Biomedical subjects

A M Timperio

Publications and source records attributed to A M Timperio.

9 recordsLinked to original sources

PubMed2003

Identification of a furazolidone metabolite responsible for the inhibition of amino oxidases.

1 Furazolidone, a drug widely used in human and veterinary medicine, exhibits inhibition of monoamine oxidase activity, as observed in the tissues of a number of different animal species, including man. The aim of the current study was to determine which of the two possible metabolites, 3-amino-2-oxazolidone (AOZ) or beta-hydroxyethylhydrazine (HEH), a well-known carcinogenic compound, is involved in the toxicological effects reported. 2 A new spectrometric method was set up to differentiate intracellular HEH from AOZ inside cells. This method works well at low pH where both AOZ and HEH are free in solution and available to react with the chemical chromophore (DAB). 3 The results confirm that furazolidone has to be metabolized in the intact cell in order to exhibit mitochondrial monoamine oxidase inhibition, whereas AOZ itself is able to exert a reversible monoamine oxidase inhibition. AOZ also inhibits bovine serum amino oxidase. On the contrary, HEH gives irreversible inhibition of both enzymes. However, the reversible nature of the AOZ inhibition with respect to HEH suggests that the two metabolites act by different mechanisms which do not require the biotransformation of AOZ to HEH. 4 Cell lysates, previously incubated with AOZ, were directly analysed and the formation of HEH from AOZ was not detected, supporting the conclusion that the amino oxidase inhibition observed on treatment with furazolidone was attributable to AOZ and not to HEH.

Animals↗

PubMed2001

Isoforms of photosystem II antenna proteins in different plant species revealed by liquid chromatography-electrospray ionization mass spectrometry.

The high selectivity offered by reversed-phase high-performance liquid chromatography on-line coupled to electrospray ionization mass spectrometry has been utilized to characterize the major and minor light-harvesting proteins of photosystem II (Lhcb). Isomeric forms of the proteins, revealed either on the basis of different hydrophobicity enabling their chromatographic separation or on the basis of different molecular masses identified within one single chromatographic peak, were readily identified in a number of monocot and dicot species. The presence of several Lhcb1 isoforms (preferably in dicots) can explain the tendency of dicot Lhcb1 to form trimeric aggregates. The Lhcb1 molecular masses ranged from 24,680 to 25,014 among different species, whereas within the same species, the isoforms differed by 14-280 mass units. All Lhcb1 proteins appear to be highly conserved among different species such that they belong to a single gene group that has several different gene family members. In all species examined, the number of isoforms corresponded more or less to the genes cloned previously. Two isoforms of Lhcb3 were found in petunia and tomato. For Lhcb6, the most divergent of all light-harvesting proteins, the greatest number of isoforms was found in petunia, tobacco, tomato, and rice. Lhcb2, Lhcb4, and Lhcb5 were present in only one form. The isoforms are assumed to play an important role in the adaptation of plants to environmental changes.

Chromatography, High Pressure Liquid↗

PubMed2001

High-performance liquid chromatography-electrospray ionization mass spectrometry using monolithic capillary columns for proteomic studies.

The use of tetrahydrofuran/decanol as porogens for the fabrication of micropellicular poly(styrene/divinylbenzene) monoliths enabled the rapid and highly efficient separation of peptides and proteins by reversed-phase high-performance liquid chromatography (RP-HPLC). In contrast to conventional, granular, porous stationary phases, in which the loading capacity is a function of molecular mass, the loadability of the monoliths both for small peptides and large proteins was within the 0.40.9-pmol range for a 60- x 0.2-mm capillary column. Lower limits of detection obtained by measuring UV-absorbance at 214 nm with a 3-nl capillary detection cell were 500 amol for an octapeptide and 200 amol for ribonuclease A. Upon reduction of the concentration of trifluoroacetic acid in the eluent from the commonly used 0.1-0.2 to 0.05%, the separation system was successfully coupled to electrospray ionization mass spectrometry (ESI-MS) at the cost of only a small decrease in separation efficiency. Detection limits for proteins with ESI-MS were in the lower femtomole range. High-quality mass spectra were extracted from the reconstructed ion chromatograms, from which the masses of both peptides and proteins were deduced at a mass accuracy of 50-150 ppm. The applicability of monolithic column technology in proteomics was demonstrated by the mass fingerprinting of tryptic peptides of bovine catalase and human transferrin and by the analysis of membrane proteins related to the photosystem II antenna complex of higher plants.

Chromatography, High Pressure Liquid↗

PubMed2000

High performance liquid chromatography-electrospray mass spectrometry for the simultaneous resolution and identification of intrinsic thylakoid membrane proteins.

In higher plants, both photosystem I (PSI) and II (PSII) consist of membrane-embedded proteins that contain more than one transmembrane alpha helix. PSI is a multiprotein complex consisting of a core complex of thirteen proteins surrounded by four different types of light harvesting antenna proteins. Up to now, the protein components of both photosystems have been characterized by SDS-PAGE and/or immunoblotting and, therefore, identification made only on the basis of electrophoretic mobility, which is sometimes not sufficient to discriminate between individual membrane proteins. This is also complicated by the fact that some proteins, such as the antenna proteins, have almost identical molecular mass and amino acid sequence, making it difficult to identify and ascertain the relative stoichiometry of the proteins. In this paper, we report the complete resolution of the antenna proteins and most of the core components of PSI from spinach, together with the identification of proteins by molecular mass, successfully deduced by the combined use of HPLC coupled on-line with a mass spectrometer equipped with an electrospray ion source (ESI-MS). The proposed RP-HPLC-ESI-MS method holds several advantages over SDS-PAGE, including better protein separation, especially for antenna proteins, mass accuracy, speed, efficiency, and the potential to reveal isomeric forms. Moreover, the molecular masses determined by HPLC-ESI-MS are in good agreement with the molecular masses of the individual components calculated on the basis of their nucleotide-derived amino acid sequences, indicating an absence of post-translational modifications in these proteins. It follows that if the method proposed is useful for these highly hydrophobic proteins, it may be of general use for any membrane proteins, where the presence of detergent for solubilization may compromise their characterization.

