RESUMO
In this work, lipid extracts from spinach membrane fragments enriched in Photosystem II (PSII) and from spinach PSII dimers were analyzed, by means of Thin Layer Chromatography (TLC) and Electro-Spray Ionization Mass Spectrometry. Cardiolipin found in association with PSII was isolated and purified by preparative TLC, then characterized by mass and mass-mass analyses. Cardiolipin structures with four unsaturated C18 acyl chains and variable saturation degrees were evidenced. Structural and functional effects of different phospholipids on PSII complexes were investigated by Fluorescence, Resonance Light Scattering and Oxygen Evolution Rate measurements. An increment of PSII thermal stability was observed in the presence of cardiolipin and phosphatidylglycerol.
Assuntos
Cardiolipinas/química , Cardiolipinas/isolamento & purificação , Complexo de Proteína do Fotossistema II/química , Spinacia oleracea/química , Análise de Variância , Cardiolipinas/metabolismo , Cromatografia em Camada Fina , Oxigênio/metabolismo , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Photosystem II (PSII) complex activity is known to decrease under strong white light illumination, and this photoinhibition phenomenon is connected to the photobleaching of the PSII photosynthetic pigments. In this work the pigment photobleaching has been studied on PSII core complexes, by observing the effects of different factors such as the aggregation state (PSII monomers and dimers were used), temperature (20 degrees C and 10 degrees C temperatures were tested) and the presence of the exogenous phospholipids (cardiolipin and phosphatidylglycerol). In particular, PSII resistance against white light stress was studied by means of UV/VIS Absorption and Fluorescence Emission measurements. It was found that PSII dimers resulted more resistant against photobleaching and that lower temperature reduces the pigment photodestruction. Moreover, the presence of phosphatidylglycerol or cardiolipin enhanced the PSII resistance to the photobleaching phenomenon, mainly at lower temperatures.
Assuntos
Fosfolipídeos/química , Fotodegradação , Fotossíntese , Complexo de Proteína do Fotossistema II/química , Pigmentos Biológicos/química , Spinacia oleracea/enzimologia , Temperatura , Dimerização , Fosfolipídeos/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Pigmentos Biológicos/metabolismo , Ligação Proteica , Spinacia oleracea/químicaRESUMO
In this work the effect of temperature and n-dodecyl-beta-d-maltoside (DM) on PSII complexes organization was investigated. An aggregation process of PSII monomers and dimers was documented at different temperatures and low DM concentration by steady-state fluorescence, absorption, circular dichroism, Rayleigh and dynamic light-scattering experiments. Measures of oxygen evolution enabled us to estimate the change in photoactivity of PSII during the aggregation. This process was found to be extensively reversed by increasing DM concentration as proved by means of steady-state fluorescence and dynamic light-scattering experiments.
Assuntos
Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/metabolismo , Dimerização , Análise Espectral , Spinacia oleracea/química , Temperatura , Água/químicaRESUMO
In this work, we performed investigations on the lipid content of higher plants (spinach) under hyperosmotic stress, by means of thin layer chromatography (TLC) and mass spectrometry. In particular, the experiments have been performed at different plant organization levels: whole leaves, freshly prepared protoplast suspension and mesophyll cells obtained by reformation of the cell wall from protoplast suspension. The results obtained showed that hyperosmotic stress induces changes in the phospholipid content depending on the different plant organization levels studied. All phospholipids showed an increment of their content in stressed whole leaves. In particular, phosphatidylglycerol (PG) redoubles its content by 1 h of osmotic shock. Different responses to hyperosmotic stress were reported for the other systems. In the case of protoplasts, an increment of PG, phosphatidylcholine (PC) and phosphatidylinositol (PI) together with biphosphatidylglycerol (BPG) and phosphatidylethanolamine (PE) content decreasing were observed in stressed sample. For PG, identified as PG (34:4) by elecrospray ionization mass spectrometry, the increment was of about 30%. In the case of cells, conversely, a decrease of PG content under osmotic stress was recorded. The results suggest an important role of phospholipids, in particular of PG, in the osmotic stress response.
Assuntos
Metabolismo dos Lipídeos , Spinacia oleracea/metabolismo , Concentração de Íons de Hidrogênio , Lipídeos/análise , Pressão Osmótica , Folhas de Planta/metabolismo , Protoplastos/metabolismo , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The interactions between chlorophyll a, and three cyclodextrins, hydroxypropyl-beta-cyclodextrin heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin and hydroxypropyl-gamma-cyclodextrin, were studied in aqueous solutions by means of absorption, emission and circular dichroism spectroscopy. Nanosecond laser flash photolysis and steady-state singlet oxygen generation experiments were performed to clarify the photoactivity of chlorophyll a in these systems. Moreover the photosensitizing activity of these complexes towards human leukemia T-lymphocytes (Jurkat cells) was tested and compared with that of the free sensitizer, chlorophyll a. The results obtained indicate that each cyclodextrin is able to carry the pigment in monomeric form inside of cells producing singlet oxygen.
