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Research Interests: Mass Spectrometry, Chemical Biology, Humans, Circular Dichroism, NF-kappa B, and 9 morePeptides, Protein-protein interaction networks, Protein Secondary Structure Prediction, Amino Acid Sequence, Protein Binding, Binding Site, Biochemistry and cell biology, Molecular Sequence Data, and binding sites
Carbonic anhydrases (CAs) catalyze with high efficiency the reversible hydration of carbon dioxide, an essential reaction for many biological processes, such as photosynthesis, respiration, renal tubular acidification, and bone... more
Carbonic anhydrases (CAs) catalyze with high efficiency the reversible hydration of carbon dioxide, an essential reaction for many biological processes, such as photosynthesis, respiration, renal tubular acidification, and bone resorption. Diatoms, which are one of the most common types of phytoplankton and are widespread in oceans, possess CAs fundamental for acquisition of inorganic carbon. Recently, in the marine diatom Thalassiosira weissflogii a novel enzyme, CDCA1, naturally using Cd in its active site, has been isolated and categorized in a new CA class, namely zeta-CA. This enzyme, which consists of three repeats (R1, R2 and R3), is a cambialistic carbonic anhydrase that can spontaneously exchange Zn or Cd at its active centre, presumably an adaptative advantage for diatoms that grow fast in the metal-poor environment of the surface ocean. In this paper we completed the characterization of this enzyme, reporting the X-ray structure of the last repeat, CDCA1-R3 in its cadmium-bound form, and presenting a model of the full length protein obtained by docking approaches. Results show that CDCA1 has a quite compact not symmetric structure, characterized by two covalently linked R1-R2 and R2-R3 interfaces and a small non-covalent R1-R3 interface. The three dimensional arrangement shows that most of the non-conserved aminoacids of the three repeats are located at the interface regions and that the active sites are far from each other and completely accessible to the substrate. Finally, a detailed inhibition study of CDCA1-R3 repeat in both cadmium- and zinc- bound form has been performed with sulfonamides and sulfamates derivatives. The results have been compared with those previously reported for other CA classes, namely alpha- and beta-classes, and correlated with the structural features of these enzymes.
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Human carbonic anhydrases (EC 4.2.1.1) IX (hCA IX) and XII (hCA XII) are two tumor-associated proteins, being overexpressed in many tumors and involved in critical processes associated with cancer progression and response to therapy. Both... more
Human carbonic anhydrases (EC 4.2.1.1) IX (hCA IX) and XII (hCA XII) are two tumor-associated proteins, being overexpressed in many tumors and involved in critical processes associated with cancer progression and response to therapy. Both are multi-domain proteins consisting of an extracellular catalytic domain (CA), a transmembrane portion (TM) and an intracytoplasmic (IC) segment. These domains have peculiar biochemical and physiological features. CA IX contains an additional proteoglycan-like (PG) domain at the N-terminus which constitutes a unique feature of this enzyme within the CA family. Starting from a brief description of the main molecular and catalytic features of both enzymes, their role in tumor physiology and their three-dimensional structure, this review describes the main classes of small molecule inhibitors, investigated between 2008 and 2013, able to inhibit these enzymes for both diagnostic and therapeutic applications. A consistent number of patents on molecules...
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A new supercritical extraction methodology was applied to extract azadirachtin A (AZA-A) from neem seed kernels. Supercritical and liquid carbon dioxide (CO(2)) were used as extractive agents in a three-separation-stage supercritical... more
A new supercritical extraction methodology was applied to extract azadirachtin A (AZA-A) from neem seed kernels. Supercritical and liquid carbon dioxide (CO(2)) were used as extractive agents in a three-separation-stage supercritical pilot plant. Subcritical conditions were tested too. Comparisons were carried out by calculating the efficiency of the pilot plant with respect to the milligrams per kilogram of seeds (ms/mo) of AZA-A extracted. The most convenient extraction was gained using an ms/mo ratio of 119 rather than 64. For supercritical extraction, a separation of cuticular waxes from oil was set up in the pilot plant. HPLC and electrospray mass spectroscopy were used to monitor the yield of AZA-A extraction.
