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Abstract: The quantization of glycated isoforms of hemoglobin has been increasingly used in clinical practice in recent years. Glycated hemoglobin is currently considered the most important measurement for long-term control of the glycemic state and it has become a reference tool for the management of diabetes. Glutathionylated hemoglobin is an increasingly clinically relevant covalent adduct of glutathione with β chain of the globin and its concentration has been correlated with oxidative stress. We have developed an innovative technique based on linear mode matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for quantitative analysis of hemoglobin species. This method was applied to the quantification of glycated and glutathionylated hemoglobin. A rigorous comparison was pursued to evaluate the analytical performances in quantifying glycated hemoglobin in comparison to an established high-performance liquid chromatography method. Our results indicated a complete equivalence between the two methods. The same analysis enabled the quantitative determination of the glutathionylated hemoglobin fraction. This isoform was investigated in an adult Italian population (184 individuals, 101 males and 83 females), indicating a bimodal distribution of this species. In fact 65.22% of screened individuals had glutathionylated hemoglobin levels lower than 0.50% while 34.78% had glutathionylated hemoglobin levels higher than 0.50%. A semiautomatic robotic procedure was developed for fast analysis of a large number of samples. This is the first report of a quantitative application of linear MALDI-TOF mass spectrometry for the determination of glutathionylated hemoglobin in blood samples. This method allows fast screening of this hemoglobin isoform, therefore opening the route to explore its specificity and sensitivity as a molecular biomarker. [Copyright &y& Elsevier]

Abstract: Detergents are crucial to the isolation of integral membrane proteins. During membrane protein purification, it is useful to accurately quantify detergent, especially if concentration steps have been used. Previously, this has been difficult and time-consuming. We present a simple, rapid, and sensitive procedure for the quantification of glycosidic and bile salt-based detergents such as dodecylmaltoside, octylglucoside, and CHAPS. The method directly quantifies sugar or cholate moieties via colorimetric reactions with phenol and sulfuric acid. A number of detergents have been screened, and the assay has been validated in the presence of commonly used reagents. In addition to determining the overall detergent concentration in solution, the procedure allows accurate quantification of specific binding of glycosidic or bile salt-based detergents to purified membrane proteins. Both the colorimetric method and the radiometric 14C method were used to determine detergent binding to two integral membrane proteins: the cytochrome cbb3 oxidase from Pseudomonas stutzeri and the turkey β-adrenergic receptor. Both methods gave similar results. After separating monomeric glycosidic detergent from micellar solutions by ultrafiltration, we used the colorimetric method to determine the concentration of monomeric detergent present. We observed that values obtained are in close agreement with previously determined critical micelle concentrations. [Copyright &y& Elsevier]

The mitochondrial permeability transition, an established mechanism for heart diseases, is a long-standing mystery of mitochondrial biology and a prime drug target for cardioprotection. Several hypotheses about its molecular nature have been put forward over the years, and the prevailing view is that permeabilization of the inner mitochondrial membrane follows opening of a high-conductance channel, the permeability transition pore, which is also called mitochondrial megachannel or multiconductance channel. The permeability transition strictly requires matrix Ca2+ and is favored by the matrix protein cyclophilin D, which mediates the inhibitory effects of cyclosporin A. Here we provide a review of the field, with specific emphasis on the possible role of the adenine nucleotide translocator and of the F-ATP synthase in channel formation, and on currently available small molecule inhibitors. While the possible mechanisms through which the adenine nucleotide translocator and the F-ATP synthase might form high-conductance channels remain unknown, reconstitution experiments and site-directed mutagenesis combined to electrophysiology have provided important clues. The hypothesis that more than one protein may act as a permeability transition pore provides a reasonable explanation for current controversies in the field, and holds great promise for the solution of the mystery of the permeability transition. • The mitochondrial permeability transition (PT) plays a role in heart diseases. • The PT is mediated by inner membrane high-conductance channel(s). • We discuss channel formation by adenine nucleotide translocator and F-ATP synthase. • We review PT inhibitors and their potential use in cardioprotection. [ABSTRACT FROM AUTHOR]

