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1.
J Agric Food Chem ; 58(17): 9407-12, 2010 Sep 08.
Article in English | MEDLINE | ID: mdl-20695638

ABSTRACT

Direct-infusion electrospray ionization-mass spectrometry [ESI(+)-MS] of several milk powder samples, confiscated by the Brazilian Federal Police, showed ions accounting for sodiated and potassiated molecules of disaccharides (m/z 365 and 381) as well as trisaccharides (m/z 527 and 543), whereas monosaccharide ions were not detected. The trisaccharide ions were not detected in samples of genuine milk powder, raising the suspicion that their presence indicates adulteration by the addition of maltodextrin. In control samples, maltose and maltotriose were hydrolyzed by alpha-glucosidase and not beta-galactosidase, whereas lactose was resistant to alpha-glucosidase but was hydrolyzed with beta-galactosidase. Samples suspected of being adulterated behaved in the same fashion, confirming the presence of maltose and maltotriose or maltodextrin. Direct-infusion ESI-MS is shown therefore to provide rapid screening of milk powder for adulteration with maltodextrin, whereas its combination with selective enzymatic hydrolysis provides highly reliable confirmation for unambiguous results.


Subject(s)
Milk/chemistry , Polysaccharides/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , alpha-Glucosidases/metabolism , Animals , Hydrolysis
2.
Analyst ; 135(10): 2533-9, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20593076

ABSTRACT

Using two desorption/ionization techniques (DESI and EASI) and Brazilian real, US$ dollar, and euro bills as proof-of-principle techniques and samples, direct analysis by ambient mass spectrometry is shown to function as an instantaneous, reproducible, and non-destructive method for chemical analysis of banknotes. Characteristic chemical profiles were observed for the authentic bills and for the counterfeit bills made using different printing processes (inkjet, laserjet, phaser and off-set printers). Detection of real-world counterfeit bills and identification of the counterfeiting method has also been demonstrated. Chemically selective 2D imaging of banknotes has also been used to confirm counterfeiting. The nature of some key diagnostic ions has also been investigated via high accuracy FTMS measurements. The general applicability of ambient MS analysis for anti-counterfeiting strategies particularly via the use of "invisible ink" markers is discussed.

3.
Analyst ; 135(4): 745-50, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20309447

ABSTRACT

Using easy ambient sonic-spray ionization mass spectrometry (EASI-MS), fast and non-destructive fingerprinting identification and aging of ballpoint pen ink writings have been performed directly from paper surfaces under ordinary ambient conditions. EASI-MS data obtained directly from the ink lines showed that pens from different brands provide typical ink chemical profiles. Accelerated ink aging has also been monitored by EASI-MS revealing contrasting degradation behaviors for six different common ink dyes. As demonstrated for Basic Violet 3, some dyes display a cascade of degradation products whose abundances increase linearly with time thus functioning as 'chemical clocks' for ink aging. Analysis of questionable documents has confirmed the ink aging capabilities of EASI-MS. The order of superimposition at a crossing point has also been determined by EASI-MS. For two superimposed ink lines, continuous EASI-MS analysis has also shown that the EASI spray is able to penetrate through the layers and therefore both ink layers could be characterized.

4.
Cytometry A ; 69(6): 515-23, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16680678

ABSTRACT

BACKGROUND: Cytochrome c release from mitochondria to cytosol is a hallmark of apoptosis and is used to characterize the mitochondria-dependent pathway of this type of cell death. Techniques currently used to measure cytochrome c release, Western blot and fluorescence microscopy of immunolabeled cells, are time-consuming and inaccurate, and the latter is still limited by sample size. METHODS: We developed a rapid and reliable technique to detect cytochrome c release during drug-induced apoptosis, using flow cytometry. Plasma membrane of apoptotic HL-60 cells and thymocytes, treated with staurosporine and dexamethasone, respectively, were selectively permeabilized by digitonin at a low concentration. The released cytochrome c was quickly washed out from cells and that which remained in the mitochondria was immunolabeled after fixing the cells. RESULTS: The fraction of cells that retained their mitochondrial cytochrome c, or the highly fluorescent cells, gradually decreased so that after 4-8 h of drug treatment almost all the cells lost their cytochrome c and emerged as a population of low fluorescent cells. This was confirmed by parallel fluorescence microscopy of cells immunolabeled for cytochrome c. CONCLUSIONS: This technique allows the analysis of cytochrome c release from mitochondria of a large number of apoptotic cells in a short period of time and is proposed as an alternative to the methods currently used for this same purpose.


