Biomedical subjects

Christian Rolando

Publications and source records attributed to Christian Rolando.

17 recordsLinked to original sources

PubMed2006

DNA damage by benzo(a)pyrene in human cells is increased by cigarette smoke and decreased by a filter containing rosemary extract, which lowers free radicals.

We found previously that the human lung benzo(a)pyrene (BP)-7,8-diol-9,10-epoxide-N(2)-deoxyguanosine (BPDE-dG) adduct concentrate in the target bronchial cells. This adduct is now considered to be critical event in tumorigenesis by BP. In this study, we investigate the contribution of cigarette smoke on the BPDE-dG formation. In a cell-free system, the amount of (-)-anti-BPDE-dG adduct increased linearly with concentration of cigarette smoke in the presence of (+)-BP-7,8-diol. Catalase and superoxide dismutase inhibited its formation by >80%. When MCF-7 cells were treated for 2 hours with the (+)-BP-7,8-diol, cigarette smoke increased dose dependently the formation of (-)-anti-BPDE-dG and decreased the cytochrome P450 (CYP)-dependent formation of (+)-r-7,t-8-dihydroxy-c-9,10-oxy-7,8,9,10-tetrahydro-BP the adduct. Then, cells were treated for up to 1 day with BP and then exposed for 2 hours with cigarette smoke. During these 2 hours, there are twice the increase in the adduct formation in cells treated with cigarette smoke compared with levels in nontreated cells due to CYP activity. Thus, cigarette smoke containing reactive oxygen species may activate the second step of BP metabolic way, leading to the formation of BPDE-dG adduct. Cigarette smoke thus seems may be in part responsible for the formation of the critical lung tumorigenic adduct. Finally, modified cigarette filter containing rosemary extract decreases by >70% of the BPDE-dG adducts level due to the cigarette smoke in MCF-7 cells. This approach may lead to decreasing lung cancer risk in addicted smokers.

Benzo(a)pyrene↗

PubMed2006

Identification of proteins in renaissance paintings by proteomics.

The presented work proposes a new methodology based on proteomics techniques to identify proteins in old art paintings. The main challenging tasks of this work were (i) to find appropriate conditions for extracting proteins from the binding media without protein hydrolysis in amino acids and (ii) to develop analytical methods adapted to the small sample quantity available. Starting from microsamples of painting models (ovalbumin-based, which is the major egg white protein, and egg-based paintings), multiple extraction solutions (HCl, HCOOH, NH3, NaOH) and conditions (ultrasonic bath, mortar and pestle, grinding resin) were evaluated. The best results were obtained using a commercial kit including a synthetic resin, mortar and pestle to grind the sample in an aqueous solution acidified with trifluoroacetic acid at 1% with additional multiple steps of ultrasonic baths. The resulting supernatant was analyzed by MALDI-TOF in linear mode to verify the efficiency of the extraction solution. An enzymatic hydrolysis step was also performed for protein identification; the peptide mixture was analyzed by nanoLC/nanoESI/Q-q-TOF MS/MS with an adapted chromatographic run for the low sample quantity. Finally, the developed methodology was successfully applied to Renaissance art painting microsamples of approximately 10 microg from Benedetto Bonfigli's triptych, The Virgin and Child, St. John the Baptist, St. Sebastian (XVth century), and Niccolo di Pietro Gerini's painting, The Virgin and Child (XIVth century), identifying, for the first time and without ambiguity, the presence of whole egg proteins (egg yolk and egg white) in a painting binder.

Egg Proteins↗

PubMed2006

High-sensitivity staining of proteins for one- and two-dimensional gel electrophoresis using post migration covalent staining with a ruthenium fluorophore.

