Biomedical subjects

Fabio C Gozzo

Publications and source records attributed to Fabio C Gozzo.

10 recordsLinked to original sources

PubMed2006

Distonoid ions.

By Yates, Bouma, and Radom's definition, distonic radical ions are those formally arising by ionization of diradicals or zwitterionic molecules (including ylides). These ions differ, therefore, from conventional radical ions by displaying the charge site and unpaired electron site (spin) localized mandatorily on separate atoms or group of atoms; that is, these sites are separated in all of their major resonance forms. Many conventional radical ions with a major resonance form in which charge and spin sites reside formally on the same atom or group of atoms display, however, high degree of discretionary (non-mandatory) charge-spin separation. By analogy with the metal/metalloid terminology, we propose that these distonic-like radical ions be classified as distonoid ions. Radical ions would, therefore, be divided into three sub-classes: conventional, distonic, and distonoid ions. B3LYP/6-311 + G(d,p) calculations for a proof-of-principle set of radical cations are used to demonstrate the existence of many types of distonoid ions with a high degree of discretionary charge-spin separation. Reliable calculations are indispensable for probing distonoid ions, since an ion that was expected to be distonoid (by the analysis of its resonance forms) is shown by the calculations to display a characteristic conventional-ion electronic distribution. Similarly to many distonic radical ions, and in sharp contrast to a conventional radical ion (ionized 1,4-dioxane), the gas-phase intrinsic bimolecular reactivity with selective neutrals of a representative distonoid ion, ionized 2-methylene 1,3-dioxolane, is found to include dual ion-radical type reactions.

Journal Article↗

PubMed2006

Spectroscopic characterization of the tumor antigen NY-REN-21 and identification of heterodimer formation with SCAND1.

Human NY-REN-21 is a C2H2 type multi-finger protein, with a SCAN domain in the N-terminal region and a predicted coil central region. It represents a putative ortholog of mouse ZFP38, a transcriptional factor that recognizes a bipartite DNA motif and is unable to form homodimers. As shown in this work, NY-REN-21 contains a SCAN domain able to form homodimers and a central region that behaves as an intrinsically disordered protein. The SCAN domain is found in 71 human proteins and its ability to form homo- and heterodimers widens the number of genes that are regulated by this group of transcription factors. NY-REN-21 interaction with SCAND1 was identified using the yeast two-hybrid system and confirmed using recombinant proteins. SCAND1 is a truncated SCAN box protein, lacking the zinc finger region and the NY-REN-21/SCAND1 heterodimer is asymmetric concerning the DNA binding region. This result indicates that NY-REN-21 can function either as a homodimer or as a heterodimer with SCAND1.

Amino Acid Sequence↗

PubMed2006

Formal gas-phase polar [4 + 1+] cycloaddition of ionized methylene to alpha-dicarbonyl compounds: synthesis of 2-unsubstituted 1,3-dioxoles.

Ion/molecule reactions of +CH2OCH2. with alpha-dicarbonyl compounds were performed via pentaquadrupole mass spectrometry. Besides the previously known [3+ + 2] 1,3-cycloaddition reaction that forms cyclic 1,3-dioxonium ions, an unprecedented reaction proceeding formally by [4 + 1+] cycloaddition of ionized methylene (CH2+.) to the alpha-dicarbonyl compounds occurs competitively, leading to the gas-phase synthesis of several ionized 2-unsubstituted 1,3-dioxoles. This novel cycloaddition reaction may therefore be added to the set of methods available for the synthesis of 1,3-dioxoles.

Journal Article↗

PubMed2006

Identification and characterization of a proteolysis-resistant fragment containing the PCI domain in the Arabidopsis thaliana INT6/eIF3e translation factor.

