Characterization of the disordered-to-α-helical transition of IA₃ by SDSL-EPR spectroscopy

Electron paramagnetic resonance (EPR) spectroscopy coupled with site-directed spin labeling (SDSL) is a valuable tool for characterizing the mobility and conformational changes of proteins but has seldom been applied to intrinsically disordered proteins (IDPs). Here, IA₃ is used as a model system demonstrating SDSL-EPR characterization of conformational changes in small IDP systems. IA₃ has 68 amino acids, is unstructured in solution, and becomes α-helical upon addition of the secondary structural stabilizer 2,2,2-trifluoroethanol (TFE). Two single cysteine substitutions, one in the N-terminus (S14C) and one in the C-terminus (N58C), were generated and labeled with three different nitroxide spin labels. The resultant EPR line shapes of each of the labels were compared and each reported changes in mobility upon addition of TFE. Specifically, the spectral line shape parameters h₍₊₁₎/h₍₀₎, the local tumbling volume (V_L), and the percent change of the h₍₋₁₎ intensity were utilized to quantitatively monitor TFE-induced conformational changes. The values of h_(+1)/h₍₀₎ as a function of TFE titration varied in a sigmoidal manner and were fit to a two-state Boltzmann model that provided values for the midpoint of the transition, thus, reporting on the global conformational change of IA₃. The other parameters provide site-specific information and show that S14C-SL undergoes a conformational change resulting in more restricted motion than N58C-SL, which is consistent with previously published results obtained by studies using NMR and circular dichroism spectroscopy indicating a higher degree of α-helical propensity of the N-terminal segment of IA₃. Overall, the results provide a framework for data analyzes that can be used to study induced unstructured-to-helical conformations in IDPs by SDSL.     

Fe bioavailability from Fe-enriched yeast biomass in growing rats

The Fe content in animal feeds is highly variable. The availability of Fe in feeds varies with the feed and the form in which Fe is present. The present study reports the effect of the addition of different concentrations of Fe from yeast biomass on Fe bioavailability and Fe level in rat liver, compared with a diet containing Fe-sulphate (Fe-sulphate) addition (control) and with a diet without any addition of Fe. Male Wistar rats were fed ad libitum for 10 days a diet with different levels of Fe-enriched yeast biomass (20, 35 and 50 mg of Fe), or Fe-sulphate diet (50 mg of Fe) or without Fe addition. Faeces and urine were collected for Fe analyses during the last 5 days of the test period. The results clearly showed a highly significant (P < 0.001) better bioavailability of Fe from Fe-enriched yeast biomass, independent of the level of Fe in the diet. This was on average 36% higher than the availability of Fe from the Fe-sulphate-enriched diet. Liver Fe storage depended on the level of Fe in the diet from yeast biomass. A significantly lower amount of Fe was found to be incorporated in the liver in the group with an inorganic source of Fe (Fe-sulphate) in the diet.     

Adverse effects of tempol on hidrosaline balance in rats with acute sodium overload

We studied the effects of tempol, an oxygen radical scavenger, on hydrosaline balance in rats with acute sodium overload. Male rats with free access to water were injected with isotonic (control group) or hypertonic saline solution (0.80 mol/l NaCl) either alone (Na group) or with tempol (Na-T group). Hydrosaline balance was determined during a 90 min experimental period. Protein expressions of aquaporin 1 (AQP1), aquaporin 2 (AQP2), angiotensin II (Ang II) and endothelial nitric oxide synthase (eNOS) were measured in renal tissue. Water intake, creatinine clearance, diuresis and natriuresis increased in the Na group. Under conditions of sodium overload, tempol increased plasma sodium and protein levels and increased diuresis, natriuresis and sodium excretion. Tempol also decreased water intake without affecting creatinine clearance. AQP1 and eNOS were increased and Ang II decreased in the renal cortex of the Na group, whereas AQP2 was increased in the renal medulla. Nonglycosylated AQP1 and eNOS were increased further in the renal cortex of the Na-T group, whereas AQP2 was decreased in the renal medulla and was localized mainly in the cell membrane. Moreover, p47-phox immunostaining was increased in the hypothalamus of Na group, and this increase was prevented by tempol. Our findings suggest that tempol causes hypernatremia after acute sodium overload by inhibiting the thirst mechanism and facilitating diuresis, despite increasing renal eNOS expression and natriuresis.     

Differential effect of lentil feeding on proteosynthesis rates in the large intestine, liver and muscle of rats

The aim of this work was to test the hypothesis that the trophic effect of lentil feeding on large intestine results from a stimulation of protein synthesis and to determine whether it interferes with protein metabolism in other splanchnic or peripheral organs. Two groups of growing Sprague Dawley male rats were pair-fed iso-caloric iso-nitrogenous balanced diets containing either cooked lentils (Lens esculenta puyensis) or casein as unique protein source. Protein synthesis rates were measured in vivo, in large intestine, liver and gastrocnemius at the postprandial state. In large intestine, protein and ribonucleic acid contents were higher in the lentil-fed group than in the control group, and the amount of proteins synthesized was also higher (+57%). By contrast, liver protein and ribonucleic acid contents as well as protein synthesis rates were significantly lower in the lentil-fed group than in the control group. In the gastrocnemius muscle protein and ribonucleic acid contents were significantly lower and the amount of protein synthesized was also lower (-18%) in the lentil fed group than in the control group. This study suggests that stimulation of protein synthesis in the large intestine is compensated for by a decrease in liver and muscle.     

Bacterial species and evolution: Theoretical and practical perspectives

A discussion of the species problem in modern evolutionary biology serves as the point of departure for an exploration of how the basic science aspects of this problem relate to efforts to map bacterial diversity for practical pursuits—for prospecting among the bacteria for useful genes and gene-products. Out of a confusing array of species concepts, the Cohesion Species Concept seems the most appropriate and useful for analyzing bacterial diversity. Techniques of allozyme analysis and DNA fingerprinting can be used to put this concept into practice to map bacterial genetic diversity, though the concept requires minor modification to encompass cases of complete asexuality. Examples from studies of phenetically definedBacillus species provide very partial maps of genetic population structure. A major conclusion is that such maps frequently reveal deep genetic subdivision within the phenetically defined specles; divisions that in some cases are clearly distinct genetic species. Knowledge of such subdivisions is bound to make prospecting within bacterial diversity more effective. Under the general concept of genetic cohesion a hypothetical framework for thinking about the full range of species conditions that might exist among bacteria is developed and the consequences of each such model for species delineation, and species identification are discussed. Modes of bacterial evolution, and a theory of bacterial speciation with and without genetic recombination, are examined. The essay concludes with thoughts about prospects for very extensive mapping of bacterial diversity in the service of future efforts to find useful products. In this context, evolutionary biology becomes the handmaiden of important industrial activities. A few examples of past success in commercializing bacterial gene-products from species ofBacillus and a few other bacteria are reviewed.