Fabio C. Gozzo

Single embryo and oocyte lipid fingerprinting by mass spectrometry

Methods used for lipid analysis in embryos and oocytes usually involve selective lipid extraction from a pool of many samples followed by chemical manipulation, separation and characterization of individual components by chromatographic techniques. Herein we report direct analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of single and intact embryos or oocytes from various species. Biological samples were simply moisturized with the matrix solution and characteristic lipid (represented by phosphatidylcholines, sphingomyelins and triacylglycerols) profiles were obtained via MALDI-MS. As representative examples, human, bovine, sheep and fish oocytes, as well as bovine and insect embryos were analyzed. MALDI-MS is shown to be capable of providing characteristic lipid profiles of gametes and embryos and also to respond to modifications due to developmental stages and in vitro culture conditions of bovine embryos. Investigation in developmental biology of the biological roles of structural and reserve lipids in embryos and oocytes should therefore benefit from these rapid MALDI-MS profiles from single and intact species.

The Biginelli Reaction with an Imidazolium–Tagged Recyclable Iron Catalyst: Kinetics, Mechanism, and Antitumoral Activity

The present work describes the synthesis, characterization, and application of a new ion-tagged iron catalyst. The catalyst was employed in the Biginelli reaction with impressive performance. High yields have been achieved when the reaction was carried out in imidazolium-based ionic liquids (BMI⋅PF6, BMI⋅NTf2, and BMI⋅BF4), thus showing that the ionic-liquid effects play a role in the reaction. Moreover, the ion-tagged catalyst could be recovered and reused up to eight times without any noticeable loss in activity. Mechanistic studies performed by using high-resolution electrospray-ionization quadrupole-time-of-flight mass (HR-EI-QTOF) spectrometry and kinetic experiments indicate only one reaction pathway and rule out the other two possibilities under the development conditions. The theoretical calculations are in accordance with the proposed mechanism of action of the iron catalyst. Finally, the 37 dihydropyrimidinone derivatives, products of the Biginelli reaction, had their cytotoxicity evaluated in assays against MCF-7 cancer cell linages with encouraging results of some derivatives, which were virtually non-toxic against healthy cell linages (fibroblasts).

Proteomic analysis of the reproductive tract fluids from tropically-adapted Santa Ines rams ☆

The present study is focused on the proteome of reproductive tract fluids from tropically-adapted Santa Ines rams. Seminal plasma, cauda epididymal (CEF) and vesicular gland fluid (VGF) proteins were analyzed by 2-D electrophoresis and mass spectrometry. Seminal plasma maps contained 302 ± 16 spots, within the 4-7 pH range. From these maps, 73 spots were identified, corresponding to 41 proteins. Ram Seminal Vesicle Proteins (RSVP) 14 and 22kDa and bodhesins 1 and 2 represented the most abundant seminal components. Other seminal proteins included clusterin, angiotensin-converting enzyme, matrix metalloproteinase-2, tissue-inhibitor of metalloproteinase-2, plasma glutamate carboxypeptidase, albumin, lactoferrin, alpha enolase, peroxiredoxin, leucine aminopeptidase, β-galactosidase, among others. Later, seminal plasma gels were run within narrow pH intervals (3.9-5.1; 4.7-5.9; 5.5-6.7), allowing the additional identification of 21 proteins not detected in 4-7 pH maps. Major proteins of CEF and VGF were albumin and transferrin, and RSVPs, respectively. Western blots confirmed that RSVPs were mainly present in VGF while bodhesins, in VGF and CEF. Based on RT-PCR, RSVP and bodhesin genes were primarily expressed in the vesicular glands. In summary, the reproductive tract fluids of Brazilian hairy rams contain several categories of proteins, with potential roles in sperm protection, capacitation, acrosome reaction and sperm-oocyte interaction.

Hemoglobin-derived peptides as novel type of bioactive signaling molecules.

Most bioactive peptides are generated by proteolytic cleavage of large precursor proteins followed by storage in secretory vesicles from where they are released upon cell stimulation. Examples of such bioactive peptides include peptide neurotransmitters, classical neuropeptides, and peptide hormones. In the last decade, it has become apparent that the breakdown of cytosolic proteins can generate peptides that have biological activity. A case in point and the focus of this review are hemoglobin-derived peptides. In vertebrates, hemoglobin (Hb) consists of a tetramer of two α- and two β-globin chains each containing a prosthetic heme group, and is primarily involved in oxygen delivery to tissues and in redox reactions (Schechter Blood 112:3927–3938, 2008). The presence of α- and/or β-globin chain in tissues besides red blood cells including rodent and human brain and peripheral tissues (Liu et al. Proc Natl Acad Sci USA 96:6643–6647, 1999; Newton et al. J Biol Chem 281:5668–5676, 2006; Wride et al. Mol Vis 9:360–396, 2003; Setton-Avruj Exp Neurol 203:568–578, 2007; Ohyagi et al. Brain Res 635:323–327, 1994; Schelshorn et al. J Cereb Blood Flow Metab 29:585–595, 2009; Richter et al. J Comp Neurol 515:538–547, 2009) suggests that globins and/or derived peptidic fragments might play additional physiological functions in different tissues. In support of this hypothesis, a number of Hb-derived peptides have been identified and shown to have diverse functions (Ivanov et al. Biopoly 43:171–188, 1997; Karelin et al. Neurochem Res 24:1117–1124, 1999). Modern mass spectrometric analyses have helped in the identification of additional Hb peptides (Newton et al. J Biol Chem 281:5668–5676, 2006; Setton-Avruj Exp Neurol 203:568–578, 2007; Gomes et al. FASEB J 23:3020–3029, 2009); the molecular targets for these are only recently beginning to be revealed. Here, we review the status of the Hb peptide field and highlight recent reports on the identification of a molecular target for a novel set of Hb peptides, hemopressins, and the implication of these peptides to normal cell function and disease. The potential therapeutic applications for these Hb-derived hemopressin peptides will also be discussed.

