Carbocysteine lysine salt monohydrate (SCMC-LYS) is a selective scavenger of reactive oxygen intermediates (ROIs)

Carbocysteine lysine salt monohydrate (SCMC-Lys) is a well-known mucoactive drug whose therapeutic efficacy is commonly related to the ability of SCMC-Lys to replace fucomucins by sialomucins. The aim of this study was to determine if SCMC-Lys could exert an anti-oxidant action by scavenging reactive oxygen intermediates (ROIs). Our results show that SCMC-Lys proved effective as a selective scavenger of hypochlorous acid (HOCl) and hydroxyl radical (OH.), this effect being related to the reactivity of the SCMC tioether group. The scavenger activity of SCMC-Lys was observed in free cellular system as well as in activated human polymorphonuclear neutrophils (PMNs). SCMC-Lys scavenger activity on HOCl was paralleled by a powerful protection from HOCl-mediated inactivation of alpha1-antitripsin (alpha1-AT) inhibitor, the main serum protease inhibitor. Production of interleukin-(IL-)8, a major mediator of PMN recruitment in inflammatory diseases, is known to be mediated by intracellular OH. SCMC-Lys significantly reduced IL-8 production on stimulated human peripheral blood mononuclear cells (PBMCs) in the same range of concentrations affecting OH. activity. It is concluded that SCMC-Lys could exert, in addition to its mucoactive capacity, an anti-oxidant action, thus contributing to the therapeutic efficacy of SCMC-Lys.     

Effect of metabolic syndrome risk factors and MMP-2 genetic variations on circulating MMP-2 levels in childhood obesity

Matrix metalloproteinase-2 is involved in the development of the adipose tissue, and associated with cardiovascular diseases. Metabolic risk factors (MRFs) and functional polymorphisms in the MMP-2 gene may affect its expression and activity. We investigated whether traditional MRFs and two MMP-2 gene polymorphisms (C^?1306T; rs243865, and C^?735T; rs2285053) affect circulating MMP-2 levels in children and adolescents, and whether MMP-2 polymorphisms and/or haplotype are associated with susceptibility to childhood obesity. We studied 114 healthy controls, 43 obese, and 83 obese with ≥3 MRFs children and adolescents. Genotypes were determined by Taqman allele discrimination assay and real-time PCR. Plasma MMP-2 was measured using zymography. We found positive correlations between MMP-2 concentrations and mean blood pressure in all children and adolescents group (r = 0.132; P < 0.05) and in obese children and adolescents (r = 0.247; P < 0.01). We found that the CC genotype for the C^?1306T polymorphism was more common in subjects with higher MMP-2 concentrations in controls (P = 0.003) and in the obese group (P = 0.013). The CT genotype (OR = 0.40; P < 0.01) and the T allele (OR = 0.48; P < 0.01) for the C^?735T polymorphism were less common in obese children and adolescents than in controls. The haplotypes distribution did not show significant differences between control and obese (P > 0.05). Ours findings show that blood pressure is associated with circulating MMP-2 concentrations, and that the CC genotype for the C^?1306T polymorphism was more common subjects (controls and obese) with higher MMP-2 concentrations, whereas the CT genotype and the T allele for the C^?735T polymorphism are less common in obesity.     

Synthesis and biological activity of tachykinin analogs containing the adamantane moiety

Summary The adamantane moiety was introduced in the tachykinin NK₂ receptor-selective agonist [-Ala⁸]-NKA(4–10) (H-Asp-Ser-Phe-Val--Ala-Leu-Met-NH₂, MEN 10210) and in different positions of the NK₂ receptor antagonist MEN 10376 (H-Asp-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys-NH₂) in order to investigate how this substitution affects their biological activity at tachykinin NK₁, NK₂ and NK₃ receptors. 1-Adamantaneacetic acid (1-Ada-CH₂COOH) was directly conjugated in the solid phase as the preformed OBt active ester to the N-terminal position of MEN 10210, obtaining MEN 10586 (1-Ada-CH₂CO-Asp-Ser-Phe-Val--Ala-Leu-Met-NH₂). The Pfp ester of adamantaneacetic acid (1) was prepared and used for the acylation of the N-terminal position of MEN 10376, yielding MEN 10606 (1-Ada-CH₂CO-Asp-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys-NH₂). Compound 1 was then used to obtain the building block Fmoc-Lys(1-Ada-CH₂CO)-OH as a modified amino acid for the synthesis of MEN 10818 [H-Asp-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys(1-Ada-CH₂CO)-NH₂]. In order to investigate the biological activity of the peptide bearing the adamantane group together with the free N-terminal amino function, we synthesised MEN 10676 [H-Asp(O-2-Ada)-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys-NH₂] using Fmoc-Asp(O-2-Ada)-OH, in which 2-adamantanole was the protecting group of the aspartate -COOH moiety during the peptide synthesis and survived the final peptide cleavage and deprotection carried out under controlled conditions. MEN 10586 showed an agonist activity comparable to that of the parent compound MEN 10210 at NK₁ and NK₂ receptors of guinea pig ileum, rabbit isolated pulmonary artery and hamster isolated trachea preparations, while it showed a 25-fold higher agonist activity at NK₃ receptors of rat isolated portal vein. The three modified antagonist analogs displayed similar or reduced affinity at NK₁, NK₂ and NK₃ receptors as compared to MEN 10376. The drop was particularly evident (>2 log units) at the NK₂ receptors of the rabbit isolated pulmonay artery.     

