Biomedical subjects

Emilio Camafeita

Publications and source records attributed to Emilio Camafeita.

11 recordsLinked to original sources

PubMed2006

Proteomic analysis of phytopathogenic fungus Botrytis cinerea as a potential tool for identifying pathogenicity factors, therapeutic targets and for basic research.

Botrytis cinerea is a phytopathogenic fungus causing disease in a substantial number of economically important crops. In an attempt to identify putative fungal virulence factors, the two-dimensional gel electrophoresis (2-DE) protein profile from two B. cinerea strains differing in virulence and toxin production were compared. Protein extracts from fungal mycelium obtained by tissue homogenization were analyzed. The mycelial 2-DE protein profile revealed the existence of qualitative and quantitative differences between the analyzed strains. The lack of genomic data from B. cinerea required the use of peptide fragmentation data from MALDI-TOF/TOF and ESI ion trap for protein identification, resulting in the identification of 27 protein spots. A significant number of spots were identified as malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The different expression patterns revealed by some of the identified proteins could be ascribed to differences in virulence between strains. Our results indicate that proteomic analysis are becoming an important tool to be used as a starting point for identifying new pathogenicity factors, therapeutic targets and for basic research on this plant pathogen in the postgenomic era.

Botrytis↗

PubMed2006

Mass spectrometry technologies for proteomics.

In the late 1980s, the advent of soft ionization techniques capable of generating stable gas phase ions from thermally unstable biomolecules, namely matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), laid the way for the development of a set of powerful alternatives to the traditional Edman chemistry for the structural characterization of peptides and proteins. The rapid protein identification capabilities that, coupled with two-dimensional gel electrophoresis, provided insights into all sorts of biological systems since the dawn of proteomics and have been exploited in the last few years for the development of more powerful and automatable gel-free strategies, mainly based on multidimensional chromatographic separations of peptides from proteolytic digests. In parallel to the evolution of ion sources, mass analysers and scan modes, the invention of new elegant biochemical strategies to fractionate or simplify highly complex mixtures, or to introduce isotopic labels in peptides in a variety of ways now makes also possible large-scale, high-coverage quantitative studies in a wide dynamic range. In this review, we provide the fundamental concepts of mass spectrometry (MS) and describe the technological progress of MS-based proteomics since its earliest days. Representative literature examples of their true power, either when employed as exploratory or as targeted techniques, is provided as well.

Mass Spectrometry↗

PubMed2006

A proteomic approach to study pea (Pisum sativum) responses to powdery mildew (Erysiphe pisi).

As a global approach to gain a better understanding of the mechanisms involved in pea resistance to Erysiphe pisi, changes in the leaf proteome of two pea genotypes differing in their resistance phenotype were analyzed by a combination of 2-DE and MALDI-TOF/TOF MS. Leaf proteins from control non-inoculated and inoculated susceptible (Messire) and resistant (JI2480) plants were resolved by 2-DE, with IEF in the 5-8 pH range and SDS-PAGE on 12% gels. CBB-stained gels revealed the existence of quantitative and qualitative differences between extracts from: (i) non-inoculated leaves of both genotypes (77 spots); (ii) inoculated and non-inoculated Messire leaves (19 spots); and (iii) inoculated and non-inoculated JI2480 leaves (12 spots). Some of the differential spots have been identified, after MALDI-TOF/TOF analysis and database searching, as proteins belonging to several functional categories, including photosynthesis and carbon metabolism, energy production, stress and defense, protein synthesis and degradation and signal transduction. Results are discussed in terms of constitutive and induced elements involved in pea resistance against Erysiphe pisi.

Ascomycota↗

PubMed2006

Proteomic and transcriptional characterization of aromatic degradation pathways in Rhodoccocus sp. strain TFB.

Rhodococcus sp. strain TFB is a versatile gram-positive bacterium able to grow on a wide variety of aromatic compounds as carbon and energy sources. Since the strain is refractory to genetic analysis, a proteomic approach was used to study the metabolic pathways involved in the catabolism of such compounds by analyzing differentially induced proteins. The most marked difference was observed when the proteome profiles of phthalate-grown cells were compared with those cultured in the presence of tetralin- or naphthalene, suggesting that different metabolic pathways are involved in the degradation of mono- and polyaromatic compounds. Comparison with the proteome of glucose-grown cells indicated that each pathway was specifically induced by the corresponding aromatic compound. A combination of proteomics and molecular biology led to the identification of 14 proteins (65-80% identical to known Pht proteins) that describe a complete pathway for the catabolism of phthalate to central metabolites via intradiol cleavage of protochatechuic acid. Chaperonins were also induced in phthalate-grown cells, indicating that growth on this compound induces a stress response. Absence of catabolite repression by glucose was observed by both transcriptional and proteome analysis, suggesting that Rhodococcus sp. strain TFB may have advantages over other tightly regulated strains in bioremediation.