Assuntos
Clorofila/química , Ciclodextrinas/química , Fármacos Fotossensibilizantes/química , Sobrevivência Celular/efeitos dos fármacos , Clorofila/toxicidade , Clorofila A , Humanos , Células Jurkat , Oxigênio/química , Análise EspectralRESUMO
Hydrophobic PbS nanocrystals (NCs) emitting in the near infrared spectral region were encapsulated in the core of micelles and in the bilayer of liposomes, respectively, to form polyethylene glycol (PEG)-grafted phospholipids. The phospholipid-based functionalization process of PbS NCs required the replacement of the pristine capping ligand at the NC surface with thiol molecules. The procedures carried out for two systems, micelles and liposomes, using PEG-modified phospholipids were carefully monitored by optical, morphological and structural investigations. The hydrodynamic diameter and the colloidal stability of both micelles and liposomes loaded with PbS NCs were evaluated using Dynamic Light Scattering (DLS) and ζ-potential experiments, and both were satisfactorily stable in physiological media. The cytotoxicity of the resulting PbS NC-loaded nanovectors was assessed by the in vitro investigation on Saos-2 cells, indicating that the toxicity of the PbS NC loaded liposomes was lower than that of the micelles with the same NC cargo, which is reasonable due to the different overall composition of the two prepared nanocarriers. Finally, the cellular uptake in the Saos-2 cells of both the NC containing systems was evaluated by means of confocal microscopy studies by exploiting a visible fluorescent phospholipid and demonstrating the ability of both luminescent nanovectors to be internalized. The obtained results show the great potential of the prepared emitting nanoprobes for imaging applications in the second biological window.
RESUMO
The aim of this study was to predict the geographic origin of lentils by using isotope ratio mass spectrometry (IRMS) in combination with chemometrics. Lentil samples from two origins, i.e. Italy and Canada, were analysed obtaining the stable isotope ratios of δ(13)C, δ(15)N, δ(2)H, δ(18)O, and δ(34)S. A comparison between median values (U-test) highlighted statistically significant differences (p<0.05) for all isotopic parameters between the lentils produced in these two different geographic areas, except for δ(15)N. Applying principal component analysis, grouping of samples was observed on the basis of origin but with overlapping zones; consequently, two supervised discriminant techniques, i.e. partial least squares discriminant analysis and k-nearest neighbours algorithm were used. Both models showed good performances with external prediction abilities of about 93% demonstrating the suitability of the methods developed. Subsequently, isotopic determinations were also performed on the protein and starch fractions and the relevant results are reported.
Assuntos
Análise de Alimentos/métodos , Lens (Planta)/química , Espectrometria de Massas , Canadá , Isótopos de Carbono/análise , Proteínas Alimentares/análise , Análise Discriminante , Geografia , Itália , Análise dos Mínimos Quadrados , Isótopos de Nitrogênio/análise , Isótopos de Oxigênio/análise , Amido/análiseRESUMO
Sweet cherries from two Italian regions, Apulia and Emilia Romagna, were analysed using electronic nose (EN) and isotope ratio mass spectrometry (IRMS), with the aim of distinguishing them according to their geographic origin. The data were elaborated by statistical techniques, examining the EN and IRMS datasets both separately and in combination. Preliminary exploratory overviews were performed and then linear discriminant analyses (LDA) were used for classification. Regarding EN, different approaches for variable selection were tested, and the most suitable strategies were highlighted. The LDA classification results were expressed in terms of recognition and prediction abilities and it was found that both EN and IRMS performed well, with IRMS showing better cross-validated prediction ability (91.0%); the EN-IRMS combination gave slightly better results (92.3%). In order to validate the final results, the models were tested using an external set of samples with excellent results.
