Effect of the β-amyloid peptide Aβ<Subscript>25–35</Subscript> and fullerene C<Subscript>60</Subscript> on the activity of enzymes in erythrocytes

The effect of the β-amyloid peptide Aβ_25–35 and fullerene C₆₀ on the activity of the cytoplasmic enzymes lactate dehydrogenase (LDH) and glutathione peroxidase (GLP), and membrane-bound phosphofructokinase (PFK) and Na⁺,K⁺-ATPase in human erythrocytes has been studied. When used in combination, the cytotoxins decrease the activity of LDH and PFK in a nonadditive manner; in this case, Aβ_25–35 protects PFK against the inhibitory effect of C₆₀. The activity of LDH, GLP, and PFK decreases within the first 2–20 min of incubation of erythrocytes with Aβ_25–35 in the absence of glucose. The addition of glucose sharply decreases the inhibitory action of Aβ_25–35 on LDH and GLP but does not affect the fourfold decrease in activity of PFK; the activity of membrane-bound Na⁺,K⁺-ATPase does not depend on the presence of glucose. Possible mechanisms of interaction of Aβ_25–35 and fullerene C₆₀ with the erythrocyte membrane and enzymes are discussed.     

Comparisons of warfarin metabolism by liver microsomes of rats treated with a series of polybrominated biphenyl congeners and by the component-purified cytochrome P-450 isozymes

R- and S-warfarin metabolite profiles (regio- and stereoselectivity) have been determined with hepatic microsomes from untreated rats and rats treated with nine individual polybrominated biphenyl (PBB) congeners, a commercial mixture of PBBs, and, for comparison with phenobarbital and 3-methylcholanthrene. The metabolic rates have been correlated with cytochrome P-450 (P-450) isozyme concentrations in the microsomes determined by immunochemical quantitation techniques (G. A. Dannan, F. P. Guengerich, L. S. Kaminsky, and S. D. Aust, (1983) J. Biol. Chem., 258, 1282–1288). The warfarin hydroxylase activities of the P-450 isozyme components of the various microsomal preparations (F. P. Guengerich, G. A. Dannan, S. T. Wright, M. V. Martin, and L. S. Kaminsky (1982) Biochemistry, 21, 6019–6030) were multiplied by the corresponding isozyme concentrations to obtain an assessment of the potential warfarin hydroxylase capacity of the microsomes, and the results were compared with actual activities. The results of these studies and comparisons indicate that substrate regio- and stereoselectivities of microsomal-bound P-450s are essentially retained on purification of the isozymes to homogeneity and reconstitution, that warfarin metabolism by microsomal preparations can be used to predict microsomal P-450 isozyme compositions, and that microsomal warfarin hydroxylase activity is greater than would be predicted based on the approx 20:1 ratio of P-450 to NADPH-P-450 reductase in the microsomes and on the known activities of constituent isozymes. Two P-450 isozymes which are induced by treatment of rats with phenobarbital appear to be more tightly linked to NADPH-P-450 reductase than does an isozyme induced by β-naphthoflavone.     

Detergents as probes of reconstituted rat liver cytochrome P-450 function

A series of 16 ionic, zwitterionic, and nonionic detergents have been used to perturb the catalytic activities of major cytochrome P-450 (P-450) forms from untreated (UT-A), phenobarbital-treated (PB-B) and beta-naphthoflavone-treated (BNF-B) rats in reconstituted systems with NADPH--P-450 reductase Detergent effects on R warfarin hydroxylase activities were correlated with detergent effects on the quaternary structures of P-450 and reductase, and on their 1:1 complexes as determined by gel exclusion chromatography using sodium cholate as a prototype detergent. The detergent concentrations used did not in most cases affect rates of NADPH-dependent reduction of cytochrome c by the reductase. With P-450 BNF-B, ionic and zwitterionic detergents enhanced warfarin hydroxylase activities at low concentrations and produced marked inhibition at higher concentrations, while nonionic detergents only inhibited. With P-450 UT-A, some nonionic and zwitterionic detergents increased rates at low concentrations and inhibited at higher concentrations. P-450 PB-B was inhibited by detergents of all three classes at low and high concentrations. The concentrations of a detergent required to affect 50% inhibition differed for the three P-450s, suggesting, together with the differential susceptibilities to detergent-mediated rate enhancing effects, that the reductase interacts functionally differently with the three P-450s. Chromatographic studies demonstrated that concentrations of sodium cholate which optimally enhanced metabolic rates with P-450 BNF-B facilitated the uptake of the P-450 into the functional reductase/P-450 complex, and higher concentrations of cholate, which completely inhibited activity, produced profound disruptions of the complex. The data have provided insight into the functional interactions required for monooxygenase activity.     