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A rapid, sensitive and inexpensive HPLC method for routine screening of beauvericin, fusaproliferin, and enniatin B(1), A(1), and B has been optimized. Detection limits were determined, ranging between 0. 5 and 3.6 ng according to the... more
A rapid, sensitive and inexpensive HPLC method for routine screening of beauvericin, fusaproliferin, and enniatin B(1), A(1), and B has been optimized. Detection limits were determined, ranging between 0. 5 and 3.6 ng according to the compound obtained after spiking samples with each mycotoxin at 10-56 microg/mL concentration range; recoveries averaging from 56 to 74% were obtained. LC-MS conditions for enniatin analyses by API electrospray technique were set up, this allowing a unique identification of three different enniatins.
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Strains of certain plant pathogenic bacteria, in particular several pathovars of Pseudomonas syringae, are known to produce cyclic lipodepsipeptides (LDPs) endowed with peculiar structural features and noticeable biological activities. In... more
Strains of certain plant pathogenic bacteria, in particular several pathovars of Pseudomonas syringae, are known to produce cyclic lipodepsipeptides (LDPs) endowed with peculiar structural features and noticeable biological activities. In this study, a mass spectrometry procedure is proposed for screening LDP-producing bacterial strains and for identifying and assessing individual LDPs. After matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) screening of thirteen P. syringae strains for LDP production, the extracts from culture filtrates of eight positive strains were subjected to electrospray mass spectrometry for the identification of LDPs. Five strains were found to produce two forms of syringomycins (SR-E and SR-G) and two forms of syringopeptin 25 (SP25A and SP25B); two strains produced SR-E, SR-G and a new form of SP22; one strain produced syringotoxin (ST) and syringostatin A (SS-A) in addition to SP25A and SP25B. The yield in culture of two major LPDs: SR-G (3.2-13.8 mg x L(-1)) and SP25A (41.6-231.5 mg x L(-1)) was assessed by and high-performance liquid chromatography with electrospray mass spectrometry (HPLC/ESI-MS) in both scan and single ion monitoring (SIM) modes. Results of this investigation showed that the mass spectrometry protocol developed here is a precise and reliable method for screening bacterial strains for LDP production and for assessing the amount of each metabolite under various culture conditions. This could be of practical value in view of potential applications, e.g. biocontrol of post-harvest fungal diseases.
Research Interests: Earth Sciences, Mass Spectrometry, Biological Sciences, Pseudomonas, Time of Flight, and 7 moreCyclic peptides, High Performance Liquid Chromatography, High Pressure Liquid Chromatography, CHEMICAL SCIENCES, Liquid Chromatography / Electrospray Ionization Mass Spectrometry, Amino Acid Sequence, and Rapid
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Aqueous solutions of lactose and lysine were refluxed for up to 4 h without pH control. Samples were collected every hour, and the reaction was monitored by measuring the pH, the optical density at 420 nm, and the relative antioxidative... more
Aqueous solutions of lactose and lysine were refluxed for up to 4 h without pH control. Samples were collected every hour, and the reaction was monitored by measuring the pH, the optical density at 420 nm, and the relative antioxidative efficiency (RAE). The greatest change in optical density and antioxidative efficiency occurred for the mixture heated for 4 h. The 4 h solution was separated into three fractions according to the molecular weights of the components and tested for RAE. The high molecular weight fraction was more colored, and it had the highest antioxidative activity. The low molecular weight fraction was separated by high-performance liquid chromatography (HPLC). RAE values were measured for each purified compound. HPLC coupled with diode array and electrospray mass spectrometry allowed a rapid screening of the solutions and a tentative identification of several peaks. Nuclear magnetic resonance analysis allowed the identification of galactosylisomaltol and pyrraline. The resonance assignments for these compounds were revised.