Assuntos
Nariz Eletrônico , Isótopos/análise , Espectrometria de Massas/métodos , Prunus avium/química , Análise Espectral/métodos , Geografia , ItáliaRESUMO
A series of modified chlorophylls (chlorophyll a, pyrochlorophyll a, Zn-pheophytin a and Zn-pheophorbide a) have been inserted into lamellar phases of sodium bis-(2-ethylhexyl)-sulfosuccinate (AOT). The role played by the different functional groups in affecting the bilayer formation and organisation has been investigated by means of the NMR quadrupolar splitting technique. Evidence is reported for the first time on the capacity of the phytyl chain of the chlorophylls to anchor the tetrapyrroles into the bilayer, favouring at the same time the regular formation of the lamellae.
Assuntos
Clorofila/análogos & derivados , Clorofila/química , Bicamadas Lipídicas/química , Ácido Fítico/química , Ácido Fítico/metabolismo , Clorofila/metabolismo , Bicamadas Lipídicas/metabolismo , Espectroscopia de Ressonância Magnética , Feofitinas/química , Feofitinas/metabolismo , Spinacia oleracea/química , Fatores de TempoRESUMO
Photosystem II is a multisubunit membrane complex which performs the water oxidation process in the higher plants. Core dimers and monomers of photosystem II have been isolated from thylakoid membranes by sucrose density gradient centrifugation. Lipids extracted from different photosystem II-enriched fractions obtained from spinach thylakoids have been analysed by thin layer chromatography. Cardiolipin is enriched throughout the purification of photosystem II complexes; in particular dimers contained two times more cardiolipin than their monomeric counterparts.
Assuntos
Cardiolipinas/química , Cardiolipinas/isolamento & purificação , Cromatografia em Camada Fina/métodos , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/isolamento & purificação , Folhas de Planta/metabolismo , Tilacoides/metabolismo , Fracionamento Químico/métodos , Spinacia oleracea/metabolismoRESUMO
The interaction of Rose Bengal (RB) in aqueous solution of LiClO4 0.1 M with alpha-cyclodextrin (alpha-CD), hydroxypropyl-beta-cyclodextrins (HP-beta-CD) and hydroxypropyl-gamma-cyclodextrins (HP-gamma-CD) were studied by spectrophotometric measurements. The presence of Induced Circular Signals and the results of the analysis of the modifications in the absorbance spectra of RB produced by the presence of CDs in solution indicate that RB forms inclusion complexes only with HP-beta-CD and with HP-gamma-CD.
Assuntos
Ciclodextrinas/química , Ciclodextrinas/efeitos da radiação , Eletroquímica/métodos , Rosa Bengala/química , Rosa Bengala/efeitos da radiação , Análise Espectral/métodos , Água/química , Dicroísmo Circular/métodos , Dimerização , Luz , Substâncias Macromoleculares , Ligação Proteica/efeitos da radiação , SoluçõesRESUMO
The regeneration method of Khorana [J. Biol. Chem. 262 (1987) 9271] has been modified in order to study the effect of endogenous archaeabacterial lipids and, in particular, of glycocardiolipin (GlyC) in the refolding and chromophore regeneration of bacteriorhodopsin (BR). BR refolding and chromophore regeneration could be obtained in the presence of endogenous lipid mixtures containing or not containing glycocardiolipin; however, the kinetics of bacteriorhodopsin regeneration in the presence of glycocardiolipin was faster than in its absence. These results show for the first time that the interaction of glycocardiolipin with bacteriorhodopsin favours its refolding from the denaturated state and the chromophore regeneration.
Assuntos
Bacteriorodopsinas/química , Bacteriorodopsinas/efeitos da radiação , Cardiolipinas/química , Cardiolipinas/efeitos da radiação , Misturas Complexas/química , Misturas Complexas/efeitos da radiação , Cinética , Luz , Lipídeos de Membrana/química , Lipídeos de Membrana/efeitos da radiação , Ligação Proteica , Desnaturação Proteica/efeitos da radiação , Dobramento de ProteínaRESUMO
In this study, non-targeted (1)H NMR fingerprinting was used in combination with multivariate statistical techniques for the classification of Italian sweet cherries based on their different geographical origins (Emilia Romagna and Puglia). As classification techniques, Soft Independent Modelling of Class Analogy (SIMCA), Partial Least Squares Discriminant Analysis (PLS-DA), and Linear Discriminant Analysis (LDA) were carried out and the results were compared. For LDA, before performing a refined selection of the number/combination of variables, two different strategies for a preliminary reduction of the variable number were tested. The best average recognition and CV prediction abilities (both 100.0%) were obtained for all the LDA models, although PLS-DA also showed remarkable performances (94.6%). All the statistical models were validated by observing the prediction abilities with respect to an external set of cherry samples. The best result (94.9%) was obtained with LDA by performing a best subset selection procedure on a set of 30 principal components previously selected by a stepwise decorrelation. The metabolites that mostly contributed to the classification performances of such LDA model, were found to be malate, glucose, fructose, glutamine and succinate.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Prunus/química , Análise Discriminante , Geografia , Itália , Análise Multivariada , Prunus/classificaçãoRESUMO
In this paper, virgin olive oils (VOOs) coming from three different geographic origins of Apulia, were analysed for free acidity, peroxide value, spectrophotometric indexes, chlorophyll content, sterol, fatty acid, and triacylglycerol compositions. In order to predict the geographical origin of VOOs, different multivariate approaches were applied. By performing principal component analysis (PCA) a modest natural grouping of the VOOs was observed on the basis of their origin, and consequently three supervised techniques, i.e., general discriminant analysis (GDA), partial least squares-discriminant analysis (PLS-DA) and soft independent modelling of class analogy (SIMCA) were used and the results were compared. In particular, the best prediction ability was produced by applying GDA (average prediction ability of 82.5%), even if interesting results were obtained also by applying the other two classification techniques, i.e., 77.2% and 75.5% for PLS-DA and SIMCA, respectively.