This paper describes the use of a ruthenium complex ((bis(2,2'-bipyridine)-4'-methyl-4-carboxybipyridine-ruthenium-N-succidimyl ester-bis(hexafluorophosphate), abbreviated below as ASCQ_Ru) commercially available and chemically pure. This new ruthenium complex ASCQ_Ru brings an activated ester, allowing the selective acylation of amino acid side chain amines for the post migration staining of proteins separated in 1-DE and 2-DE. The protocol used is a simple three-step protocol fixing the proteins in the gel, staining and then washing, as no lengthy destaining step is required. First the critical staining step was optimized. Although in solution the best described pH for acylating proteins with this reagent is phosphate buffer at pH 7.0, we found that best medium for in-gel staining is unbuffered ACN/water solution (20/80 v/v). The two other steps are less critical and classical conditions are satisfactory: fixing with 7% acetic acid/10% ethanol solution and washing four times for 10 min with water. Sensitivity tests were performed using 1-DE on protein molecular weight markers. We obtained a higher sensitivity than SYPRO Ruby with a detection limit of 80 pg of protein per well. However, contrary to SYPRO Ruby, ASCQ_Ru exhibits a logarithmic dependency on the amount of protein. The dynamic range is similar to SYPRO Ruby and is estimated between three and four orders of magnitude. Finally, the efficiency of the post migration ASCQ_Ru staining for 2-D gel separation is demonstrated on the whole protein extract from human colon carcinoma cells lines HCT 116. ASCQ_Ru gave the highest number of spot detected compared to other common stains Colloidal CBB, SYPRO Ruby and Deep Purple.

Cell Line, Tumor↗

PubMed2005

An open design microfabricated nib-like nanoelectrospray emitter tip on a conducting silicon substrate for the application of the ionization voltage.

This paper describes a novel emitter tip having the shape of a nib and based on an open structure for nano-electrospray ionization mass spectrometry (nanoESI-MS). The nib structure is fabricated with standard lithography techniques using SU-8, an epoxy-based negative photoresist. The tip is comprised of a reservoir, a capillary slot and a point-like feature, and is fabricated on a silicon wafer. We present here a novel scheme for interfacing such nib tips to MS by applying the ionization voltage directly onto the semi-conductor support. The silicon support is in direct contact with the liquid to be analyzed at the reservoir and microchannel level, thus allowing easy use in ESI-MS. This scheme is especially advantageous for automated analysis as the manual step of positioning a metallic wire into the reservoir is avoided. In addition, the analysis performance was enhanced compared with the former scheme, as demonstrated by the tests of standard peptides (gramicidin S, Glu-fibrinopeptide B). The limit of detection was determined to be lower than 10(-2) microM. Due to their enhanced performance, these microfabricated sources might be of great interest for analysis requiring very high sensitivity, such as proteomics analysis using nanoESI-MS.

Journal Article↗

PubMed2005

Polyphenols deriving from chalcones: investigations of redox activities.

The redox properties of a series of hydroxychalcones (a group of polyphenols abundantly present in plants) were investigated by cyclic voltammetry. As for many polyphenols, their beneficial properties have been mainly related to their antioxidant activities, which in turn are directly associated to their redox behavior. Two types of radicals can be produced that are localized on either one of the two aromatic systems. Their thermodynamic and kinetic parameters were extracted and compared to the predictions of density functional theory calculations. When at least one OH is present on each ring, their behaviors are dominated by the conjugated system: phenolic ring A-double bond-ketone, which is the only one to be oxidized. However, the redox properties of this conjugated system are strongly influenced by the presence of ring B. When an OH is present on ring B, an important feature is the existence of strong hydrogen bonding that remains almost unmodified even when ring A is oxidized. It does not considerably change the thermodynamics of ring A but strongly increases the rigidity of the molecule that remains planar under the neutral, anionic, or radical forms. Oxidation potentials of the phenolates range between 0.1 and 0.2 V versus a saturated calomel electrode, which correspond to species that are very easy to oxidize and lead to the rapid formation of nonradical species, underlining the potential antioxidant properties of these molecules.

Algorithms↗

PubMed2004

Monoliths for microfluidic devices in proteomics.

We report here on the preparation of monolithic capillary columns in view to their integration in a microsystem for on-chip sample preparation before their on-line analysis by electrospray and mass spectrometry (ESI-MS). These monolithic columns are based on polymer materials and consist of reverse phases for peptide separation and/or desalting. They were prepared using lauryl methacrylate (LMA), ethylene dimethacrylate (EDMA) as well as a suitable porogenic mixture composed of cyclohexanol and ethylene glycol. The resulting stationary phases present thus a C12-functionality. The LMA-based columns were first prepared in a capillary format using capillary tubing of 75 microm i.d. and tested in nanoLC-MS experiments for the separation of a commercial Cytochrome C digest composed of 12 peptidic fragments whose isoelectric point values and hydrophobic character cover a wide range. The LMA-based columns were capable of separating the peptidic fragments and their performances were seen to be similar as those of standard commercial columns dedicated to proteomic purposes with calculated separation efficiencies up to 145 x 10(3) plates/m. Monolithic LMA-based phases were then successfully polymerized in microchannels fabricated using the negative photoresist SU-8. After the polymerization, the systems were seen to withstand the pressures applied during the nanoLC-MS separation tests that were carried out in the same conditions as for the monolithic capillary columns. The pressure drop during these tests of the in-microchannel monoliths was as high as 50 bar; however, the separation was not as good as for a capillary format which could be accounted for by the monolith dimensions.