Amino Acid Sequence↗

PubMed2006

Two-dimensional electrophoresis protein profile of the phytopathogenic fungus Botrytis cinerea.

Botrytis cinerea is a phytopathogenic fungi causing disease in a number of important crops. It is considered a very complex species in which different populations seem to be adapted to different hosts. In order to characterize fungal virulence factors, a proteomic research was started. A protocol for protein extraction from mycelium tissue, with protein separation by 2-DE and MS analysis, was optimised as a first approach to defining the B. cinerea proteome. Around 400 spots were detected in 2-DE CBB-stained gels, covering the 5.4-7.7 pH and 14-85 kDa ranges. The averages of analytical and biological coefficients of variance for 64 independent spots were 16.1% and 37.5%, respectively. Twenty-two protein spots were identified by MALDI-TOF or ESI IT MS/MS, with some of them corresponding to forms of malate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. Two more spots matched a cyclophilin and a protein with an unknown function.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2006

Modifications in the human T cell proteome induced by intracellular HIV-1 Tat protein expression.

The effects of the human immunodeficiency virus type 1 (HIV-1) Tat protein on cellular gene expression were analysed using a Jurkat cell line that was stably transfected with tat gene in a doxycycline-repressible expression system. Expressed Tat protein (aa 1-101) was proved to present basically a nuclear localisation, and to be fully functional to induce HIV LTR transactivation. Tat expression also resulted in protection from Tunicamycin-induced apoptosis as determined by DNA staining and TUNEL assays. We applied proteomics methods to investigate changes in differential protein expression in the transfected Jurkat-Tat cells. Protein identification was performed using 2-D DIGE followed by MS analysis. We identified the down-regulation of several cytoskeletal proteins such as actin, beta-tubulin, annexin II, as well as gelsolin, cofilin and the Rac/Rho-GDI complex. Down-expression of these proteins could be involved in the survival of long-term reservoirs of HIV-infected CD4+ T cells responsible for continuous viral production. In conclusion, in addition to its role in viral mRNA elongation, the proteomic approach has provided insight into the way that Tat modifies host cell gene expression.

HIV-1↗

PubMed2006

Structural analysis of the human respiratory syncytial virus phosphoprotein: characterization of an alpha-helical domain involved in oligomerization.

Human respiratory syncytial virus (HRSV) phosphoprotein (P), an essential cofactor of the viral polymerase, is much shorter (241 aa) than and has no sequence similarity to P of other paramyxoviruses. Nevertheless, bioinformatic analysis of HRSV P sequence revealed a modular organization, reminiscent of other paramyxovirus Ps, with a central structured domain (aa 100-200), flanked by two intrinsically disordered regions (1-99 and 201-241). To test the predicted structure experimentally, HRSV P was purified from cell extracts infected with recombinant vaccinia virus or HRSV. The estimated molecular mass of P by gel filtration (approximately 500 kDa) greatly exceeded the theoretical mass of a homotetramer, proposed as the oligomeric form of native P. Nevertheless, the profile of cross-linked products obtained with purified P resembled that reported by others with P purified from bacteria or mammalian cells. Thus, the shape of HRSV P probably influences its elution from the gel filtration column, as reported for other paramyxovirus Ps. Digestion of purified HRSV P with different proteases identified a trypsin-resistant fragment (X) that reacted with a previously characterized monoclonal antibody (021/2P). N-terminal sequencing and mass spectrometry analysis placed the X fragment boundaries (Glu-104 and Arg-163) within the predicted structured domain of P. Cross-linking and circular dichroism analyses indicated that fragment X was oligomeric, with a high alpha-helical content, properties resembling those of the multimerization domain of Sendai and rinderpest virus P. These results denote structural features shared by HRSV and other paramyxovirus Ps and should assist in elucidation of the HRSV P structure.

Cell Line↗

PubMed2005

Overexpression of the multidrug efflux pumps MexCD-OprJ and MexEF-OprN is associated with a reduction of type III secretion in Pseudomonas aeruginosa.

