C. Di Ilio

Antileukotriene Drugs: Clinical Application, Effectiveness and Safety

Cysteinyl leukotrienes (Cys-LTs) are potent proinflammatory mediators derived from arachidonic acid through the 5-lypoxigenase (5-LO) pathway. They exert important pharmacological effects by interaction with at least two different receptors: Cys-LT(1) and Cys-LT(2). By competitive binding to the Cys-LT(1) receptor, leukotriene receptor antagonist drugs such as montelukast, zafirlukast, and pranlukast, block the effects of Cys-LTs and alleviate the symptoms of many chronic diseases, especially bronchial asthma and allergic rhinitis. Evidence obtained by randomized clinical trials as also by direct experience derived from patients suffering from asthma and allergic rhinitis justifies a broader role for leukotrienes receptor antagonists (LTRAs). Recently published studies and case reports have demonstrated beneficial effects of LTRAs on other diseases commonly associated with asthma (exercise induced asthma, rhinitis, chronic obstructive pulmonary disease, interstitial lung disease, chronic urticaria, atopic dermatitis, allergic fungal disease, nasal polyposis, and paranasal sinus disease) as well as other diseases not connected to asthma (migraine, respiratory syncytial virus postbronchiolitis, systemic mastocytosis, cystic fibrosis, pancreatitis, vulvovaginal candidiasis, cancer, atherosclerosis, eosinophils cystitis, otitis media, capsular contracture, and eosinophilic gastrointestinal disorders). The aim of this review is to show the most recent applications and effectiveness in clinical practice of the LTRAs.

Reversible inactivation of tyrosine amino transferase from guinea pig liver by thiol and disulfide compounds

Abstract Tyrosine aminotransferase from guinea pig liver is strongly inactivated by a variety of natural thiols and disulfides. L-cysteine was used as a model compound in the study of inactivation. Inactivation is due to the disulfide produced by spontaneous oxidation of thiol during incubation. Binding studies with [ 35 S]-cysteine revealed simultaneous incorporation of [ 35 S] into tyrosine aminotransferase and loss of enzyme activity. The reversibility demonstrates that the inactivation is the result of the formation of mixed disulfide between the disulfide and the sulfhydryl group of tyrosine aminotransferase. Some features of the enzyme active site are showed by the inactivation reaction.

Evaluation of the role of two conserved active-site residues in beta class glutathione S-transferases.

Glutathione S-transferases (GSTs) normally use hydroxy-group-containing residues in the N-terminal domain of the enzyme for stabilizing the activated form of the co-substrate, glutathione. However, previous mutagenesis studies have shown that this is not true for Beta class GSTs and thus the origin of the stabilization remains a mystery. The recently determined crystal structure of Proteus mirabilis GST B1-1 (PmGST B1-1) suggested that the stabilizing role might be fulfilled in Beta class GSTs by one or more residues in the C-terminal domain of the enzyme. To test this hypothesis we mutated His(106) and Lys(107) of PmGST B1-1 to investigate their possible role in the enzymes catalytic activity. His(106) was mutated to Ala, Asn and Phe, and Lys(107) to Ala and Arg. The effects of the replacement on the activity, thermal stability and antibiotic-binding capacity of the enzyme were examined. The results are consistent with the involvement of His(106) and Lys(107) in interacting with glutathione at the active site but these residues do not contribute significantly to catalysis, folding or antibiotic binding.

Isoelectric focusing of brain cortex GSH S-transferase activity in mammals: Evidence that polymorphism is absent in man

Specific activities of GSH S-transferase toward different model substrates were determined in the cytosol prepared from rat, guinea pig, rabbit, mouse, sheep, beef, pig and human brain cortex. The GSH S-transferase composition of the eight mammalian brain cortices was studied by using density gradient isoelectric focusing technique. Human brain cortex GSH S-transferase was resolved into a single peak of activity centered at pH 4.6, whereas the supernatants of all other mammals consisted of more than one enzymatic form. The kinetic properties of all forms isolated were compared.

Activities of enzymes associated with the metabolism of glutathione in fetal rat liver and placenta.