Two different Xylella fastidiosa strains circulating in Italy: phylogenetic and evolutionary analyses

Xylella fastidiosa, a bacterial species infecting a broad range plants, includes five subspecies, fastidiosa, multiplex, pauca, mulberry and sandyi. In Europe, Xylella was isolated in olive trees in southern Italy (Apulia region) during the year 2013. The aim of the present study was to apply phylogenetic and evolutionary analysis to trace the possible origin and way of the entrance of Xylella fastidiosa in Italy. All the genomes available for Xylella fastidiosa spp were downloaded from NCBI. A phylogeographic analysis was performed using BEAST. X. fastidiosa strains belonging to X. fastidiosa subsp. pauca and subsp. sandyi have been reported to infect olive trees and coffee plants, respectively. The phylogeographic analysis also revealed and confirmed these two different ways of provenience X. fastidiosa subsp. pauca from Costa Rica and X. fastidiosa subsp sandyi from California Phylogeny have been an important tool to validate and support the recent hypothesis for X. fastidiosa pauca provenience.     

Matrix metalloproteinase (MMP)-2 gene polymorphisms affect circulating MMP-2 levels in patients with migraine with aura

Matrix metalloproteinases (MMP) are involved in the disruption of blood–brain barrier (BBB) during migraine attacks. In the present study, we hypothesized that two functional polymorphisms (C^− 1306T and C^− 735T) in MMP-2 gene and MMP-2 haplotypes are associated with migraine and modify MMP-2 and tissue inhibitor of MMP (TIMP)-2 levels in migraine. Genotypes for MMP-2 polymorphisms were determined by real time-PCR using Taqman allele discrimination assays. Haplotypes were inferred using the PHASE program. Plasma MMP-2 and TIMP-2 concentrations were measured by gelatin zymography and ELISA, respectively, in 148 healthy women without history of migraine and in 204 women with migraine (153 without aura; MWA, and 51 with aura; MA). Patients with MA had higher plasma MMP-2 concentrations and MMP-2/TIMP-2 ratios than patients with MWA and controls (P < 0.05). While MMP-2 genotype and haplotype distributions for the polymorphisms were similar among the groups (P > 0.05), we found that the CC genotype for C^− 735T polymorphism and the CC haplotype were associated with higher plasma MMP-2 concentrations in MA group (P < 0.05). Our findings may help to understand the role of MMP-2 and its genetic variants in the pathophysiology of migraine and to identify a particular group of migraine patients with increased MMP-2 levels that would benefit from the use of MMP inhibitors.     

Chiasma Repatterning Across a Chromosomal Hybrid Zone Between Chromosomal Races of Mus Musculus Domesticus

Chiasma number and distribution were analysed in male house mice from a karyotypic hybrid zone between the CD race (2n=22) and the standard race (2n=40) located in central Italy. Chiasma repatterning occurs across the transect. The overall trend produces a diminution of chiasmata in the mice with CD chromosomal background. The progressive reduction of chiasmata indicates that genes could pass from one race to another in an asymmetrical way: from metacentric races to the standard population.     

Monitoring of low density lipoprotein oxidation by low-level chemiluminescence

A method for monitoring low-density lipoprotein (LDL) oxidation by low-level chemiluminescence (LL-CL) is described in this study. The kinetic indices obtained with this procedure, in particular lag-time and K value (related to prooxidant activity of Cu²⁺ bound to LDL) are compared with those of the established UV-absorbing conjugated diene assay. The correlation of lag-time values obtained by LL-CL and conjugated diene assay was very high both in the case of Cu²⁺- and peroxyl-radical-mediated oxidation (r = 0.99). By using the transient free radical scavenging activity of butylated hydroxytoluene, a calibration of LL-CL for lipid peroxyl radical and termination rate was obtained. The spectral analysis of LL-CL from oxidizing LDL shows a maximum peak between 420 and 500 nm, corresponding to the emission of triplet carbonyl compounds. LL-CL allows continuous and direct monitoring of LDL oxidation as extraction and derivatization of lipid peroxidation products are not required. Moreover, some limitations of UV spectroscopy such as by absorbing compounds need not be considered. Therefore, the present procedure represents a simple and convenient tool for continuous monitoring of LDL oxidation which may be applied to mechanistic and clinical studies.     

Inhibition of Francisella tularensis LVS infection of macrophages results in a reduced inflammatory response: evaluation of a therapeutic strategy for intracellular bacteria

Francisella tularensis is an intracellular pathogen and is able to invade several different cell types, in particular macrophages, most commonly through phagocytosis. A flow cytometric assay was developed to measure bacterial uptake, using a fluorescein isothiocyanate-labelled anti-F. tularensis lipopolysaccharide antibody in conjunction with antibodies to cell surface markers, in order to determine the specific cell phenotypes that were positive for the bacteria. Several phagocytic inhibitors were evaluated in macrophage cell lines and a lung homogenate assay to determine whether the uptake of F. tularensis strain LVS could be altered. Our data show that cytochalasin B, LY294002, wortmannin, nocodazole, MG132 and XVA143 inhibitors reduced LVS uptake by >50% in these assays without having significant cytotoxic effects. Furthermore, a reduction in the inflammatory cytokines monocyte chemoattractant protein-1, interleukin-6 and tumour necrosis factor-α was found in the supernatant of lung tissue infected with LVS when the inhibitory compounds were present. Similarly, there was an alteration in bacterial uptake and a reduction in the inflammatory cytokine response following the administration of wortmannin to LVS-infected mice. Although wortmannin treatment alone did not correlate with the enhanced survival of LVS-infected mice, these inhibitors may have utility in combination therapeutic approaches or against other intracellular pathogens that use phagocytic mechanisms to enter their optimal niche.