Assuntos
Óleos de Plantas/química , Clorofila/análise , Análise Discriminante , Ácidos Graxos/análise , Geografia , Itália , Análise dos Mínimos Quadrados , Análise Multivariada , Azeite de Oliva , Análise de Componente Principal , Controle de Qualidade , Triglicerídeos/análiseRESUMO
Cyclodextrins, cyclic oligosaccharides composed of amylose subunits, are known to interact with mycotoxins. The interactions may be useful to analytical chemists by altering the properties of the mycotoxin of interest, namely the chromatographic properties, electrophoretic properties, fluorescence, or absorption of these fungal metabolites. Practical applications of these effects have been the incorporation of cyclodextrins into high-performance liquid chromatography and capillary electrophoresis methods for mycotoxin detection. Specific mycotoxins include those with a native fluorescence such as the aflatoxins, ochratoxin A (OTA) and zearalenone (ZEN) as well as those that can be rendered fluorescent through derivatization, such as T-2 toxin. The literature describing the applications of cyclodextrins in mycotoxin analysis is reviewed and an attempt to extend the use of cyclodextrins to the detection of labelled T-2 toxin is presented. Twenty cyclodextrins were evaluated for their ability to enhance the fluorescence emission of T-2 toxin derivatized with pyrene-1-carbonyl cyanide (T2-Pyr). This evaluation revealed that heptakis (2,6-di-O-methyl)-beta-cyclodextrin (DIMEB), in particular, enhanced T2-Pyr fluorescence. DIMEB was used as a buffer modifier in a capillary electrophoresis-laser-induced fluorescence (CE-LIF) method for detecting T-2 in maize. Because of the effects that certain cyclodextrins have, especially under aqueous conditions, they may make useful additives for a variety of mycotoxin analytical methods.
Assuntos
Ciclodextrinas/química , Micotoxinas/análise , Espectrometria de Fluorescência/métodos , Micotoxinas/químicaRESUMO
In a rapid 51Cr release assay, peripheral blood mononuclear cells from 12 healthy donors did not lyse the hepatitis B virus deoxyribonucleic-acid-transfected human hepatoma cell line 2.2.15, but under the same experimental conditions they did lyse K562 cells. Peripheral blood mononuclear cells from 10 out of 16 patients with chronic active hepatitis B exhibited cytotoxic activity against 2.2.15 cells in the presence of a relatively reduced natural killer cell activity to the K562 cell target. Enhancement of the cytotoxic activity to 2.2.15 cells was statistically significant in the group of patients being treated with leukocyte alpha-interferon. The activity was not influenced by the degree of human leukocyte antigen type matching between effector and target, and was enhanced by depletion of T-cells and by in vitro interferon treatment. These results therefore support the concept of a natural killer-like cell activated by clinical administration of interferon in chronic active hepatitis B patients. This cell effector was lytic for the virus B negative HEP-G2 cells also. However, T-cells purified from a few patients failed to lyse the HEP-G2 while lysing the 2.2.15 target, thus indicating that a preferential recognition of the virus-infected target may be exerted by certain T-lymphocyte subsets. The use of the human leukocyte antigen type defined, highly differentiated, hepatitis B virus releasing 2.2.15 cell line as target for fresh lymphocytes in this cytolytic assay did not disclose cytolytic T-cells in an obvious way. Further manipulation of this system perhaps using T-cell clones may be the next step to exploit the investigative possibilities offered by the availability of the 2.2.15 cell target.