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... Nevertheless, since several F. proliferatum strains could produce FB 1 , BEA, and FP (Logriecoet al., 1995; Moretti et al., 1996), and since these toxins occurred at significant level in several ... Macchia, L.; Di Paola, R.;... more
... Nevertheless, since several F. proliferatum strains could produce FB 1 , BEA, and FP (Logriecoet al., 1995; Moretti et al., 1996), and since these toxins occurred at significant level in several ... Macchia, L.; Di Paola, R.; Fornelli, F.; Nenna, S.; Moretti, A.; Napoletano, R.; Logrieco, A ...
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Fusaproliferin (FP) is a sesterterpene mycotoxin produced by some species of Fusarium, common contaminants of maize. Toxicity assays on Artemia salina L. brine shrimp larvae using FP and its derivatives showed that toxic activity of the... more
Fusaproliferin (FP) is a sesterterpene mycotoxin produced by some species of Fusarium, common contaminants of maize. Toxicity assays on Artemia salina L. brine shrimp larvae using FP and its derivatives showed that toxic activity of the metabolite increased after acetylation of the ...
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Carbonic anhydrase IX (CA IX) is a tumor associated protein, since it is highly expressed in a multitude of carcinomas, while it is present in a limited number of normal tissues. It is a multi-domain protein consisting of an N-terminal... more
Carbonic anhydrase IX (CA IX) is a tumor associated protein, since it is highly expressed in a multitude of carcinomas, while it is present in a limited number of normal tissues. It is a multi-domain protein consisting of an N-terminal proteoglycan-like (PG) domain, a catalytic domain, a trans-membrane portion (TM) and an intracytoplasmatic (IC) segment. These domains have peculiar biochemical and physiological features. Among these, only the PG domain is unique among the CA family. This review focuses on the most recent molecular and catalytic features uncovered of this enzyme, the role of its different domains in tumor physiology, and its three dimensional structure which has recently been solved. In addition, we present recent advances in the development of antibodies and small inhibiting molecules able to target CA IX for diagnostic and therapeutic applications.
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The X-ray crystal structure of the fluorescent antitumor sulfonamide carbonic anhydrase (CA, EC, 4.2.1.1) inhibitor (4-sulfamoylphenylethyl)thioureido fluorescein (1) in complex with the cytosolic isoform hCA II is reported, together with... more
The X-ray crystal structure of the fluorescent antitumor sulfonamide carbonic anhydrase (CA, EC, 4.2.1.1) inhibitor (4-sulfamoylphenylethyl)thioureido fluorescein (1) in complex with the cytosolic isoform hCA II is reported, together with a modeling study of the adduct of 1 with the tumor-associated isoform hCA IX. Its binding to hCA II is similar to that of other benzesulfonamides, with the ionized sulfonamide coordinated to the Zn2+ ion within the enzyme active site, and also participating in a network of hydrogen bonds with residues Thr199 and Glu106. The scaffold of 1 did not establish polar interactions within the enzyme active site but made hydrophobic contacts (<4.5 A) with Gln92, Val121, Phe131, Val135, Leu198, Thr199, Thr200, and Pro202. The substituted 3-carboxy-amino-phenyl functionality was at van der Waals distance from Phe131, Gly132, and Val135. The bulky tricyclic fluorescein moiety was located at the rim of the active site, on the protein surface, and strongly interacted with the alpha-helix formed by residues Asp130-Val135. All these interactions were preserved in the hCA IX-1 adduct, but the carbonyl moiety of the fluorescein tail of 1 participates in a strong hydrogen bond with the guanidine moiety of Arg130, an amino acid characteristic of the hCA IX active site. This may account for the roughly 2 times higher affinity of 1 for hCA IX over hCA II and may explain why in vivo the compound specifically accumulates only in hypoxic tumors overexpressing CA IX and not in the normal tissues. The compound is in clinical studies as an imaging tool for acute hypoxic tumors.