins both involved in maturation and transport of MHC class I complexes to the plasma membrane, were noticed. These findings correlate with the decreased surface expression of MHC class I molecules described in E5-expressing cells, HPV-positive cervical lesions and cervical carcinomas. These results stress the value of the proteomic approach, as used here in the experimental design, which allows the correlation of changes in host gene expression with biological functions of viral genes.

Histocompatibility Antigens↗
PubMed2006

Impairment of mitochondrial anti-oxidant defence in SOD1-related motor neuron injury and amelioration by ebselen.

There is now compelling evidence of mitochondrial dysfunction in motor neuron disease (MND), but the molecular basis of these abnormalities is unknown. It is also unclear whether the observed mitochondrial dysfunction plays a central role in disease pathogenesis, and if so, whether its amelioration might present therapeutic opportunities. We adopted a candidate generation approach using proteomics to screen for changes in mitochondrial protein expression in a well-validated cell-culture model of superoxide dismutase 1 (SOD1) related familial MND (fMND). Changed proteins were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy. Protein candidates included apoptotic regulators, anti-oxidants and components of the electron transport chain. Confirmatory Western blotting was performed, and validated protein expression changes were further investigated. Peroxiredoxin 3 (Prx3), a mitochondrial thioredoxin-dependent hydroperoxidase, is downregulated in the presence of mutant SOD1 in both our cell-culture model and in the spinal cord mitochondria of mutant SOD1 transgenic mice. We confirm the expression of Prx3 within the mitochondria of spinal motor neurons in mouse and humans by immunohistochemistry. Using quantitative real-time PCR (Q-PCR), we show that Prx3 is also downregulated in spinal motor neurons from patients with both sporadic (sMND) and SOD1-related fMND. In a disease characterized by oxidative stress, this represents a potentially important deficit in mitochondrial anti-oxidant defence. Recent evidence suggests that oxidative stress from aberrant copper chemistry may not play a major part in the pathogenesis of SOD1-related fMND. From the results of this study we propose disruption of mitochondrial anti-oxidant defence as an alternative mechanism whereby mutant SOD1 may generate oxidative stress within motor neurons. We further demonstrate that ebselen, an anti-oxidant drug already safely used in human studies and that acts as a Prx mimic, is able to ameliorate the toxicity of mutant SOD1 in our cell-culture model. We conclude by showing that ebselen is capable of inducing transcription of the anti-oxidant response element (ARE) and postulate that ebselen may act both by the transcriptional upregulation of anti-oxidant proteins, and directly as an anti-oxidant in its own right.

Animals↗
PubMed2002

Positive and negative feedback: striking a balance between necessary antagonists.

Most biological regulation systems comprise feedback circuits as crucial components. Negative feedback circuits have been well understood for a very long time; indeed, their understanding has been the basis for the engineering of cybernetic machines exhibiting stable behaviour. The importance of positive feedback circuits, considered as "vicious circles", has however been underestimated. In this article, we give a demonstration based on degree theory for vector fields of the conjecture, made by René Thomas, that the presence of positive feedback circuits is a necessary condition for autonomous differential systems, covering a wide class of biologically relevant systems, to possess multiple steady states. We also show ways to derive constraints on the weights of positive and negative feedback circuits. These qualitative and quantitative results provide, respectively, structural constraints (i.e. related to the interaction graph) and numerical constraints (i.e. related to the magnitudes of the interactions) on systems exhibiting complex behaviours, and should make it easier to reverse-engineer the interaction networks animating those systems on the basis of partial, sometimes unreliable, experimental data. We illustrate these concepts on a model multistable switch, in the context of cellular differentiation, showing a requirement for sufficient cooperativity. Further developments are expected in the discovery and modelling of regulatory networks in general, and in the interpretation of bio-array hybridization and proteomics experiments in particular.

Animals↗
PubMed2002

High-throughput global peptide proteomic analysis by combining stable isotope amino acid labeling and data-dependent multiplexed-MS/MS.