Subject(s)
Apoptosis , Cell Membrane Permeability , Cytochromes c/analysis , Flow Cytometry/methods , Fluorescent Antibody Technique/methods , Mitochondria/metabolism , Animals , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Membrane Permeability/drug effects , Cytochromes c/immunology , Cytochromes c/metabolism , Dexamethasone/metabolism , Dexamethasone/pharmacology , Digitonin/metabolism , Digitonin/pharmacology , Female , HL-60 Cells , Humans , Mitochondria/enzymology , Rats , Staurosporine/metabolism , Staurosporine/pharmacology
5.
Toxicology ; 219(1-3): 124-32, 2006 Feb 15.
Article in English | MEDLINE | ID: mdl-16343726

ABSTRACT

Statins (3-hydroxy-3-methylglutaryl-CoA reductase inhibitors) are used in the treatment of hypercholesterolemic patients to reduce risk of cardiovascular diseases because of their cholesterol lowering action. Other lipid independent protective actions of statins have been reported. However, some adverse side effects have, also, been described. We report, here, that liver mitochondria isolated from hypercholesterolemic LDL receptor knockout mice treated during 15 days with therapeutic doses (100 mg/kg, p.o.) of lovastatin presented a higher susceptibility to develop membrane permeability transition (MPT). In experiments in vitro, lovastatin-induced MPT in a dose-dependent manner (10-80 microM) by a mechanism sensitive to cyclosporin A (cyclophilin sequestrant), dithiothreitol (reducing agent), adenine nucleotide carrier inhibitor (ADP), catalase (H2O2 reductant) and EGTA (calcium chelator). In agreement with the inhibition of the mitochondrial swelling by dithiothreitol, lovastatin, also, decreased the content of total mitochondrial membrane protein thiol groups. Simvastatin had similar effects on mitochondria; however, pravastatin, a hydrophilic statin, had a weaker effect in inducing MPT. In conclusion, statins can act directly on mitochondria either in vivo or in vitro inducing permeability transition, which is a process involved in cell death.


Subject(s)
Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Mitochondria, Liver/drug effects , Mitochondria, Muscle/drug effects , Adenosine Diphosphate/pharmacology , Animals , Catalase/pharmacology , Chelating Agents/pharmacology , Cholesterol/blood , Dithiothreitol/pharmacology , Egtazic Acid/pharmacology , Electrophysiology , Hindlimb/metabolism , In Vitro Techniques , Lovastatin/pharmacology , Mice , Mice, Knockout , Mitochondrial Swelling/drug effects , Permeability/drug effects , Phenazines , Proteins/metabolism , Receptors, LDL/genetics , Sulfhydryl Compounds/metabolism , Sulfhydryl Reagents/pharmacology
6.
FASEB J ; 19(2): 278-80, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15569776

ABSTRACT

Atherosclerotic disease remains a leading cause of death in westernized societies, and reactive oxygen species (ROS) play a pivotal role in atherogenesis. Mitochondria are the main intracellular sites of ROS generation and are also targets for oxidative damage. Here, we show that mitochondria from atherosclerosis-prone, hypercholesterolemic low-density lipoprotein (LDL) receptor knockout mice have oxidative phosphorylation efficiency similar to that from control mice but have a higher net production of ROS and susceptibility to develop membrane permeability transition. Increased ROS production was observed in mitochondria isolated from several tissues, including liver, heart, and brain, and in intact mononuclear cells from spleen. In contrast to control mitochondria, knockout mouse mitochondria did not sustain a reduced state of matrix NADPH, the main source of antioxidant defense against ROS. Experiments in vivo showed faster liver secretion rates and de novo synthesis of triglycerides and cholesterol in knockout than in control mice, suggesting that increased lipogenesis depleted the reducing equivalents from NADPH and generated a state of oxidative stress in hypercholesterolemic knockout mice. These data provide the first evidence of how oxidative stress is generated in LDL receptor defective cells and could explain the increased LDL oxidation, cell death, and atherogenesis seen in familiar hypercholesterolemia.