The PCI domain comprises approx 200 amino acids and is found in subunits of the eukaryotic translation initiation factor 3 (eIF3), the 26S proteasome and the COP9/signalosome complexes. The PCI domain is involved in protein-protein interaction, and mouse INT6 truncated proteins lacking the PCI domain show cell malignanttransforming activity. In this work, the Arabidopsis thaliana INT6/eIF3e (AtINT6) protein was dissected using limited proteolysis, and a protease-resistant fragment containing the PCI domain was identified. Based on mass spectrometry analyses of the protease-resistant fragments and on secondary structure prediction, AtINT6-truncated proteins were cloned and expressed in Escherichia coli. Stability studies using thermal unfolding followed by circular dichroism revealed a midpoint transition temperature of 44 degrees C for the full-length AtINT6 protein, whereas the truncated proteins comprising residues 125-415 (AtINT6TR2) and 172-415 (AtINT6TR3) showed transition temperatures of 49 and 58 degrees C, respectively. AtINT6TR3 contains the PCI domain with additional amino acids at the N and C termini. It shows high solubility, and together with the high thermal stability, should facilitate further characterization of the PCI domain structure, which is important to understand its function in protein- protein interaction.

Amino Acid Motifs↗

PubMed2005

Identification, molecular cloning and functional characterization of an octaprenyl pyrophosphate synthase in intra-erythrocytic stages of Plasmodium falciparum.

Isoprenoids play important roles in all living organisms as components of structural cholesterol, steroid hormones in mammals, carotenoids in plants, and ubiquinones. Significant differences occur in the length of the isoprenic side chains of ubiquinone between different organisms, suggesting that different enzymes are involved in the synthesis of these side chains. Whereas in Plasmodium falciparum the isoprenic side chains of ubiquinone contain 7-9 isoprenic units, 10-unit side chains are found in humans. In a search for the P. falciparum enzyme responsible for the biosynthesis of isoprenic side chains attached to the benzoquinone ring of ubiquinones, we cloned and expressed a putative polyprenyl synthase. Polyclonal antibodies raised against the corresponding recombinant protein confirmed the presence of the native protein in trophozoite and schizont stages of P. falciparum. The recombinant protein, as well as P. falciparum extracts, showed an octaprenyl pyrophosphate synthase activity, with the formation of a polyisoprenoid with eight isoprenic units, as detected by reverse-phase HPLC and reverse-phase TLC, and confirmed by electrospray ionization and tandem MS analysis. The recombinant and native versions of the enzyme had similar Michaelis constants with the substrates isopentenyl pyrophosphate and farnesyl pyrophosphate. The recombinant enzyme could be competitively inhibited in the presence of the terpene nerolidol. This is the first report that directly demonstrates an octaprenyl pyrophosphate synthase activity in parasitic protozoa. Given the rather low similarity of the P. falciparum enzyme to its human counterpart, decaprenyl pyrophosphate synthase, we suggest that the identified enzyme and its recombinant version could be exploited in the screening of novel drugs.

Alkyl and Aryl Transferases↗

PubMed2004

Gaseous supramolecules of imidazolium ionic liquids: "magic" numbers and intrinsic strengths of hydrogen bonds.

