Search

While it is widely appreciated that the denatured state of a protein is a heterogeneous conformational ensemble, there is still debate over how this ensemble changes with environmental conditions. Here, we use single-molecule chemo-mechanical unfolding, which combines force and urea using the optical tweezers, together with traditional protein unfolding studies to explore how perturbants commonly used to unfold proteins (urea, force, and temperature) affect the denatured-state ensemble. We compare the urea m-values, which report on the change in solvent accessible surface area for unfolding, to probe the denatured state as a function of force, temperature, and urea. We find that while the urea- and force-induced denatured states expose similar amounts of surface area, the denatured state at high temperature and low urea concentration is more compact. To disentangle these two effects, we use destabilizing mutations that shift the Tm and Cm. We find that the compaction of the denatured state is related to changing temperature as the different variants of acyl-coenzyme A binding protein have similar m-values when they are at the same temperature but different urea concentration. These results have important implications for protein folding and stability under different environmental conditions.

The present article investigates in laboratory conditions the effect of cyclic freezing and thawing (CFT) on characteristics of the neat bitumen and bitumens modified with different polymers, as well as stone mastic asphalt concretes prepared with the use of the neat and the modified bitumens. Maximal and minimal temperature in cycles of freezing and thawing (FT) is accepted equal to +20 &deg, C and &minus, 18 &plusmn, 2 &deg, C respectively. Characteristics of low temperature resistance of bitumens (stiffness S and m-value) have been determined on bending beam rheometer at the temperatures of &minus, 24 &deg, C, &minus, 30 &deg, C, and &minus, 36 &deg, C. Strength at direct tension of the stone mastic asphalt concretes has been evaluated in the device TRAVIS (Infratest Ltd., Brackenheim, Germany) at the temperatures of 0 &deg, 10 &deg, 20 &deg, C. Resistance of stone mastic asphalt concretes to rutting has been determined on a Hamburg wheel tracking machine. The characteristics of the bitumens have been determined in the initial condition and after 25 and 50 cycles of FT, and the characteristics of stone mastic asphalt concretes, in the initial condition and after 50 cycles of FT. The results obtained experimentally have shown that CFT impacts essentially on the investigated characteristics of bitumens and stone mastic asphalt concretes. Modification with polymers improves the mechanical characteristics of the bitumens and decreases the effect of CFT. It is also found out that when selecting a bitumen for specific climatic conditions it is necessary to take into account both the number of cycles of FT and the characteristic low temperature.

Objective. Semicarbazide-sensitive amine oxidase (SSAO) is widely expressed in adipose tissue, where it may contribute to stimulation of glucose transport via GLUT4 recruitment. We tested the relationships of soluble SSAO, as reflected by its plasma activity, with insulin sensitivity and indices of body fat, and determined whether insulin is involved in regulating plasma SSAO activity. Material and methods. In 24 non-diabetic subjects, the relationships of plasma SSAO activity with insulin sensitivity (M-value and free fatty acid (FFA) suppression during a 3-h hyperinsulinemic (8.3 mu U kg(-1) s(-1)), euglycemic clamp), body mass index (BMI 25.5 +/- 3.1 kg m(-2)), waist- hip ratio and fat mass were assessed. In 16 subjects, the effect of insulin infusion, administered at a rate of 8.3 mu U kg(-1) s(-1) during 3 h, followed by 3-h insulin infusion at a high rate of 41.7 mu U kg(-1) s(-1) on plasma SSAO activity was determined. In the other 8 subjects, the response of plasma SSAO activity to 24- h insulin infused at 8.3 mu U kg(-1) s(-1) was assessed. Results. There were no relationships ( all p > 0.10) of plasma SSAO activity (215 +/- 60 mu U L-1) with the M-value or with any indices of body fat and FFA before and after insulin suppression. Plasma SSAO activity changed by 27.2 (95% CI, -14.5 to + 0.2)% after 3 h (NS) and decreased by 10.1 (95% CI, 19.2 to 1)% after 6 h of insulin infusion ( p

Background/Aim. Methylglyoxal is a physiologic metabolite involved in the post-translational protein modification as precursor of advanced glycation endproducts (AGE), which are related to degenerative alterations of tissues in diabetes. Its physiologic concentration is low ; however, in pathophysiologic conditions it may rise significantly. The aim of the study was to examine a hypothesis that methylglyoxal production is related to glycemic fluctuation. Patients and methods. Methylglyoxal was measured by the HPLC method in 41 diabetic patients, in correlation to daily glucose profile, fasting glucose as well as early (HbA1c) and advanced glycation products. Results. Methylglyoxal was in parallel analyzed in whole blood and plasma samples of the same individual. A significantly higher concentration was measured in plasma than in whole blood samples of both controls (349 55 vs 520 41 nmol/l ; p=0.0002) and diabetic patients (408 130 vs 741 140 nmol/l ; p=0.0000). The 24-h glycemia variability was expressed as M value (a quantitative index of diurnal glucose fluctuation). Elevated methylglyoxal production was observed in patients with M value >20, yielding a highly significant correlation between M value and methylglyoxal level (whole blood: r=0.5, p=0.000 ; plasma: r=0.35, p=0.012). In three cases of morning hypoglycemia, a relationship was observed between fasting plasma glucose and elevated concentration of methylglyoxal. Conclusion. A discrepancy between whole blood and plasma levels of methylglyoxal indicates that glucose-derived triosephosphates are not an exclusive source of methylglyoxal, but that some amounts may also be generated from other metabolic sources, probably from ketone bodies. Significant elevation of methylglyoxal recorded in patients with marked glycemic fluctuation was closely related to M values, an index of diurnal blood glucose deviation from the optimal glycemic control. Glucose fluctuation including hyperglycemic and hypoglycemic episodes through an increased production of toxic aldehydes may be a part of the mechanisms that promote tissue damage in diabetes.