Biomedical subjects
Publications and source records attributed to D Fabris.
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Acquired drug resistance is one of the most important problems in cancer chemotherapy. One of the mechanisms proposed to contribute to this phenomenon is the sequestration of alkylating agents by metallothionein (MT) in vivo. In this study cadmium-induced human bladder tumor T24 cells were exposed to the therapeutic agents chlorambucil and melphalan. MT-2a, was shown by capillary electrophoresis to comprise 56% of the MT isoforms in induced cells, and drug adducts of MT-2a were isolated and characterized by HPLC and electrospray ionization mass spectrometry. One to four equivalents of drug were found to be covalently adducted. Major binding sites on metallothionein were located in the C-terminal domain by peptide mapping, consistent with previous studies in vitro.
Electrospray mass spectrometry is used at pH 8.0 in combination with accurate mass measurements to confirm the multiplicity of insulin in stable noncovalent complexes with Zn(II) ions. Determination of the nature and number of ligands involved in Zn(II) chelation is in agreement with crystal and solution structures. Counting the number of ligands participating in each center allows deduction of the geometric configuration of the ligand field and gives indirect information about the conformational state of the insulin monomers in solution.
The literature reports that traumatic spondylolisthesis of L5 is an uncommon lesion. The authors report their experience of three cases of this particular fracture-dislocation of the lumbosacral spine. They stress the importance of certain radiographic signs in the diagnosis: namely, the presence of unilateral multiple fracture of the transverse lumbar apophysis. As far as the treatment is concerned, they state the need for an open reduction and an internal segmental fixation by posterior approach. A preoperative MRI study appears mandatory in order to evaluate the integrity of the L5-S1 disc. In the event of a traumatic disruption of the disc, they state the importance of posterior interbody fusion by means of a strut graft carved from the ilium or, in case of iliac wing fracture (which is not uncommon in these patients), by means of interbody cages.
Sarcoplasmic reticulum vesicles were incubated with the maleimide-directed probe ThioGlo1, resulting in ATPase inactivation. Reacted ThioGlo1, revealed by its enhanced fluorescence, was found to be associated with the cytosolic but not with the membrane-bound region of the ATPase. The dependence of inactivation on ThioGlo1 concentration suggests derivatization of approximately four residues per ATPase, of which Cys(364), Cys(498), and Cys(636) were identified in prominently fluorescent peptide fragments. These cysteines reside within the phosphorylation and nucleotide-binding region of the ATPase. Accordingly, protection is observed in the presence of ATP, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-AMP), or an fluoroisothiocyanate label of Lys(515). Furthermore, protection is observed in the presence of vanadate (or decavanadate), but not in the presence of phosphate. Labeling occurs equally well in the presence or in the absence of Ca(2+) and thapsigargin, excluding a role of the E1-to-E2 transition in the protective effect of vanadate. It is concluded that protection by vanadate is due to formation of a pentacoordinated orthovanadate complex at the phosphorylation site, corresponding to a stable transition state analog of the phosphorylation reaction, with intermediate characteristics of the EP1 and EP2 states. The lack of protection by phosphate is attributed to instability of its complex with the enzyme (EP2). These findings are discussed with respect to different structural images obtained from diffraction studies of ATPase in the presence or in the absence of Ca(2+) and/or decavanadate (Ogawa et al., 1998, Biophys. J. 75:41-52).
Acquired drug resistance is one of the most important problems in cancer chemotherapy. One of the proposed mechanisms for these phenomena is the sequestration of alkylating agents by metallothionein in vivo. This research shows that metallothionein can covalently sequester phosphoramide mustard, the active form of cyclophosphamide in vitro. On-line electrospray mass spectrometry reveals that it is phosphoramide, not nornitrogen mustard that alkylates metallothionein, although the metallothionein/nornitrogen mustard adduct was isolated as the major adduct. Tandem mass spectrometric experiments were performed on an isolated drug-modified tryptic peptide. The alkylation occurred predominantly at Cys48 of metallothionein. These results provide further evidence that overexpression of metallothionein can detoxify the active form of the drugs.
The capabilities of electrospray ionization mass spectrometry are demonstrated for monitoring the flux of metal ions out of and into the metalloprotein rabbit liver metallothionein and, in one example, chlorambucil-alkylated metallothionein. Metal ion transfers may be followed as the reactions proceed in situ to provide kinetic information. More uniquely to this technique, metal ion stoichiometries may be determined for reaction intermediates and products. Partners used in these studies include EDTA, carbonic anhydrase, a zinc-bound hexamer of insulin, and the core domain of bacteriophage T4 gene 32 protein, a binding protein for single-stranded DNA.
The research reported here demonstrates covalent binding to the metal-binding protein metallothionein (MT) by the therapeutic nitrogen mustard mechlorethamine. The most surprising aspect of this interaction is the selectivity of the alkylating agent for specific residues of MT. A combination of MS and proteolytic and enzymatic methods was used to deduce specific locations of mechlorethamine alkylation. These experiments indicated that alkylation occurs predominantly in the carboxyl domain of MT, with one molecule of mechlorethamine covalently cross-linking two cysteine residues. Electrospray MS revealed the retention of all seven metal ions in the cross-linked MT/mechlorethamine adducts, highlighting the uniqueness of this protein. Computerized docking experiments supported the hypothesis that selective binding precedes selective alkylation, and the structure of the drug indicates the minimal structural requirements for this binding. These results support the idea that MT overexpressed in tumor cells contributes to the inactivation of anticancer drugs.
