Oxidized bases in the ribosome's peptidyl transferase center and their effects on translation

The ribosome is central to protein biosynthesis and the focus of extensive research. Recent biochemical and structural studies, especially detailed crystal structures and high resolution Cryo-EM in different functional states have broadened our understanding of the ribosome and its mode of action. However, the exact mechanism of peptide bond formation and how the ribosome catalyzes this reaction is not yet understood. Also, consequences of direct oxidative stress to the ribosome and its effects on translation have not been studied. So far, no conventional replacement or even removal of the peptidyl transferase center's bases has been able to affect in vitro translation. Significant contribution to the catalytic activity seems to stem from the ribose-phosphate backbone, specifically 2'OH of A2451. Using the technique of atomic mutagenesis, novel unnatural bases can be introduced to any desired position in the 23S rRNA, surpassing conventional mutagenesis and effectively enabling to alter single atoms in the ribosome. Reconstituting ribosomes in vitro using this approach, we replaced universally conserved PTC bases with synthetic counterparts carrying the most common oxidations 8-oxorA, 5-HOrU and 5-HOrC. To investigate the consequent effects on translation, the chemically engineered ribosomes were studied the in various functional assays. Incorporation of different oxidized bases into the 70S ribosome affected the ribosomes in different ways. Depending on the nucleobase modified, the reconstituted ribosomes exhibited radical deceleration of peptide bond formation, decrease of synthesis efficiency or even an increase of translation rate. These results may further our understanding of the residues involved in the peptide bond formation mechanism, as well as the disease-relevant effects of oxydative stress on the translation machinery.

Oxidized bases in the ribosome's peptidyl transferase center and their effects on translation

The ribosome is central to protein biosynthesis and the focus of extensive research. Recent biochemical and structural studies, especially detailed crystal structures and high resolution Cryo-EM in different functional states have broadened our understanding of the ribosome and its mode of action. However, the exact mechanism of peptide bond formation and how the ribosome catalyzes this reaction is not yet understood. Also, consequences of direct oxidative stress to the ribosome and its effects on translation have not been studied. So far, no conventional replacement or even removal of the peptidyl transferase center's bases has been able to affect in vitro translation. Significant contribution to the catalytic activity seems to stem from the ribose-phosphate backbone, specifically 2'OH of A2451. Using the technique of atomic mutagenesis, novel unnatural bases can be introduced to any desired position in the 23S rRNA, surpassing conventional mutagenesis and effectively enabling to alter single atoms in the ribosome. Reconstituting ribosomes in vitro using this approach, we replaced universally conserved PTC bases with synthetic counterparts carrying the most common oxidations 8-oxorA, 5-HOrU and 5-HOrC. To investigate the consequent effects on translation, the chemically engineered ribosomes were studied the in various functional assays. Incorporation of different oxidized bases into the 70S ribosome affected the ribosomes in different ways. Depending on the nucleobase modified, the reconstituted ribosomes exhibited radical deceleration of peptide bond formation, decrease of synthesis efficiency or even an increase of translation rate. These results may further our understanding of the residues involved in the peptide bond formation mechanism, as well as the disease-relevant effects of oxydative stress on the translation machinery.

Circulating oxidized low-density lipoprotein (LDL) is associated with risk factors of the metabolic syndrome and LDL size in clinically healthy 58-year-old men (AIR study).

OBJECTIVES Hypothetically the atherogenic effect of the metabolic syndrome may be mediated through the increased occurrence of small LDL-particles which are easily modified to atherogenic oxidized LDL (ox-LDL). The aim of this study was to test this concept by examining the association between circulating ox-LDL, LDL-particle size, and the metabolic syndrome. DESIGN AND RESULTS A population-based sample of clinically healthy 58-year-old men (n = 391) was recruited. Ox-LDL was measured by ELISA (specific monoclonal antibody, mAb-4E6) and LDL-particle size by gradient gel electrophoresis. The results showed that ox-LDL significantly correlated to factors constituting the metabolic syndrome; triglycerides (r = 0.43), plasma insulin (r = 0.20), body mass index (r = 0.20), waist-to-hip ratio (r = 0.21) and HDL (r = -0.24); (P < 0.001). Ox-LDL correlated also to LDL-particle size (r = -0.42), Apo-B (r = 0.70), LDL (r = 0.65); (P < 0.001) and, furthermore, with Apo A-1 (r = -0.13) and heart rate (r = 0.13); (P < 0.01). CONCLUSION The metabolic syndrome was accompanied by high plasma ox-LDL concentrations compared with those without the syndrome. Ox-LDL levels were associated with most of the risk factors constituting the metabolic syndrome and was, in addition related to small LDL-particle size. To our knowledge the present study is the first one to demonstrate that circulating ox-LDL levels are associated with small LDL-particle size in a population representative sample of clinically healthy middle-aged men. The high degree of intercorrelation amongst several factors makes it difficult to clarify the independent role of any specific factor.