Molting-specific downregulation of C. elegans body-wall muscle attachment sites: The role of RNF-5 E3 ligase

Repeated molting of the cuticula is an integral part of arthropod and nematode development. Shedding of the old cuticle takes place on the surface of hypodermal cells, which are also responsible for secretion and synthesis of a new cuticle. Here, we use the model nematode Caenorhabditis elegans to show that muscle cells, laying beneath and mechanically linked to the hypodermis, play an important role during molting. We followed the molecular composition and distribution of integrin mediated adhesion structures called dense bodies (DB), which indirectly connect muscles to the hypodermis. We found the concentration of two DB proteins (PAT-3/β-integrin and UNC-95) to decrease during the quiescent phase of molting, concomitant with an apparent increase in lateral movement of the DB. We show that levels of the E3-ligase RNF-5 increase specifically during molting, and that RNF-5 acts to ubiquitinate the DB protein UNC-95. Persistent high levels of RNF-5 driven by a heatshock or unc-95 promoter lead to failure of ecdysis, and in non-molting worms to a progressive detachment of the cuticle from the hypodermis. These observations indicate that increased DB dynamics characterizes the lethargus phase of molting in parallel to decreased levels of DB components and that temporal expression of RNF-5 contributes to an efficient molting process.     

Drug resistance in Trypanosoma brucei spp., the causative agents of sleeping sickness in man and nagana in cattle

Drug resistance in pathogenic trypanosomes threatens successful control of fatal sleeping sickness in man and hinders economic livestock production in sub-Saharan Africa. We report on the occurrence and development of drug resistance, and discuss the genetic basis of such resistance in Trypanosoma brucei. Understanding these mechanisms at the molecular level will enable improved management of existing drugs and provide valuable clues to the development of new trypanocides.     

Sensitivity of respiratory metabolism and efficiency to foliar nitrogen during growth and maintenance

Scaling of respiration from the leaf to the canopy level currently depends on identification of physiological parameters that are tightly linked to respiration and that can readily be determined. Several recent studies have helped provide guides to predicting whole canopy respiration on the basis of foliar nitrogen (N). This approach is potentially powerful owing to the well‐described patterns of allocation of N that follow interception of radiation. In the present study, we investigated the sensitivity of the N–respiration correlation to environmental and developmental factors, in order to evaluate its usage for attempts to scale respiration to the organism and ecosystem level. We studied fully expanded, 1 and 2‐year‐old, and current‐year needles from canopies of Pinus radiata that had been treated (unthinned, thinned and thinned+fertilized treatments) in ways likely to induce a wide range of growth and respiratory responses. We examined respiration in detail during the growth period in spring and again at the end of summer, using calorespirometric methods (combined measurements of CO₂ and heat rates) to determine the respiration rates , instantaneous enthalpic growth rates (R_SGΔH_B, a measure of the conservation of electrons in anabolic products) and the enthalpy conversion efficiency (η_H) of needles differing in age. A general linear model revealed that was positively correlated with needle N, but this correlation was strongly dependent on the season and the needle age – indicating an important physiological difference between expanding young needles and fully expanded old needles. Furthermore, the strength of the correlation between needle N and respiration was comparatively weak for the current year, expanding foliage, indicating that factors other than foliage N significantly influenced the respiration of young needles. The analysis of instantaneous growth rates revealed two general processes. Older, nonexpanding foliage showed considerable rates of enthalpic growth (increases in enthalpy) that was mainly caused by the increment of lignin during secondary growth. Secondly, canopy development appeared dynamic and to be optimized according to environmental drivers and constraints – such as light and water availability. In late spring, needle extension slowed in the upper, but not the lower canopy, because the upper canopy appeared to be affected first by the onset of drought stress in late spring. Growth rates were reduced in the upper canopy despite greater rates of respiration, indicating higher demand of ATP for the maintenance of protein and for export of sugars. Consequently, the enthalpy conversion efficiency and enthalpic N productivity (enthalpic growth per unit N) were comparatively poor indicating advanced development of needles in the upper canopy. We suggest that the growth and maintenance paradigm of respiration is, at best, only moderately useful when applied to whole trees, and is not valid at the cellular level or that of the plant organ. A different concept, namely that of respiratory efficiency, seems a more suitable way to represent respiration in carbon (C) balance models and should help provide a better mechanistic understanding of how respiration affects the C conversion efficiency of plants, and ultimately the net primary productivity of ecosystems.