The structural gene for glutathione transferase (XlGSTS1-1) in the amphibia Xenopus laevis has been cloned from an embryo library and its nucleotide sequence has been determined. Open reading frame analysis indicated that xlgsts1 gene encodes the smallest protein of sigma class GST so far identified as being composed of only 194 amino acid residues. The recombinant XlGSTS1-1 shows a narrow range of substrate specificity as well as a significantly lower 1-chloro-2,4-dinitrobenzene conjugation capacity than that of squid sigma class GST. To compare the structural and functional differences between the squid and amphibian enzymes, several site-specific mutations were introduced in XlGSTS1-1, i.e., Ser100Asn, Phe102Tyr, Trp143Leu, Phe146Leu, and Trp148Cys. The results obtained indicate that Trp143 and Trp148 are more important determinants for the structural stability of XlGSTS1-1 rather than for its substrate specificity. [Copyright &y& Elsevier]

Over the years, the potential pathogenicity of Acanthamoeba for humans and animals has gained increasing attention from the scientific community. More than 24 species belong to this genus, however only some of them are causative agents of keratitis and encephalitis in humans. Due to technical difficulties in diagnosis, these infections are likely to be under-detected. The introduction of 18S rDNA amplification for the identification of Acanthamoeba has dramatically enhanced diagnosis performances, but the attestation of genotyping requires supplementary sequencing-based procedures. In this study, 15 Acanthamoeba strains were collected and grown on nutrient agar media. Each strain was genotyped by end-point PCR assay for the amplification of the 18S rDNA gene and the genotype was assigned by sequencing analysis through neighbor joining phylogenetic tree. In order to optimize standardization of the MALDI-TOF MS assay, we established the collection time point at the cystic phase. Two strains of each genotype were randomly chosen to customize the biotyper database. For all strains, 24 spectral measurements were acquired and submitted to identification and cluster analysis of spectra. The obtained results highlighted the correct identification of Acanthamoeba strains and the overlapping of spectra dendrogram clusters to the 18S genotype assignations. In conclusion, the MALDI-TOF MS Biotyper revealed the capability to identify and genotype the Acanthamoeba strains, providing a new frontier in the diagnostic identification of amaebae and in taxonomic and phylogenetic studies. [ABSTRACT FROM AUTHOR]

Abstract: Tobacco smoke exposure is the cause of exaggerated inflammatory responses and tissue destruction leading to chronic bronchitis and emphysema. A number of studies have used biochemical and immunological technologies to identify biomarkers of severity, risk and pharmacological target of disease. Recently, genomic and proteomic studies have been carried out to explore tobacco smoke-induced lung damage mechanisms. Eight of these studies, including 81 healthy non-smokers, 138 healthy smokers and 24 smokers with COPD, had open platform generated data available online and were reviewed in order to identify markers of smoke-induced damage by analyzing differential gene and protein expression in healthy individuals exposed to tobacco smoke in comparison with chronic obstructive pulmonary disease (COPD) smokers and healthy non-smokers. To this end the Ingenuity Pathways Analysis, a web-based application enables identifying the main biological functions and pathways, was used. The pathway most significantly associated with healthy smokers was the Nrf2-mediated Oxidative Stress Response (p-value < 0.01): out of the 22 genes/proteins identified in healthy smokers, 19 were up-regulated and three down-regulated, compared to non-smokers. Interestingly, four genes/proteins of the same pathway were differentially regulated in COPD, one up-regulated and three down-regulated, compared to healthy smokers. Moreover, in the comparison between COPD and healthy smokers, our analysis showed that the most relevant pathway was the Mitochondrial Dysfunction (p-value < 0.01) with 12 differentially regulated genes/proteins. This data-mining review supports the notion that Nrf2-regulated anti-oxidant genes play a central role in protection against tobacco smoke toxic effects and may be amenable to use as COPD risk biomarkers. Furthermore, this review suggests that mitochondrial dysfunction may be involved in the development of COPD. [ABSTRACT FROM AUTHOR]

Abstract: Anti-oxidants are paradoxically much lower in plasma than inside cells even blood is comparably exposed to the oxidative stress. ‘In vitro’ models suggest a critical role of albumin as substitutive anti-oxidant in plasma but no proof for this role is available ‘in vivo.’ Herein, we demonstrate by LC/MS/MS that plasma albumin undergoes massive oxidation in primary nephrotic syndrome, involving stable sulphonation of the free SH of Cys 34 with +48Da increase in exact mass of the protein (ESI-MS) and formation of a fast moving isoform in the pH range between 5 and 7. Physical–chemical experiments with DSC and fluorescence spectra indicate a thermal stabilization of the structure upon oxidation. This is the first demonstration of massive oxidation of albumin ‘in vivo’ that reflects a functional role of the protein. Free radicals should be implicated in the pathogenesis of proteinuria in human FSGS. [Copyright &y& Elsevier]