Mechanistic Studies on Lewis Acid Catalyzed Biginelli Reactions in Ionic Liquids: Evidence for the Reactive Intermediates and the Role of the Reagents

This paper describes the use of common Lewis acids supported in imidazolium-based ionic liquids as the catalysts to promote the Biginelli reaction. The ionic liquid effect and the reaction mechanism are discussed on the basis of nuclear magnetic resonance (NMR), electrospray ionization mass spectrometry (ESI-MS), and theoretical calculations. Indeed, the results showed that the ionic medium plays a fundamental role in the synthesis of biologically active dihydropyrimidinones due to the stabilization of the charged intermediates proposed in the mechanism. When conducted in an ionic liquid as solvent, the reaction mechanism is more complex than in other Lewis acid catalyzed Biginelli reactions.

Proteomic analysis of papaya fruit ripening using 2DE-DIGE

Papayas have a very short green life as a result of their rapid pulp softening as well as their susceptibility to physical injury and mold growth. The ripening-related changes take place very quickly, and there is a continued interest in the reduction of postharvest losses. Proteins have a central role in biological processes, and differential proteomics enables the discrimination of proteins affected during papaya ripening. A comparative analysis of the proteomes of climacteric and pre-climacteric papayas was performed using 2DE-DIGE. Third seven proteins corresponding to spots with significant differences in abundance during ripening were submitted to MS analysis, and 27 proteins were identified and classified into six main categories related to the metabolic changes occurring during ripening. Proteins from the cell wall (alpha-galactosidase and invertase), ethylene biosynthesis (methionine synthase), climacteric respiratory burst, stress response, synthesis of carotenoid precursors (hydroxymethylbutenyl 4-diphosphate synthase, GcpE), and chromoplast differentiation (fibrillin) were identified. There was some correspondence between the identified proteins and the data from previous transcript profiling of papaya fruit, but new, accumulated proteins were identified, which reinforces the importance of differential proteomics as a tool to investigate ripening and provides potentially useful information for maintaining fruit quality and minimizing postharvest losses.

Clonostachys rosea BAFC3874 as a Sclerotinia sclerotiorum antagonist: mechanisms involved and potential as a biocontrol agent.

Aims:  To establish the modes of action of the antagonistic fungal strain Clonostachys rosea BAFC3874 isolated from suppressive soils against Sclerotinia sclerotiorum and to determine its potential as a biocontrol agent.

Redox Control of 20S Proteasome Gating

UNLABELLED The proteasome is the primary contributor in intracellular proteolysis. Oxidized or unstructured proteins can be degraded via a ubiquitin- and ATP-independent process by the free 20S proteasome (20SPT). The mechanism by which these proteins enter the catalytic chamber is not understood thus far, although the 20SPT gating conformation is considered to be an important barrier to allowing proteins free entrance. We have previously shown that S-glutathiolation of the 20SPT is a post-translational modification affecting the proteasomal activities. AIMS The goal of this work was to investigate the mechanism that regulates 20SPT activity, which includes the identification of the Cys residues prone to S-glutathiolation. RESULTS Modulation of 20SPT activity by proteasome gating is at least partially due to the S-glutathiolation of specific Cys residues. The gate was open when the 20SPT was S-glutathiolated, whereas following treatment with high concentrations of dithiothreitol, the gate was closed. S-glutathiolated 20SPT was more effective at degrading both oxidized and partially unfolded proteins than its reduced form. Only 2 out of 28 Cys were observed to be S-glutathiolated in the proteasomal α5 subunit of yeast cells grown to the stationary phase in glucose-containing medium. INNOVATION We demonstrate a redox post-translational regulatory mechanism controlling 20SPT activity. CONCLUSION S-glutathiolation is a post-translational modification that triggers gate opening and thereby activates the proteolytic activities of free 20SPT. This process appears to be an important regulatory mechanism to intensify the removal of oxidized or unstructured proteins in stressful situations by a process independent of ubiquitination and ATP consumption. Antioxid. Redox Signal. 16, 1183-1194.

On the use of 2,1,3-benzothiadiazole derivatives as selective live cell fluorescence imaging probes

Newly designed 2,1,3-benzothiadiazole-containing fluorescent probes with four excited state intramolecular proton transfer (ESIPT) sites were successfully tested in live cell-imaging assays using a confluent monolayer of human stem-cells (tissue). All tested dyes were compared with the commercially available DAPI and gave far better results.

Chemical cross-linking with a diazirine photoactivatable cross-linker investigated by MALDI- and ESI-MS/MS

Crystallography and nuclear magnetic resonance are well-established methods to study protein tertiary structure and interactions. Despite their usefulness, such methods are not applicable to many protein systems. Chemical cross-linking of proteins coupled with mass spectrometry allows low-resolution characterization of proteins and protein complexes based on measuring distance constraints from cross-links. In this work, we have investigated cross-linking by means of a heterobifunctional cross-linker containing a traditional N-hydroxysuccinimide (NHS) ester and a UV photoactivatable diazirine group. Activation of the diazirine group yields a highly reactive carbene species, with potential to increase the number of cross-links compared with homobifunctional, NHS-based cross-linkers. Cross-linking reactions were performed on model systems such as synthetic peptides and equine myoglobin. After reduction of the disulfide bond, the formation of intra- and intermolecular cross-links was identified and the peptides modified with both NHS and diazirine moieties characterized. Fragmentation of these modified peptides reveals the presence of a marker ion for intramolecular cross-links, which facilitates identification.