It is of interest to test the hypothesis that induced metallothionein (MT) acts in acquired drug resistance by covalent sequestration. In this study MT was incubated in vitro with chlorambucil (CHB) under conditions where only 1:1 covalent adducts were formed. The proteolytic products of these adducts were analyzed by HPLC and mass spectrometry to reveal two major sites of modification. These were the sulfur atoms of cysteines 33 and 48, which cochelate the same metal atom in native MT. The time course of the reaction was followed using on-line electrospray ionization with a double-focusing mass spectrometer. These experiments showed that drug-modified MT binds seven metal ions, as does the unmodified protein. Molecular docking experiments showed that the selectively of drug binding is influenced by the presence of the aziridinium ion in the drug structure and complementary charge densities in the protein structure.
Interaction of chlorambucil and the glutathione-depleted human placenta pi class glutathione S-transferase (pi GST) results in the formation of a complex between the drug and the protein at physiological pH. This complex is not formed in the presence of glutathione or S-hexylglutathione. Molecular mass measurement of the reaction product using matrix-assisted laser desorption mass spectrometry indicates that one molecule of chlorambucil cross-links two subunits of the homodimeric protein. A combination of enzymic proteolysis, high performance liquid chromatography, and mass spectrometry reveals that chlorambucil alkylation occurs at cysteine 47 of one subunit and cysteine 101 of the second subunit. This result supports the idea that conformational changes occur in glutathione-depleted pi GST, which allow the bifunctional tether of chlorambucil to cross-link the two subunits of the protein.
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Oxidants targeted toward inactivation of the nucleocapsid zinc finger protein are under development as antiviral agents, especially for use against human immunodeficiency virus. In the present study, electrospray ionization-mass spectrometry is used to follow in situ the progress of the reactions of 2,2'-dithiodipyridine and disulfiram with recombinant nucleocapsid protein p7 (Ncp7) from human immunodeficiency virus-1 at pH 7.4. Both reagents react with the two zinc fingers in the protein, resulting in the ejection of two zinc ions and the formation of oxidized apo-Ncp7 with three intramolecular disulfide bonds. The ejection of zinc by 2,2'-dithiodipyridine occurs in two steps. Alkylation of unreacted cysteine residues with N-ethylmaleimide after a 2-min reaction with 2,2'-dithiodipyridine reveals that the carboxyl-terminal zinc finger is disrupted first. Cys-49, Cys-36, and, to a lesser extent, Cys-39 are all shown to be target residues for initial electrophilic attack. In the reaction of disulfiram with Ncp7, ejection of the two zinc ions also occurs in two steps; however, the fully oxidized apo-Ncp7 is formed more rapidly. Thus, after a 40-min reaction, 45% of native Ncp7 is oxidized by 2,2'-dithiodipyridine, whereas 75% is oxidized by disulfiram.
Electroblotting proteins separated by gel electrophoresis provides a suitable support for further manipulations and analysis of small amounts of relatively pure samples. On-membrane digestion, peptide mapping by mass spectrometry, and database searching offer sensitive and fast tools to identify the analyte. By providing sequence information, tandem mass spectrometry can go a step further, confirming the database identification, solving problems connected with post-translational modifications and sequence variations, or supplying the stretches of internal sequence necessary to synthesize an oligonucleotide probe for gene isolation. The viability of this approach was successfully evaluated using different tandem mass spectrometric techniques: metastable decomposition in a matrix-assisted laser desorption/ionization (MALDI) time-of-flight instrument with a curved-field reflectron; low energy collision-induced dissociation in a MALDI quadrupole ion trap mass spectrometer; and high energy collision-induced dissociation in a high-performance four-sector mass spectrometer with massive cluster-impact ionization.
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The fragmentation of the melittin 4+ ion has been studied over a range of collision energies from 28 eV to 12 keV in a four-sector mass spectrometer. Patterns of fragmentation and energy conversion were found to be analogous to those observed for singly charged peptides. High numbers of collisions were not required for activation at low energies.
For many years, all the internal fixation devices available for the surgical treatment of thoracolumbar fractures [Harrington rods (HR); Luque rods (LR); Hartshill rectangle (HTR); Roy Camille plates (RCP)] were not always able to distribute all the reductive strains (distraction, compression, derotation), or to guarantee solid stabilization without external supports. In some cases (with HR), only distraction forces might be applied, but stability of the assembly was very poor. Otherwise (with LR, HTR, or RCP), stability was well achieved using segmental fixation systems (with SSW, screws), but reduction was possible only by deflecting the spine towards the rods (or plates), by winging the sublaminar wires (or screws). Restoration of normal sagittal alignment was also very difficult to obtain. The use of internal fixation systems for reduction of thoracolumbar fractures when distraction or compression forces are needed are essential for the spinal surgeon and the patient. In some cases of thoracolumbar fractures (seat belt; flexion-distraction), in fact, the use of distraction forces is not suitable, leading only to overdistraction, not to reduction. The introduction of Cotrel-Dubousset instrumentation (CDI) in 1983, and its subsequent use in spinal traumatology, seems to answer all these unresolved questions.