Subject(s)
Antioxidants/metabolism , Arteriosclerosis/metabolism , Mitochondria/chemistry , Oxidative Stress/physiology , Animals , Arteriosclerosis/pathology , Brain/metabolism , Female , Hypercholesterolemia , Ion Channels/chemistry , Leukocytes, Mononuclear/metabolism , Male , Mice , Mice, Knockout , Mitochondria, Liver/chemistry , Mitochondrial Membrane Transport Proteins , Mitochondrial Permeability Transition Pore , Myocardium/chemistry , Reactive Oxygen Species/metabolism , Receptors, LDL/deficiency , Spleen/cytology
7.
J Biol Chem ; 277(45): 42802-7, 2002 Nov 08.
Article in English | MEDLINE | ID: mdl-12207028

ABSTRACT

Overexpression of the antiapoptotic Bcl-2 protein enhances the uptake of fluorimetric dyes sensitive to mitochondrial membrane potential, suggesting that Bcl-2 changes the mitochondrial proton gradient. In this study, we performed calibrated measurements of mitochondrial respiration, membrane potential, deltapH, and intramitochondrial [K+] in digitonin-permeabilized PC12 and GT1-7 neural cells that either do not express human Bcl-2 (control transfectants) or that were transfected with and overexpressed the human bcl-2 gene to evaluate whether Bcl-2 alters mitochondrial inner membrane ion transport. We found that although Bcl-2-overexpressing cells exhibit higher fluorescence responses to membrane potential, pH, and K+-sensitive dyes, this increased response is due to an enhanced accumulation of these dyes and not an increased mitochondrial membrane potential, deltapH, or [K+]. This result is supported by the presence of equal respiratory rates in Bcl-2+ and Bcl-2- cells. Possible structural alterations in Bcl-2+ mitochondria that could account for increases in fluorescent dye uptake were evaluated using flow cytometry particle sizing and light scattering determinations. These experiments established that Bcl-2-overexpressing mitochondria present both increased volume and structural complexity. We suggest that increased mitochondrial volume and structural complexity in Bcl-2+ cells may be related to many of the effects of this protein involved in the prevention of cell death.


Subject(s)
Genes, bcl-2 , Hydrogen-Ion Concentration , Mitochondria/physiology , Mitochondria/ultrastructure , Proto-Oncogene Proteins c-bcl-2/genetics , Animals , Cell Death/physiology , Cell Line , Gene Expression Regulation , Gene Expression Regulation, Neoplastic , Humans , Hypothalamus , Intracellular Membranes/physiology , Membrane Potentials , Neurons/cytology , PC12 Cells , Pheochromocytoma , Potassium/metabolism , Rats
8.
J Bioenerg Biomembr ; 34(3): 157-63, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12171065

ABSTRACT

Pure singlet molecular oxygen (1O2) generated by thermal decomposition of the 3,3'-(1,4-naphthylidene) dipropionate endoperoxide (NDPO2), inhibited respiration of isolated rat liver mitochondria supported by NADH-linked substrates or succinate, but not by N,N,N,N-tetramehyl-p-phenylene-diamine (TMPD)/ascorbate. Under the latter conditions, mitochondria treated with 2.7 mM NDPO2 exhibited a decrease in transmembrane potential (deltapsi) in manner dependent on NDPO2 exposure time. This process was sensitive to the mitochondrial permeability transition inhibitors EGTA, dithiothreitol, ADP, and cyclosporin A. The presence of deuterium oxide (D2O), that increases 1O2 lifetime, significantly enhanced NDPO2-promoted mitochondrial pereabilization. In addition, NDPO2-induced mitochondrial permeabilization was accompanied by DTT or ADP-sensitive membrane protein thiol oxidation. Taken together, these results provide evidence that mitochondrial permeability transition induced by chemically generated singlet oxygen is mediated by the oxidation of membrane protein thiols.


Subject(s)
Mitochondria, Liver/drug effects , Singlet Oxygen/pharmacology , Animals , Cell Respiration/drug effects , Electron Transport/drug effects , Intracellular Membranes/drug effects , Membrane Potentials/drug effects , Membrane Proteins/metabolism , Mitochondria, Liver/ultrastructure , Naphthols/pharmacology , Permeability/drug effects , Rats , Rats, Wistar , Singlet Oxygen/chemistry , Sulfhydryl Compounds/metabolism
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