Amino Acid Sequence↗

PubMed2004

Two-dimensional maps and databases of the human macrophage proteome and secretome.

Macrophages exert a crucial, but still incompletely known, role in complex disorders such inflammatory, immunological, and infectious diseases. A differential proteomic approach should help to elucidate the macrophage dysfunctions involved in these diseases. With this goal in mind, we established the first two-dimensional maps of the human macrophage proteome and secretome. Intracellular and secreted proteins were extracted from monocyte-derived macrophages obtained from healthy donors (n = 16), and separated by two-dimensional gel electrophoresis. Silver-stained gels were analyzed using Progenesis software. A high level of between-gel reproducibility was obtained, allowing us to generate two patterns specific of the macrophage proteome and secretome, respectively. A total of 127 and 66 distinct intracellular and secreted polypeptide spots, corresponding to 100 and 38 different proteins, respectively, were identified by matrix assisted laser desorption/ionisation-mass spectrometry. The two-dimensional reference maps and databases resulting from this study confirm that macrophages are involved in a wide range of biological functions, and that they provide a useful tool for a wide array of investigators involved in macrophage biology, allowing to investigate the macrophage protein changes associated with various disorders or environmental stimuli.

Databases, Protein↗

PubMed2004

A novel nib-like design for microfabricated nanospray tips.

We present here novel tips for nanoelectrosray ionization-mass spectrometry (ESI-MS) applications. These ionization sources have a planar geometry in the shape of a nib. Their functioning is based on a principle much akin to that of a fountain pen in that fluids are actuated by capillarity. Once a voltage is applied, an electrospray is formed at the nib tip. The nib fabrication relies on micromachining techniques using the epoxy-based negative photoresist SU-8 and a double exposure photolithographic process. Two types of nib-like sources were fabricated; they were made either conductive by metallization with a nickel layer or non-conductive but hydrophilic by covering them with a SiO(2) layer. In the latter case, the HV was applied via a Pt wire inserted into the reservoir feature of the nib. The nib-like sources were tested on an ion trap mass spectrometer using Gramicidin S samples at concentrations as low as 1 microM and ionization voltages as low as 1.2 kV. We have observed a good overall stability of the spray during the tests with no marked decrease in the signal intensity even under extreme conditions.

Journal Article↗

PubMed2003

Effect of catechin O-methylated metabolites and analogues on human LDL oxidation.

The effects of catechin metabolites and methylated analogues on LDL oxidation were studied in vitro using either a water-soluble initiator or copper ions to induce lipid peroxidation. Direct addition of catechin O-methylated analogues to the oxidation mixture led to a clear protective effect during lag phase and for the metabolites during both lag and propagation phases. The structure-activity relationships obtained with these selectively O-methylated compounds allowed determination of catechin active moietie: the catechol B-ring. Based on physical chemical studies, these results suggest that the mechanism implied in the scavenging properties of flavan-3-ols is not only hydrogen transfer, as generally described, but mainly an electronic transfer from the phenolate, and that 3'- and 4'-O-methylcatechin seem, moreover, to act as amphiphilic chain-breaking antioxidants. However, the plasma concentration of flavan-3-ols necessary to protect LDL is far greater than those usually found in human plasma. Therefore, the data do not support a direct physiological relevance of flavan-3-ols as antioxidants in lipid processes. Future research should focus on other effects besides simple antioxidant ones.

Antioxidants↗

PubMed2003

A planar microfabricated nanoelectrospray emitter tip based on a capillary slot.