Electrospray ionization mass spectrometry (ESI-MS) is found to gently and efficiently transfer small to large as well as singly to multiply charged [X+]n[A-]m supramolecules of imidazolium ion (X+) ionic liquids to the gas phase, and to reveal "magic numbers" for their most favored assemblies. Tandem mass spectrometric experiments (ESI-MS/MS) were then used to dissociate, via low-energy collision activation, mixed and loosely bonded [A- - - -X- - - -A']- and [X- - - -A- - - -X']+ gaseous supramolecules, as well as their higher homologues, and to estimate and order via Cooks' kinetic method (CKM) and B3LYP/6-311G(d,p) calculations the intrinsic solvent-free magnitude of hydrogen bonds. For the five anions studied, the relative order of intrinsic hydrogen-bond strengths to the 1-n-butyl-3-methylimidazolium ion [X1]+ is: CF3CO2- (zero) > BF4- (-3.1) > PF6- (-10.0) > InCl4- (-16.4) and BPh4- (-17.6 kcal mol(-1)). The relative hydrogen-bond strength for InCl4- was measured via CKM whereas those for the other anions were calculated and used as CKM references. A good correlation coefficient (R=0.998) between fragment ion ratios and calculated hydrogen-bond strengths and an effective temperature (Teff) of 430 K demonstrate the CKM reliability for measuring hydrogen-bond strengths in gaseous ionic liquid supramolecules. Using CKM and Teff of 430 K, the intrinsic hydrogen-bond strengths of BF4- for the three cations investigated is: 1-n-butyl-3-methyl-imidazolium ion (0) > 1,3-di-[(R)-3-methyl-2-butyl]-imidazolium ion (-2.4) > 1,3-di-[(R)-alpha-methylbenzyl]-imidazolium ion (-3.0 kcal mol(-1)). As evidenced by "magic" numbers, greater stabilities are found for the [(X1)2(BF4)3]- and [(X1)5A4]+ supramolecules (A not equal InCl4-).

Journal Article↗

PubMed2004

The methylerythritol phosphate pathway is functionally active in all intraerythrocytic stages of Plasmodium falciparum.

Two genes encoding the enzymes 1-deoxy-D-xylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase have been recently identified, suggesting that isoprenoid biosynthesis in Plasmodium falciparum depends on the methylerythritol phosphate (MEP) pathway, and that fosmidomycin could inhibit the activity of 1-deoxy-D-xylulose-5-phosphate reductoisomerase. The metabolite 1-deoxy-D-xylulose-5-phosphate is not only an intermediate of the MEP pathway for the biosynthesis of isopentenyl diphosphate but is also involved in the biosynthesis of thiamin (vitamin B1) and pyridoxal (vitamin B6) in plants and many microorganisms. Herein we report the first isolation and characterization of most downstream intermediates of the MEP pathway in the three intraerythrocytic stages of P. falciparum. These include, 1-deoxy-D-xylulose-5-phosphate, 2-C-methyl-D-erythritol-4-phosphate, 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol, 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol-2-phosphate, and 2-C-methyl-D-erythritol-2,4-cyclodiphosphate. These intermediates were purified by HPLC and structurally characterized via biochemical and electrospray mass spectrometric analyses. We have also investigated the effect of fosmidomycin on the biosynthesis of each intermediate of this pathway and isoprenoid biosynthesis (dolichols and ubiquinones). For the first time, therefore, it is demonstrated that the MEP pathway is functionally active in all intraerythrocytic forms of P. falciparum, and de novo biosynthesis of pyridoxal in a protozoan is reported. Its absence in the human host makes both pathways very attractive as potential new targets for antimalarial drug development.

Animals↗

PubMed2004

Electrospray mass and tandem mass spectrometry of homologous and isomeric singly, doubly, triply and quadruply charged cationic ruthenated meso-(phenyl)m-(meta- and para-pyridyl)n (m + n = 4) macrocyclic porphyrin complexes.