Glutathione S-transferase, glutathione peroxidase, glutathione reductase and gamma-glutamylcysteine synthetase activities were measured in fetal rat liver and placenta supernatant at the 16th and 20th days of pregnancy. Compared with adult liver, low activities were found in both fetal liver and placenta. Both selenium-dependent and selenium-independent glutathione peroxidase activities were present in fetal liver, but only the selenium-dependent activity augmented as parturition advanced. Selenium-independent glutathione peroxidase was found to be absent in placenta. The progress of gestation is accompanied by a significant increase in conjugating capacity toward 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene and a significant decrease toward 1,2-epoxy-3-(p-nitrophenoxy)propane in fetal liver. Glutathione S-transferase activity in rat placenta diminished from day 15 to day 20 of gestation. The elevation of enzymatic activities involved in the synthesis and recovery of glutathione, which takes place in fetal liver and placenta, was thought to be adaptively responsive to the changes that occurred in glutathione-consuming enzymes.

Glutathione-related enzyme activities and lipoperoxide levels in human internal mammary artery and ascending aorta. Relations with serum lipids.

The relation among glutathione-related enzyme activities, thiobarbituric acid-reactive substances of the human aorta and internal mammary artery, and serum lipids was studied in 40 male patients undergoing coronary revascularization. Glutathione peroxidase and glutathione reductase activities were significantly higher in the internal mammary artery, whereas glutathione transferase activity was elevated in the aortic wall. Moreover, non-selenium-dependent glutathione peroxidase activity was detectable only in the aorta. The levels of thiobarbituric acid-reactive substances were significantly higher in the aorta. A positive correlation was found among the activity of glutathione peroxidase, glutathione reductase, and thiobarbituric acid-reactive substances in the internal mammary artery and total cholesterol, low density lipoprotein cholesterol, and triglycerides. In the aortic wall, a positive correlation among the activity of glutathione peroxidase, glutathione transferase, thiobarbituric acid-reactive substances, and the previously mentioned serum lipids was evident. In contrast, high density lipoprotein cholesterol was inversely related to enzymatic activities and thiobarbituric acid-reactive substances in both the internal mammary artery and aorta. In conclusion, significant differences in the levels of glutathione-related enzyme activities and thiobarbituric acid-reactive substances in the internal mammary artery and aorta were found, suggesting a different ability of the two tissues to counteract oxidative stress: the glutathione-related antioxidant properties and the level of lipid peroxidation in the arterial tissue seem to be specifically influenced by serum lipids.

Potentiometric Determination of Glutathione and Glutathione Transferase Activity

Abstract A new method of determination of Glutathione Transferase activity (GST) and Glutathione (GSH) in solution has been developed. The determination was performed by detection of ions (H+, Cl−, F−) produced during the GST catalyzed reaction using ion-selective electrodes. The Michaelis constants of GST π from human placenta toward GSH, 1-Chloro-2, 4-dinitrobenzene (CDNB) and 1-Fluoro-2, 4-dinitrobenzene (FDNB) obtained potentiometrically were similar to those calculated with the classical spectrophotometric method. The use of pH electrode gave the best results for GST activity (1 pH unit slope per 0.1 unit of enzyme in 1 ml of solution; detection limit of 0.001 unit/ml). Different purification steps of GST TI from human placenta followed by this method gave results comparable to the spectrophotometric one. The best calibration curve for GSH (2 × M detection limit) was obtained following the production of F- ions with a fixed amount of enzyme at a concentration of 0.2 u/ml. A calibration obtained perfo...

Glutathione-related enzyme activities in pregnant rat liver.

The levels of GSH-related enzyme activities during pregnancy were determined. A significant increase in Selenium-dependent GSH peroxidase and GSH S-transferase E activity was observed. A concomitant increase in γ-glutamylcysteine synthetase was measured, which indicated an increased ability to synthesize the tripeptide.

Characterization of glutathione transferase from Gammarus italicus

1. By using affinity chromatography and chromatofocusing analysis at least two major glutathione transferases, named GST II and GST III can be isolated from Gammarus italicus. 2. GST II has an isoelectric point at pH 5.0 and is composed of two subunits with an apparent molecular mass of 28 KDa. 3. GST III which has an isoelectric point at pH 4.6 was found to be an heterodimer of 27 KDa and 28 KDa. 4. The 28 KDa subunit cross-reacted in immunoblotting analysis with antisera raised against pi class GST, whereas none of the antisera raised against alpha, mu and pi class GSTs cross-reacted with the 27 KDa subunit.

Glutathione transferase activity during Bufo bufo development

High levels of glutathione transferase activity were measured during the development of the embryos of Bufo bufo including unfertilized eggs. After stage 4 glutathione transferase activity gradually decreased until stage 25 when the minimum was reached. No change in the number of isozymes was noted during development according to isoelectric focusing analysis performed on the cytosolic fractions of selected stages.