The Pseudomonas aeruginosa genome contains several different multidrug resistance (MDR) efflux pumps. Overproduction of these pumps reduces susceptibility to a variety of antibiotics. Some recently published works have analyzed the effect of the overproduction of MDR efflux pumps on bacterial virulence. Here we have studied the effect of overproduction of the efflux pumps MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY on type III secretion (T3S) in P. aeruginosa. The type III secretion system (T3SS) is used by P. aeruginosa to deliver toxins directly into the cytoplasm of the host cell. Our data indicate that overexpression of either MexCD-OprJ or MexEF-OprN is associated with the impairment of T3S in P. aeruginosa. No effect on overexpression of either MexAB-OprM or MexXY was detected. The observed defect in T3S was due to a lack of expression of genes belonging to the T3SS regulon. Transcription of this regulon is activated by ExsA in response to environmental signals. Overexpression of this transcriptional regulator complemented the defect in T3S observed in the MexCD-OprJ- and MexEF-OprN-overproducing strains. Taken together, these results suggest that overproduction of either MexCD-OprJ or MexEF-OprN is associated with a reduction in the transcription of the T3SS regulon due to the lack of expression of the exsA gene, encoding the master regulator of the system. The relevance of potential metabolic and quorum-sensing imbalances due to overexpression of MDR pumps associated with this phenotype is also discussed.

Bacterial Outer Membrane Proteins↗

PubMed2003

Antibodies reactive to heat shock protein 90 induce oligodendrocyte precursor cell death in culture. Implications for demyelination in multiple sclerosis.

Oligodendrocyte precursor cells (OPCs) are extremely efficient at remyelination. These cells persist in the adult human central nervous system and can proliferate. However, the failure to remyelinate is a pathological characteristic of the human demyelinating disease multiple sclerosis (MS), which suggests that these cells are ineffective in this disorder. This paper reports that IgG antibodies in the cerebrospinal fluid (CSF) of MS patients specifically recognize an antigen on OPCs in culture. Control patients were found not to possess these antibodies. The antigen was immunoprecipitated in cell extracts from cultures with purified IgG from MS CSF. Peptide mass fingerprinting identified it as the beta type of heat shock protein 90 (Hsp90). Two-dimensional electrophoresis and immunoblot showed that this antigen in fact corresponds to two specific isoforms of Hsp90beta. Several control assays using monoclonal and polyclonal anti-Hsp90 antibodies confirmed the specific expression of Hsp90 on OPCs. Labeling OPCs in vivo with MS CSF and anti-Hsp90 antibodies and subsequent immunofluorescence confocal microscopy located the antigen on the cell surface. The binding of CSF antibodies from MS patients to the OPC surface led to complement activation and significant extinction of the OPC population. These results suggest that OPCs may be a target of anti-Hsp90 antibodies in MS patients and that this could prevent remyelination.

Antibodies↗

PubMed2002

An anti-ICAM-2 (CD102) monoclonal antibody induces immune-mediated regressions of transplanted ICAM-2-negative colon carcinomas.

Monoclonal antibodies (mAbs) can mediate antitumor effects by indirect mechanisms involving antiangiogenesis and up-regulation of the cellular immune response rather than by direct tumor cell destruction. From mAbs raised by immunization of rats with transformed murine endothelial cells, a mAb (EOL4G8) was selected for its ability to eradicate a fraction of established colon carcinomas that did not express the EOL4G8-recognized antigen. The antigen was found to be ICAM-2 (CD102). Antitumor effects of EOL4G8, which required a functional T-cell compartment, were abrogated by depletion of CD8(+) cells and correlated with antitumor CTL activity, whereas only a mild inhibition of angiogenesis was observed. Interestingly, we found that EOL4G8 acting on endothelial ICAM-2 markedly enhances leukotactic factor activity-1-independent adhesion of immature dendritic cells to endothelium-an effect that is at least in part mediated by DC-SIGN (CD209).

Amino Acid Sequence↗

PubMed2001

The capsid protein of a plant single-stranded RNA virus is modified by O-linked N-acetylglucosamine.

Plum pox virus (PPV) is a member of the Potyvirus genus of plant viruses. Labeling with UDP-[3H]galactose and galactosyltransferase indicated that the capsid protein (CP) of PPV is a glycoprotein with N-acetylglucosamine terminal residues. Mass spectrometry analysis of different PPV isolates and mutants revealed O-linked N-acetylglucosamination, a modification barely studied in plant proteins, of serine and/or threonine residues near the amino end of PPV CP. CP of PPV virions is also modified by serine and threonine phosphorylation, as shown by Western blot analysis with anti-phosphoserine and anti-phosphothreonine antibodies. Thus, "yin-yang" glycosylation and phosphorylation may play an important role in the regulation of the different functions in which the potyviral CP is involved.

Acetylglucosamine↗