Aging and some pathological conditions are associated with the accumulation of altered (inactive or less active) forms of enzymes. It was suggested that these agerelated alterations ref lect spontaneous changes in protein conformation and͞or posttranslational modifications (e.g., oxidation). Because changes in protein conformations are often associated with changes in surface hydrophobicity, we have examined the effects of aging and oxygen radicaldependent oxidation on the hydrophobicity of rat liver proteins. As a measure of hydrophobicity, the increase in f luorescence associated with the binding of 8-anilino-1-naphthalene-sulfonic acid to hydrophobic regions on the proteins was used. By this criterion, the hydrophobicity of liver proteins of 24-month-old rats was 15% greater than that of 2-month-old animals. Exposure of liver proteins to a metal-catalyzed oxidation system (ascorbate͞Fe(II)͞H 2 O 2 ) or a peroxyl radical generating system, 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) led to increases of 2% or 30% in surface hydrophobicity, respectively. Treatment of liver proteins with the metal-catalyzed oxidation system led to a significant increase in reactive carbonyl content and to conversion of methionine residues to methionine sulfoxide residues. Treatment with AAPH led also to oxidation of methionine, tyrosine, and tryptophan residues and to the precipitation of some proteins. Dityrosine was detected in AAPH-treated protein, both the precipitate and supernatant fraction. The oxidation-dependent increase of hydrophobicity was correlated with an increase in the levels of methionine sulfoxide and dityrosine. These results suggest that oxidative modification of proteins may be responsible for the agerelated increase of protein surface hydrophobicity in vivo, and that the oxidation of methionine by an oxidative system may be an important event for the change of protein conformation.Covalent modification of proteins by oxidative systems have been implicated in various physiological and pathological conditions, such as aging, ischemia repurfusion and inflammatory disorders [for reviews, see Oliver et al. (1,2), and Stadtman (3, 4)]. These disorders are often associated with the accumulation of altered (inactive or less active) forms of various enzymes [for reviews, see Rothstein (5) and Oliver et al. (6)]. The age-related alterations may reflect spontaneous changes in protein conformations (7,8) or in protein oxidative modifications by reactive oxygen species (ROS) [for reviews, see Stadtman (9)]. The latter possibility is consistent with the facts that (i) many enzymes that accumulate as altered forms during aging are highly susceptible to modification by ROS; (ii) the ROS-mediated modifications of an enzyme can provoke changes in thermostability (10) and susceptibility to proteolytic degradation (10) similar to those that occur during aging (11); (iii) the level of oxidatively damaged protein, as measured by the presence of reactive carbonyl groups, increases with age (2). By whatever mechan...

Metal-catalyzed oxidation results in loss of function and structural alteration of proteins. The oxidative process affects a variety of side amino acid groups, some of which are converted to carbonyl compounds. Spectrophotometric measurement of these moieties, after their reaction with 2,4-dinitrophenylhydrazine, is a simple, accurate technique that has been widely used to reveal increased levels of protein carbonyls in aging and disease. We have initiated studies aimed at elucidating the chemical nature of protein carbonyls. Methods based on gas chromatography͞mass spectrometry with isotopic dilution were developed for the quantitation of glutamic and aminoadipic semialdehydes after their reduction to hydroxyaminovaleric and hydroxyaminocaproic acids. Analysis of model proteins oxidized in vitro by Cu 2؉ ͞ascorbate revealed that these two compounds constitute the majority of protein carbonyls generated. Glutamic and aminoadipic semialdehydes were also detected in rat liver proteins, where they constitute Ϸ60% of the total protein carbonyl value. Aminoadipic semialdehyde was also measured in protein extracts from HeLa cells, and its level increased as a consequence of oxidative stress to cell cultures. These results indicate that glutamic and aminoadipic semialdehydes are the main carbonyl products of metal-catalyzed oxidation of proteins, and that this reaction is a major route leading to the generation of protein carbonyls in biological samples.

The aim of this study was to survey the bacterial diversity of Amblyomma maculatum Koch, 1844, and characterize its infection with Rickettsia parkeri. Pyrosequencing of the bacterial 16S rRNA was used to determine the total bacterial population in A. maculatum. Pyrosequencing analysis identified Rickettsia in A. maculatum midguts, salivary glands, and saliva, which indicates successful trafficking in the arthropod vector. The identity of Rickettsia spp. was determined based on sequencing the rickettsial outer membrane protein A (rompA) gene. The sequence homology search revealed the presence of R. parkeri, Rickettsia amblyommii, and Rickettsia endosymbiont of A. maculatum in midgut tissues, whereas the only rickettsia detected in salivary glands was R. parkeri, suggesting it is unique in its ability to migrate from midgut to salivary glands, and colonize this tissue before dissemination to the host. Owing to its importance as an emerging infectious disease, the R. parkeri pathogen burden was quantified by a rompB-based quantitative polymerase chain reaction (qPCR) assay and the diagnostic effectiveness of using R. parkeri polyclonal antibodies in tick tissues was tested. Together, these data indicate that field-collected A. maculatum had a R. parkeri infection rate of 12–32%. This study provides an insight into the A. maculatum microbiome and confirms the presence of R. parkeri, which will serve as the basis for future tick and microbiome interaction studies.