We report on the fabrication and testing of planar nib-like structures for nanoelectrospray ionization-mass spectrometry (nanoESI-MS) applications. The micro-nib structures were fabricated on silicon substrates using the negative photoresist SU-8; they include capillary slots with widths of 8 and 16 microm. A suitable wafer cleaving step made the nib-like structures overhang the edge of a silicon substrate to provide a robust interface for nanoESI-MS applications; this freeing of the nib tip from the wafer surface created a point-like structure that is essential to establish an electrospray. The micro-nib sources were successfully tested on an LCQ Deca XP+ ion trap mass spectrometer using peptide samples at concentrations down to 1 microM. The high voltage was applied using a platinum wire inserted in the sample reservoir upstream to the capillary slot. A Taylor cone was clearly seen at the nib tip. The micro-nibs performed well at voltages as low as 0.8 kV; such performances are state-of-the-art with respect to current micromachined ESI-MS interfaces and are conditions comparable to those used for standard emitter tips. In addition, we clearly observed the influence of the micro-nib slot width on the ionization performances: the narrower the slot, the better the performances.

Gramicidin↗

PubMed2003

Two-dimensional microfabricated sources for nanoelectrospray.

The idea of a novel two-dimensional (2D) nanoelectrospray ionization emitter tip with the shape of a nib is explored here. This novel planar design is studied as an alternative to the needle-like standard emitter tips that suffer from a lack of reproducibility and robustness and from an inherent incompatibility with high-throughput analysis. The composition of the micro-nib sources is analogous to the working of a simple fountain pen, with a liquid reservoir linked to a micro-nib tip from which the sample is electrosprayed via a capillary slot. The micro-nib prototypes described here were fabricated using microtechnology techniques and using the epoxy-based negative photoresist SU-8. The resulting free-standing micro-nib structure was supported by a silicon wafer. We present here two series of such micro-nib sources, the latter series exhibiting improved characteristics such as a 8 micro m source width of the nib tip. They were tested in mass spectrometry experiments on an ion trap mass spectrometer (LCQ Deca XP+, Thermo Finnigan) using standard peptide samples having concentrations down to 1 micro M and with a high voltage (HV) supply around 1 kV for the second series of micro-nib sources. In addition to the stability of the spray, the obtained mass spectra showed the reliability of these sources for peptide analysis; the signal of the spectra was as intense and the signal-to-noise ratio (S/N) as high as that obtained with the use of standard emitter tips.

Gramicidin↗

PubMed2002

Free radical chemistry of flavan-3-ols: determination of thermodynamic parameters and of kinetic reactivity from short (ns) to long (ms) time scale.

The physical chemistry and the free radical chemistry of the most abundant polyphenolic flavan-3-ols in food, catechin, its methylated metabolites, and several methylated analogues, have been investigated by laser flash photolysis and cyclic voltammetry studies. Two independent phenoxyl radicals formed upon oxidation of flavan-3-ols have been characterized and identified unambiguously: a short-lived resorcinol-like radical characterized by an absorption band at lambda = 495 nm and a long-lived catechol-like transient absorbing at lambda = 380 nm. The determination of all the thermodynamic constants of each phenolic function of flavan-3-ols, namely, redox potential (E degrees (3') = 0.13(5) V/SCE, E degrees (4') = 0.11(0) V/SCE, E degrees (5) = 0.28(5) V/SCE) and microscopic dissociation constants (pK(a3') = 9.02, pK(a4') = 9.12, pK(a5) = 9.43, pK(a7) = 9.58) were performed. These values are discussed and compared to the prediction of the density functional theory calculations made on the different species catechin, catechin phenoxyl, and catechin phenolate for each phenolic site.

Catechin↗

PubMed2002

Characterization of archaeological beeswax by electron ionization and electrospray ionization mass spectrometry.

To better detect and identify beeswax in ancient organic residues from archaeological remains, we developed a new analytical methodology consisting of the analysis of (i) the trimethylsilylated organic extract by GC/MS and (ii) the crude extract by ESI-MS. Selective scanning modes, such as SIM or MRM, permit separate quantification of each chemical family (fatty acids, monoesters, monohydroxyesters, and diesters) and allow an improvement in sensitivity and selectivity, allowing the crude extract to be treated without further purification. GC/MS (SIM) was revealed to be a powerful method for the detection of components, with a detection limit down to a total lipid extract in the range of approximately 50 ng in a complex matix, such as archaeological degraded material, whereas ESI-MS/MS is instead used for the detection of nonvolatile biomarkers. Identification by GC/MS (SIM) and ESI-MS/ MS (MRM) of more than 50 biomarkers of beeswax in an Etruscan cup at the parts-per-million level provides the first evidence for the use of this material by the Etruscans as fuel or as a waterproof coating for ceramics.