Ten homologous or isomeric singly, doubly, triply and quadruply charged cationic macrocyclic complexes I-Va, bn+ (n = 1-4) formed by the coordination of [Ru(bipy)2Cl]+ to the pyridyl N-atoms of a series of meso-(phenyl)m-(meta or para-pyridyl)n-porphyrins (m + n = 4) were transferred to the gas phase and structurally characterized by electrospray ionization (ESI) mass (MS) and tandem mass (MS/MS) spectrometry. Previously known to be stable in solution and in the solid state, I-Va, bn+ are found to constitute also a new class of stable, long-lived multiply charged gas-phase ions with spatially separated charge sites. Increasing intramolecular electrostatic repulsion from Ia, b+ to IVa, b3+ facilitates in-source and tandem collision-induced dissociation (CID). However, for the quadruply charged ions Va, b4+, electrostatic repulsion is alleviated mainly by ion pairing with the CF3SO3- counterion forming the salt clusters [Va,b/CF3SO3]3+ and [Va,b/(CF3SO3)2]2+ with reduced charge states. Ion-pairing that yields [IVa,b/CF3SO3]2+ is also observed as a minor ESI process for the triply charged ions IVa, b3+. The gaseous ions I-Va, bn+ (n = 2, 3 or 4) dissociate by sequential 'charge partitioning' with the formation of two cationic fragments by the release of [Ru(bipy)2Cl]+. The meta (a) and para (b) isomers and the positional isomers II2+ and III2+ display nearly identical ESI-MS and ESI-MS/MS spectra. ESI-MS/MS of I-Va, bn+ shows that the Ru-py(P) is, intrinsically, the weakest bond since this bond breaks preferentially upon CID.

Cations↗

PubMed2003

The kinetic method as a structural diagnostic tool: ionized alpha-diketones as loosely one-electron bonded diacylium ion dimers.

The kinetic method is used to corroborate the description of ground state ionized alpha-diketones as loosely electron-bonded acylium ion dimers: R(1)-C=O(+)---e(-)---(+)O=C-R(2). The abundance ratio of both the acylium ion fragments R(1)CO(+) and R(2)CO(+) (summed to those of their respective secondary fragments) formed upon low energy (5 eV) collision-induced dissociation of several ionized alpha-diketones is found to correlate linearly with the ionization energies (IEs) of the corresponding R(1)CO(.) and R(2)CO(.) free radicals as predicted by density functional theory calculations at the B3LYP/6-311++G(d,p) level. However, when these abundances are taken from 70 eV electron ionization mass spectra, lower and sometimes inverted ratios (2,3-pentanedione and 2,3-hexanedione) are observed. Inverted ratios are also observed via charge-exchange mass spectrometry/mass spectrometry (MS/MS) experiments for ionized 2,3-pentanodione formed with relatively high internal energies. Ionized alpha-diketones are found to display an effective temperature of 1705 K, which indicates an intermediate loosely-bonded nature. B3LYP/6-311++G(d,p) optimized geometries and charge and spin densities also corroborate the description of ground state ionized alpha-diketones as loosely electron-bonded diacylium ion dimers.

Journal Article↗

PubMed2002

Novel natural peptide substrates for endopeptidase 24.15, neurolysin, and angiotensin-converting enzyme.

Endopeptidase 24.15 (EC; ep24.15), neurolysin (EC; ep24.16), and angiotensin-converting enzyme (EC; ACE) are metallopeptidases involved in neuropeptide metabolism in vertebrates. Using catalytically inactive forms of ep24.15 and ep24.16, we have identified new peptide substrates for these enzymes. The enzymatic activity of ep24.15 and ep24.16 was inactivated by site-directed mutagenesis of amino acid residues within their conserved HEXXH motifs, without disturbing their secondary structure or peptide binding ability, as shown by circular dichroism and binding assays. Fifteen of the peptides isolated were sequenced by electrospray ionization tandem mass spectrometry and shared homology with fragments of intracellular proteins such as hemoglobin. Three of these peptides (PVNFKFLSH, VVYPWTQRY, and LVVYPWTQRY) were synthesized and shown to interact with ep24.15, ep24.16, and ACE, with K(i) values ranging from 1.86 to 27.76 microm. The hemoglobin alpha-chain fragment PVNFKFLSH, which we have named hemopressin, produced dose-dependent hypotension in anesthetized rats, starting at 0.001 microg/kg. The hypotensive effect of the peptide was potentiated by enalapril only at the lowest peptide dose. These results suggest a role for hemopressin as a vasoactive substance in vivo. The identification of these putative intracellular substrates for ep24.15 and ep24.16 is an important step toward the elucidation of the role of these enzymes within cells.

Amino Acid Sequence↗