Chromatography, High Pressure Liquid↗

PubMed2000

Resolution and identification of the protein components of the photosystem II antenna system of higher plants by reversed-phase liquid chromatography with electrospray-mass spectrometric detection.

Reversed-phase liquid chromatography (RPLC) was interfaced to mass spectrometry (MS) with an electrospray ion (ESI) source for the separation and accurate molecular mass determination of the individual intrinsic membrane proteins that comprise the photosystem II (PS II) major light-harvesting complex (LHC II) and minor (CP24, CP26 and CP29) antenna system, whose molecular masses range between 22,000 and 29,000. PS II is a supramolecular complex intrinsic of the thylacoid membrane, which plays the important role in photosynthesis of capturing solar energy, and transferring it to photochemical reaction centers where energy conversion occurs. The protein components of the PS II major and minor antenna systems were extracted from spinach thylacoid membranes and separated using a butyl-silica column eluted by an acetonitrile gradient in 0.05% (v/v) aqueous trifluoroacetic acid. On-line electrospray MS allowed accurate molecular mass determination and identification of the protein components of PS II major and minor antenna system. The proposed RPLC-ESI-MS method holds several advantages over sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the conventional technique for studying membrane proteins, including a better protein separation, mass accuracy, speed and efficiency.

Chromatography, Liquid↗

PubMed1996

Random amplified polymorphic DNA (RAPD) interpretation requires a sensitive method for the detection of amplified DNA.

The random amplified polymorphic DNA technique (RAPD) has found wide use in molecular genetics because of its speed and ease of use. For various reasons, with this method the amplified DNA fragments are produced at different concentrations between genotypes and even between polymerase chain reaction (PCR) runs. Since the detection of the multiple amplified fragments is performed routinely by agarose gel, and seldom by acrylamide gel electrophoresis, we have found that by capillary zone electrophoresis (CZE), which is more sensitive and accurate than gel electrophoresis, it is possible to unequivocally detect amplified fragments even at low concentration, avoiding polymorphism misinterpretation. CZE is also useful to make more potentially polymorphic fragments evident per random primer used, with obvious economical benefits.

Animals↗

PubMed1996

Capillary electrophoresis of closely related intrinsic thylakoid membrane proteins of the photosystem II light-harvesting complex (LHC II).

The electrophoretic migration behavior of three closely related hydrophobic intrinsic membrane proteins of the photosystem II light-harvesting complex (LHC II) was investigated in free solution capillary electrophoresis at pH 8.0-10 with running electrolyte solutions containing either anionic, zwitter-ionic or nonionic detergents. The complete and repeatable separation of these proteins was accomplished with a running electrolyte solution of 25 mM Tris/192 mM glycine, pH 8.8, containing either sodium dodecyl sulfate or n-octyl beta-D-glucopyranoside at concentration up to 5.0 and 7.0 mM, respectively. Migration times and resolution of the individual LHC II intrinsic membrane proteins were sensitive to the type of detergent. The effect of detergent concentration on the electrophoretic behavior of the LHC II proteins was also investigated. Electroelution of the LHC II components separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to isolate these intrinsic membrane proteins, which were then injected onto the capillary electrophoresis system for peak identification.

Chloroplasts↗

PubMed1990

Anticoagulant activity with dose-related response of glycoconjugates from bovine sublingual gland.

In this study we extracted glycoconjugates from bovine sublingual gland and attempted to investigate anticoagulant properties on blood coagulation of humans. To this purpose we performed the thromboelastographic technique and selective hemocoagulation screening tests. In particular, findings from screening tests indicated the occurrence of hemostatic effects on thrombin time (TT) and activated partial thromboplastin time (aPTT) with a response in a dose-related manner, whereas significant changes were not found on prothrombin time (PT).

Animals↗

PubMedDate not supplied

Metal binding to Pseudomonas aeruginosa azurin: a kinetic investigation.

The interaction between azurin from Pseudomonas aeruginosa and Ag(I), Cu(II), Hg(II), was investigated as a function of protein state, i.e. apo-, reduced and oxidised azurin. Two different metal binding sites, characterized by two different spectroscopic absorbancies, were detected: one is accessible to Ag(I) and Cu(II) but not to Hg(II); the other one binds Ag(I) and Hg(II) but not copper. When added in stoichiometric amount, Ag(I) shows high affinity for the redox center of apo-azurin, to which it probably binds by the -SH group of Cys112; it can displace Cu(I) from reducedazurin, while it does not bind to the redox center of oxidizedazurin. Kinetic experiments show that Ag(I) binding to the reduced form is four times faster than binding to the apo-form. This result suggests that metal binding requires a conformational rearrangement of the active site of the azurin. Interaction of Ag(I) or Hg(II) ions to the second metal binding site, induces typical changes of UV spectrum and quenching of fluorescence emission.

Azurin↗