Animals↗

PubMed2002

Daily melatonin supplementation in mice increases atherosclerosis in proximal aorta.

Considerable evidence supports the hypothesis that LDL oxidation plays an important role in atherosclerosis. Even though high melatonin doses inhibit LDL oxidation in vitro, the effect of melatonin on atherosclerosis has never been studied. We have demonstrated that the feeding of hypercholesterolemic mice with an atherogenic diet supplemented with melatonin highly increases the surface of atherosclerotic lesions in the proximal aorta. These observations occur without detectable lipidic or glucidic phenotype alteration. Melatonin treatment increased highly the sensitivity of atherogenic lipoprotein to Cu(2+) and gamma-radiolysis generated oxyradical ex vivo oxidation during the fasting period. Moreover, these altered lipoproteins were less recognized by the LDL receptor metabolic pathway of murine fibroblasts while they transferred many more cholesteryl esters to murine macrophages. This study suggests that caution should be taken as regards high melatonin dosage in hypercholesterolemic patients.

Administration, Oral↗

PubMed2002

Identification of archaeological adhesives using direct inlet electron ionization mass spectrometry.

Adhesives made from natural substances such as resins, tars, and waxes are found during excavations on archaeological sites dating back to prehistoric periods. Until now, their analysis was mainly performed by gas chromatography, possibly coupled to mass spectrometry, after extraction, purification, and derivatization of the samples. To minimize sampling and sample preparation of ancient organic remains, which are often preserved in tiny amounts, we have directly analyzed archaeological samples from Bronze and Iron Age periods by direct inlet electron ionization mass spectrometry. A series of contemporary natural and synthetic substances, including pine and pistacia resins, birch bark tar, beeswax, and plant oils, possibly used for adhesive fabrication during ancient times, was also investigated with the same technique as reference materials. Despite the complexity of their chemical composition, pine resin and birch bark tar were clearly identified in archaeological samples. Furthermore, mass spectrometry has been shown to be efficient for the identification of glues made of a mixture of beeswax, presenting a series of mass spectral peaks assigned to long-chain esters, and birch bark tar, whose mass spectrum presents characteristic peaks of lupane compounds. The intentional mixing of birch bark tar and beeswax during prehistory is reported here for the first time.

Adhesives↗

PubMed2002

(-)-Epigallocatechin (EGC) of green tea induces apoptosis of human breast cancer cells but not of their normal counterparts.

(-)-Epigallocatechin (EGC), one of green tea polyphenols, has been shown to inhibit growth of cancer cells. However its mechanism of action is poorly known. We show here that EGC strongly inhibited the growth of breast cancer cell lines (MCF-7 and MDA-MB-231) but not that of normal breast epithelial cells. The inhibition of breast cancer cell growth was due to an induction of apoptosis, without any change in cell cycle progression. MCF-7 cells are known to express a wild-type p53 whereas MDA-MB-231 cells express a mutated p53. The fact that EGC induced apoptosis in both these cell lines suggests that the EGC-triggered apoptosis is independent of p53 status. Moreover, neutralizing antibodies against the death receptor Fas and inhibitors of caspases, such as caspase-8 and -10, efficiently inhibited the EGC-triggered apoptosis. In addition, immunoblotting revealed that EGC treatment was correlated with a decrease in Bcl-2 and an increase in Bax level. These results suggest that EGC-triggered apoptosis in breast cancer cells requires Fas signaling.

Apoptosis↗

PubMedDate not supplied

Lignification in poplar plantlets fed with deuterium-labelled lignin precursors.

Lignification was investigated in wild-type (WT) and in transgenic poplar plantlets with a reduced caffeic acid O-methyl-transferase (COMT) activity. Coniferin and syringin, deuterated at their methoxyl, were incorporated into the culture medium of microcuttings. The gas chromatography-mass spectrometry (GC-MS) analysis of the thioacidolysis guaiacyl (G) and syringyl (S) lignin-derived monomers revealed that COMT deficiency altered stem lignification. GC-MS analysis proved that the deuterated precursors were incorporated into root lignins and, to a lower extent, in stem lignins without major effect on growth and lignification. Deuterium from coniferin was recovered in G and S lignin units, whereas deuterium from syringin was only found in S units, which further establishes that the conversion of G to S lignin precursors may occur at the level of p-OH cinnamyl alcohols.

Cinnamates↗