Circulating oxidized LDL is associated with the occurrence of echolucent plaques in the carotid artery in 61-year-old men.

OBJECTIVE The aim of this study was to elucidate the relationship between the echogenicity of carotid artery plaques and the following risk factors: circulating oxLDL, hsCRP, the metabolic syndrome (MetS), and several of the traditional cardiovascular (CV) risk factors. MATERIAL AND METHODS A cross-sectional population-based study of 513 sixty-one-year-old men. The levels of circulating oxLDL were determined in plasma samples by sandwich ELISA utilizing a specific murine monoclonal antibody (mAb-4E6). High-sensitivity CRP was measured in plasma by ELISA. Plaque occurrence, size and echogenicity were evaluated from B-mode ultrasound registrations in the carotid arteries. Plaque echogenicity was assessed based on a four-graded classification scale. RESULTS A higher frequency of echolucent carotid plaques was observed with increasing levels of oxLDL and systolic blood pressure (p = 0.008 and p = 0.041, respectively). Subjects with the MetS had a significantly higher frequency of echogenic plaques than subjects without the MetS (p = 0.009). In a multiple logistic regression analysis, oxLDL turned out to be independently associated with echolucent carotid plaques. CONCLUSIONS The occurrence of echolucent carotid plaques was associated with oxLDL and systolic blood pressure, and oxLDL was associated with echolucent carotid plaques independently of systolic blood pressure.

Circulating oxidized low-density lipoprotein is associated with echolucent plaques in the femoral artery independently of hsCRP in 61-year-old men.

OBJECTIVES The aim of the study was to test the hypothesis that circulating markers of inflammation (high-sensitive C-reactive protein, hsCRP) and oxidative modification of lipids (oxidized low-density lipoprotein, oxLDL) were associated with the occurrence of echolucent rather than echogenic femoral artery plaques in a cross-sectional population based cohort of 513, 61-year-old men. BACKGROUND The relationships between circulating oxLDL, hsCRP and the occurrence of echolucent plaques in the femoral artery have not previously been investigated. METHODS The levels of circulating oxLDL and hsCRP were determined in plasma by ELISA. Plaque occurrence, size and echogenicity were measured by B-mode ultrasound in the right femoral artery. Assessment of plaque echogenicity was based on the classification (grades 1-4) proposed by Gray-Weale et al. RESULTS A higher frequency of echolucent femoral plaques was observed in subjects with the metabolic syndrome and current smokers (p=0.01 and p<0.001, respectively) as well as with increasing levels of oxLDL and hsCRP (p=0.002 and p=0.005, respectively). In a multiple logistic regression analysis oxLDL and current smokers turned out to be independent associated with the presence of echolucent femoral artery plaques. CONCLUSIONS The results of the present study support our hypothesis that circulating oxLDL is a marker of an unstable echolucent plaque phenotype in the femoral artery in man.

Rapid carbon nanotubes suspension in organic solvents using organosilicon polymers

A strategy for a simple dispersion of commercial multi-walled carbon nanotubes (MWCNTs) using two organosilicones, polycarbosilane SMP10 and polysilazane Ceraset PSZ20, in organic solvents such as cyclohexane, tetrahydrofuran (THF), m-xylene and chloroform is presented. In just a few minutes the combined action of sonication and the presence of Pt(0) catalyst is sufficient to obtain a homogeneous suspension, thanks to the rapid hydrosilylation reaction between SiH groups of the polymer and the CNT sidewall. The as-produced suspensions have a particle size distribution <1μm and remain unchanged after several months. A maximum of 0.47 and 0.50mg/ml was achieved, respectively, for Ceraset in THF and SMP10 in chloroform. Possible applications as polymeric and ceramic thin films or aerogels are presented.