In this work, we describe the application of a stable isotope amino acid (lysine) labeling in conjunction with data-dependent multiplexed tandem mass spectrometry (MS/MS) to facilitate the characterization and identification of peptides from proteomic (global protein) digests. Lysine auxotrophic yeast was grown in the presence of 13C-labeled or unlabeled lysine and combined after harvesting in equal proportions. Endoproteinase LysC digestion of the cytosolic fraction produced a global proteomic sample, consisting of heavy/light labeled peptide pairs. Then data-dependent multiplexed-MS/MS was applied to simultaneously select and dissociate only labeled peptide ion pairs. The approach allows differentiation between N-terminal (e.g., b-type ions) and C-terminal fragment ions (e.g., y-type ions) in resulting tandem mass spectra, as well as the capability of differentiation between near-isobaric glutamine and lysine residues. We also describe the utility of peptide composition and fragment information to support peptide identifications and examine the potential application of lysine labeling for differential quantitative protein analysis.

Amino Acids↗
PubMed2006

A matrix-assisted laser desorption/ionization compatible reagent for tagging tryptophan residues.

Chemical tagging of amino acids is an important tool in proteomics analysis, and has been used to introduce isotope labels and mass defect labels into proteolytic peptides by derivatization of cysteine or lysine residues. Here, we present a new reagent with chemical specificity for tryptophan residues. Previously, 2-nitrobenzenesulfenyl chloride has been used as a highly specific reagent for labeling tryptophan residues. We show that this tag undergoes UV dissociation during matrix assisted laser desorption/ionization (MALDI). The multiplicity of photofragments increases the difficulty of characterizing the derivatization products. To overcome this problem, we have synthesized a new reagent, 2-(trifluoromethyl)benzenesulfenyl chloride, which is shown to react quantitatively with tryptophan in peptides and proteins. Most significantly, it exhibits high photostability in MALDI-Fourier transform mass spectrometry analyses.

Bombesin↗
PubMed2005

Improved diagnostic approaches to infection/sepsis detection.

Sepsis is a major healthcare problem from the perspective of mortality and economics. Advances in diagnostic detection of infection and sepsis have been slow, but recent advances in both soluble biomarker detection and quantitative cellular measurements promise the availability of improved diagnostic techniques. Though the promise of cytokine measurements reaching clinical practice have not matured, procalcitonin levels are currently available in many countries and appear to offer enhanced diagnostic distinction between bacterial and viral etiologies. Cellular diagnostics is poised to enter clinical laboratory practice in the form of neutrophil CD64 measurements, which offer superior sensitivity and specificity to conventional laboratory assessment of sepsis. Neutrophil CD64 expression is negligible in the healthy state. However, it increases as part of the systemic response to severe infection or sepsis. The combination of cellular proteomics, as in the case of neutrophil CD64 quantification, and selected soluble biomarkers of the inflammatory response, such as procalcitonin or triggering receptor expressed on myeloid cells (TREM)-1, is predicted to remove the current subjectivity and uncertainty in the diagnosis and therapeutic monitoring of infection and sepsis.

Biomarkers↗
PubMed2003

From the top down: towards a predictive biology of signalling networks.

True quantitative and predictive biology requires the ability to interpret increasingly complex datasets in new ways to reveal underlying functional interactions. This has come a step closer with two recent articles that describe a 'top-down' modelling approach to reconstructing functional networks from microarray data.

Computer Simulation↗
PubMed2005

Incorporation of selenomethionine into induced intracytoplasmic membrane proteins of Rhodobacter species.

Efficient multiple- or single-wavelength anomalous dispersion (MAD/SAD) techniques that use tunable X-ray sources at third-generation synchrotrons exploit the anomalous scattering of certain heavy atoms for determination of experimental phases. Development of methods for the in vivo substitution of methionine by selenomethionine (SeMet) has revolutionized the process for determination of structures of soluble proteins in recent years. Herein, we report methods for biosynthetic incorporation of SeMet into induced intracytoplasmic membrane proteins of two species of the Rhodobacter genus of purple non-sulfur photosynthetic bacteria. Amino acid analysis of a membrane protein complex that was purified to homogeneity determined that the extent of SeMet incorporation was extensive and approached quantitative replacement. Diffraction-quality crystals were obtained from SeMet-labeled membrane proteins purified from 2 l of culture. These methods augment the potential utility of photosynthetic bacteria and their inducible membrane systems for the production of foreign membrane proteins for structure determination.

Cell Membrane↗