X-ray crystallographic and kinetic investigations of 6-sulfamoyl-saccharin as a carbonic anhydrase inhibitor.

6-Sulfamoyl-saccharin was investigated as an inhibitor of 11 α-carbonic anhydrase (CA, EC 4.2.1.1) isoforms of human (h) origin, hCA I-XIV, and X-ray crystallographic data were obtained for its adduct with hCA II, the physiologically dominant isoform. This compound possesses two potential zinc-binding groups, the primary sulfamoyl one and the secondary, acylatedsulfonamide. Saccharin itself binds to the Zn(II) ion from the CA active site coordinating with this last group, in deprotonated (SO2N(-)CO) form. Here we explain why 6-sulfamoyl-saccharin, unlike saccharin, binds to the metal ion from the hCA II active site by its primary sulfonamide moiety and not the secondary one as saccharin itself. Our study is useful for shedding new light to the structure-based drug design of isoform-selective CA inhibitors of the sulfonamide type.

Carbonic anhydrase IX as a target for designing novel anticancer drugs.

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.

Recent advances in research on the most novel carbonic anhydrases, CA XIII and XV.

The carbonic anhydrase (CA) enzyme family consists of thirteen active isozymes in mammals. The most recently characterized members of this family are cytosolic CA XIII and membrane-bound CA XV. This article describes recent advances in the CA family, especially CA XIII and XV. We have also included catalytic activity data on human CA XIII and mouse CA XV. Additionally, the inhibition constants of acetazolamide toward these isozymes were determined to be k(cat) = 1.5 x 10(5) s(-1), k(cat)/K(M) = 1.1 x 10(7) M(-1) s(-1) and K(I) = 16 nM for human CA XIII and k(cat) = 4.7 x 10(5) s(-1), k(cat)/K(M) = 3.3 x 10(7) M(-1) s(-1) and K(I) = 72 nM for mouse CA XV. Although the activity of CA XIII is the second lowest reported thus far for any of the human CAs, it may have a role in maintaining the acid-base balance in the kidney and the gastrointestinal and reproductive tracts. CA XV is an exceptional enzyme, as it seems to be active in numerous species, such as rodents, birds and fish, but is absent from humans and chimpanzees. Mouse CA XV is a moderately active enzyme, suggesting that it may play a physiological role at least in the kidney. It is likely that other isozymes have substituted for this protein in humans. In addition to the novel data on CA XIII and XV, we present the catalytic activities as well as inhibition constants of acetazolamide for all mammalian CA isozymes in this review.

Characterization of phenolic compounds in virgin olive oil and their effect on the formation of carcinogenic/mutagenic heterocyclic amines in a model system.

Mutagenic heterocyclic amines (HAs) are formed at low levels during cooking of meat and fish, and some of them are considered to be possible human carcinogens. The formation of HAs may be affected by the presence of synthetic or naturally occurring antioxidants. In the present study the effect of virgin olive oil (VOO) phenolic compounds, identified and quantified by LC-MS, on the formation of HAs in a model system was evaluated. An aqueous solution of creatinine, glucose, and glycine was heated in the presence of two samples of VOO differing only in the composition of phenolic compounds. The addition of VOO to the model system inhibited the formation of 2-amino-3-methylimidazo[4,5-f]quinoxaline (IQx), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx) by between 30 and 50% compared with the control. Fresh-made olive oil, which contained a high amount of dihydroxyphenylethanol derivatives, inhibited HA formation more than a 1-year-old oil did. The inhibition of HA formation was also verified using phenolic compounds extracted from VOO.

Analysis of bacterial lipodepsipeptides by matrix-assisted laser desorption/ionisation time-of-flight and high-performance liquid chromatography with electrospray mass spectrometry.

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.

Simultaneous determination of beauvericin, enniatins, and fusaproliferin by high performance liquid chromatography.

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.

