Biomedical subjects

Nelson Guerreiro

Publications and source records attributed to Nelson Guerreiro.

5 recordsLinked to original sources

PubMed2006

Optimization and evaluation of surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry for protein profiling of cerebrospinal fluid.

Cerebrospinal fluid (CSF) potentially carries an archive of peptides and small proteins relevant to pathological processes in the central nervous system (CNS) and surrounding brain tissue. Proteomics is especially well suited for the discovery of biomarkers of diagnostic potential in CSF for early diagnosis and discrimination of several neurodegenerative diseases. ProteinChip surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) is one such approach which offers a unique platform for high throughput profiling of peptides and small proteins in CSF. In this study, we evaluated methodologies for the retention of CSF proteins < 20 kDa in size, and identify a strategy for screening small proteins and peptides in CSF. ProteinChip array types, along with sample and binding buffer conditions, and matrices were investigated. By coupling the processing of arrays to a liquid handler reproducible and reliable profiles, with mean peak coefficients of variation < 20%, were achieved for intra- and inter-assays under selected conditions. Based on peak m/z we found a high degree of overlap between the tested array surfaces. The combination of CM10 and IMAC30 arrays was sufficient to represent between 80-90% of all assigned peaks when using either sinapinic acid or alpha-Cyano-4-hydroxycinnamic acid as the energy absorbing matrices. Moreover, arrays processed with SPA consistently showed better peak resolution and higher peak number across all surfaces within the measured mass range. We intend to use CM10 and IMAC30 arrays prepared in sinapinic acid as a fast and cost-effective approach to drive decisions on sample selection prior to more in-depth discovery of diagnostic biomarkers in CSF using alternative but complementary proteomic strategies.

Journal Article↗

PubMed2005

T cell receptor (TCR) interaction with haptens: metal ions as non-classical haptens.

Haptens are classified as low molecular chemicals with an intrinsic potential to covalently modify proteins, and many of them are strong inducers of contact hypersensitivity (CHS). CHS is T cell mediated, and hapten-specific T cells have been shown to interact with hapten-modified, MHC-associated peptides. However, the most common contact sensitizer in the industrialized world is nickel. In contrast to classical haptens, nickel ions do not form covalent bonds to proteins, but rather become caught in reversible coordination complexes. We here review work demonstrating that some T cells, indeed, may react to such Ni complexes on the MHC/peptide-surface absolutely comparable to other haptens. In other cases, Ni ions unlike classical haptens, may activate T cells by crosslinking their receptors to MHC molecules, independent of the nature of the associated peptide. Moreover, Ni-interacting proteins appear to make use of the reversibility of Ni-binding, and to mediate the transfer of Ni-ions to the receptor-MHC interphase. We have demonstrated such properties for human serum albumin (HSA) as well as for transferrin and identified numerous new Ni-binding proteins in human B-cell lines or dendritic cells by affinity purification and mass spectroscopy. These proteins include a notable number of known heat shock proteins and chaperones, implying that Ni may functionally interfere with these stress proteins.

Animals↗

PubMed2005

Subproteomic analysis of metal-interacting proteins in human B cells.

Metal-protein interactions are vitally important in all living organisms. Metalloproteins, including structural proteins and metabolic enzymes, participate in energy transfer and redox reactions or act as metallochaperones in metal trafficking. Among metal-associated diseases, T cell mediated allergy to nickel (Ni) represents the most common form of human contact hypersensitivity. With the aim to elucidate disease-underlying mechanisms such as Ni-specific T cell activation, we initiated a proteomic approach to identify Ni-interacting proteins in human B cells. As antigen presenting cells, B cells are capable of presenting MHC-associated Ni-epitopes to T cells, a prerequisite for hapten-specific T cell activation. Using metal-affinity enrichment, 2-DE and MS, 22 Ni-interacting proteins were identified. In addition to known Ni-binding molecules such as tubulin, actin or cullin-2, we unexpectedly discovered that at least nine of these 22 proteins belong to stress-inducible heat shock proteins or chaperonins. Enrichment was particularly effective for the hetero-oligomeric TRiC/CCT complex, which is involved in MHC class I processing. Blue Native/SDS electrophoresis analysis revealed that Ni-NTA-beads specifically retained the complete protein machinery, including the associated chaperonin substrate tubulin. The apparent Ni-affinity of heat shock proteins suggests a new function of these molecules in human Ni allergy, by linking innate and adaptive immune responses.

B-Lymphocytes↗

PubMed2004

Probing for pH-regulated proteins in Sinorhizobium medicae using proteomic analysis.

To elucidate the mechanisms of pH response in an acid-tolerant Sinorhizobium medicae strain we have identified acid-activated gene transcription and now complement this approach by using a proteomic analysis to identify the changes that occur following exposure to acidity. Protein profiles of persistently or transiently acid-stressed S. medicae cells were compared to those grown in pH neutral, buffered media. Fifty pH-regulated proteins were identified; N-terminal sequences for 15 of these were obtained using the Edman degradation. Transient acid exposure downregulated GlnA and GlnK and upregulated a hypothetical protein. Continuing acid exposure downregulated ClpP, an ABC transporter, a hypothetical protein, a lipoprotein, the Trp-like repressor WrbA1 and upregulated DegP, fructose bisphosphate aldolase, GroES, malate dehydrogenase and two hypothetical proteins. These findings implicate proteolytic, chaperone and transport processes as key components of pH response in S. medicae.

Amino Acid Sequence↗

PubMedDate not supplied

Toxicogenomics in drug development.

Toxicogenomics represents the merging of toxicology with technologies that have been developed, together with bioinformatics, to identify and quantify global gene expression changes. It represents a new paradigm in drug development and risk assessment, which promises to generate a wealth of information towards an increased understanding of the molecular mechanisms that lead to drug toxicity and efficacy, and of DNA polymorphisms responsible for individual susceptibility to toxicity. Gene expression profiling, through the use of DNA microarray and proteomic technologies will aid in establishing links between expression profiles, mode of action and traditional toxic endpoints. Such patterns of gene expression, or 'molecular fingerprints' could be used as diagnostic or predictive markers of exposure, that is characteristic of a specific mechanism of induction of that toxic or efficacious effect. It is anticipated that toxicogenomics will be increasingly integrated into all phases of the drug development process particularly in mechanistic and predictive toxicology, and biomarker discovery. This review provides an overview of the expression profiling technologies applied in toxicogenomics. and discusses the promises as well as the future challenges of applying this discipline to the drug development process.

Animals↗