A comparison of color formation and maillard reaction products of a lactose-lysine and lactose-N(alpha)-acetyllysine model system.

The formation of color and Maillard reaction products in two model systems consisting of lactose and lysine or N(alpha)-acetyllysine has been investigated. During heating, the blockage of the N(alpha) group of lysine determined a faster color and antioxidative ability development compared to the system with free lysine. This is combined to a greater amount of melanoidin formation in the acetylated lysine system, while in the free lysine system a higher amount of pyrraline and hydroxymethyl furfural were detected. The pattern of low molecular weight products suggests that 3-deoxyglucosone and 1-deoxyglucosone degradation pathways are favored for free lysine and N(alpha)-acetyllysine, respectively. Whole data allow us to hypothesize that in a lactose-N(alpha)-acetyllysine model system the formation of colored high molecular weight polymer proceeds faster because less material is dispersed in reaction pathways, mainly the Strecker degradation, which leads to small and intermediate molecular weight products.

Convenient synthesis of lactuloselysine and its use for LC-MS analysis in milk-like model systems.

The synthesis of the Amadori product lactuloselysine [N(epsilon)-(1-deoxy-D-lactulosyl-1)-L-lysine] was obtained starting from FMOC-lysine-OH (N(alpha)-9-fluorenylmethoxy-carbonyl-N(epsilon)H(2)-L-lysine-OH) and lactose. Compound identity was confirmed by MALDI-ToF, electrospray, and NMR analysis. A selective LC-MS procedure which allowed the detection of lactuloselysine up to 10 ng mL(-)(1) was set up and used to follow the formation of the compound in a lactose-lysine model system; quantification of this molecule after complete enzymatic hydrolysis of whey-proteins from milk samples was also performed.

Extraction of azadirachtin A from neem seed kernels by supercritical fluid and its evaluation by HPLC and LC/MS.

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.

LC/MS analysis and antioxidative efficiency of Maillard reaction products from a lactose-lysine model system.

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.

Polyclonal antibodies against fusaproliferin.

Fusaproliferin (FP), a toxic metabolite of the world-wide maize pathogens Fusarium proliferatum and Fusarium subglutinans, was recently found to be a natural contaminant of maize. Its toxic activity on haematopoietic human cell lines and its teratogenic effects on chicken embryos has been recently proved. Therefore a sensitive, rapid, and inexpensive screening test to detect FP in agricultural commodities is necessary to protect human health. FP-hemiglutarate conjugated to modified bovine serum albumin was synthesized, characterized, and used as an antigen for raising polyclonal antibodies by immunizing rabbits. Indirect and competitive ELISA and immunoblotting analyses were performed to determine antibody specificity towards the mycotoxin. The determination of 10 micrograms of free FP/mL was achieved using antibodies purified by means of affinity chromatography on a FP-lysine-Sepharose column. This unsatisfactory detection limit is due to high background values; thus, this method is not competitive with traditional UV-HPLC methods.

Identification of a beta-lactoglobulin lactosylation site.

Thermal treatment of milk leads to non-enzymatic glycosylation of proteins through Maillard reaction. Free NH2 groups of basic amino acids react with the reducing carbonyl group of lactose forming the so-called Amadori products. Electrospray mass spectrometry analysis shows that beta-lactoglobulin (beta-LG), the major whey protein, undergoes lactosylation under industrial thermal treatment. In order to investigate the specificity of reactive sites for lactose binding the analysis of trypsin hydrolysates of beta-LG isolated from different industrial milks was performed. Results demonstrate that Lys-100 is a preferential lactosylation site of beta-LG during industrial milk treatment. These results were confirmed by an analysis of the three-dimensional model of the protein which showed that Lys-100 had the highest solvent accessibility and proximity to another amino group making Lys-100 the best candidate to lactosylation. Lys-47, previously identified by other authors, showed a good proximity to another Lys residue, but an intermediate level of exposition to solvent.