Structural and thermodynamic characterization of the DNA binding properties of a triple alanine mutant of MATalpha2

Triply mutated MATalpha2 protein, alpha2-3A, in which all three major groove-contacting residues are mutated to alanine, is defective in binding DNA alone or in complex with Mcm1 yet binds with MATa1 with near wild-type affinity and specificity. To gain insight into this unexpected behavior, we determined the crystal structure of the a1/alpha2-3A/DNA complex. The structure shows that the triple mutation causes a collapse of the alpha2-3A/DNA interface that results in a reorganized set of alpha2-3A/DNA contacts, thereby enabling the mutant protein to recognize the wild-type DNA sequence. Isothermal titration calorimetry measurements reveal that a much more favorable entropic component stabilizes the a1/alpha2-3A/DNA complex than the alpha2-3A/DNA complex. The combined structural and thermodynamic studies provide an explanation of how partner proteins influence the sequence specificity of a DNA binding protein.     

Accurate quantification of basal plasma levels of 3-nitrotyrosine and 3-nitrotyrosinoalbumin by gas chromatography-tandem mass spectrometry

Measurement of 3-nitro-L-tyrosine (NO(2)Tyr) and protein-related 3-nitro-L-tyrosine in human plasma is associated with numerous methodological problems which result in highly divergent basal plasma levels often ranging within two orders of magnitude. Recently, we have described an interference-free GC-tandem MS-based method for NO(2)Tyr which yielded the lowest basal plasma NO(2)Tyr levels reported thus far. This method was extended to quantify protein-associated 3-nitrotyrosine and in particular 3-nitrotyrosinated albumin (NO(2)TyrALB) in human plasma. NO(2)TyrALB and albumin (ALB) were extracted from plasma by affinity column extraction and digested enzymatically at neutral pH. 3-Nitro- L-[2H(3)]tyrosine was used as internal standard. In plasma of 18 healthy young volunteers the molar ratio of NO(2)TyrALB to albumin-derived tyrosine (TyrALB), i.e. NO(2)TyrALB/TyrALB, was determined to be 1.55+/-0.54x1:10(6) (mean+/-SD). The plasma concentration of NO(2)TyrALB was estimated as 24+/-4 nM. The NO(2)Tyr plasma levels in these volunteers were determined to be 0.73+/-0.53 nM. In the same volunteers, NO(2)TyrALB/TyrALB, NO(2)TyrALB and NO(2)Tyr were measured 15 days later and the corresponding values were determined to be 1.25+/-0.58x1:10(6), 25+/-6 nM and 0.69+/-0.16 nM. For comparison, NO(2)Tyr and NO(2)TyrALB were measured in six plasma samples from healthy volunteers by GC-MS and GC-tandem MS. Different values were found for NO(2)Tyr, i.e. 5.4+/-2.8 versus 2.7+/-1.5 nM, and comparable values for NO(2)TyrALB/TyrALB, i.e. 0.5+/-0.2x1:10(6) versus 0.4+/-0.1x1:10(6), by these methods. The ratio of the values measured by GC-MS to those measured by GC-tandem MS were 2.9+/-3.1 for NO(2)Tyr and 1.2+/-0.2 for NO(2)TyrALB/TyrALB. The present GC-tandem MS method provides accurate values of NO(2)Tyr and NO(2)TyrALB in human plasma.     

Inverse response of leukocyte heat shock proteins and DNA damage to exercise and heat

Elevated ambient temperature may exert an additional impact on the exercise-induced expression of heat shock proteins (HSP) and DNA damage in leukocytes. The protective functions of HSP include antioxidative and antiapoptotic effects and may prevent damage to DNA. Twelve athletes completed a continuous run (75% VO2max) on the treadmill, six at 28 degrees C and six at 18 degrees C room temperature. Leukocyte expression of HSP27 and inducible HSP70 was analyzed on mRNA- (RT-PCR) and protein-level (flow cytometry), while DNA damage was quantified by the comet assay. High ambient temperature induced an additional accumulation of HSP-mRNA and -protein in leukocytes compared with the exercise-induced expression at 18 degrees C. HSP27 showed a special heat sensitivity. Surprisingly, the increase of DNA damage was less pronounced after exercise at 28 degrees C compared to 18 degrees C although heat shock in vitro clearly induced DNA damage. The inverse relation between HSP and DNA damage may indicate functions of HSP which protect against exercise-induced DNA-damage in terms of thermotolerance or apoptosis.     

First asymmetric synthesis of (-)-lintetralin via intramolecular Friedel-Crafts-type cyclization

The asymmetric synthesis of an aryltetralin lignan, (-)-lintetralin, was achieved with an overall yield of 29% with seven steps. Key features of the synthesis are an asymmetric Strecker reaction, a diastereoselective Michael addition of the lithiated amino nitrile product to 5H-furan-2-one, and an intramolecular carbocationic cyclization to provide the desired ring skeleton with the correct configuration.     

The spectrin repeat: a structural platform for cytoskeletal protein assemblies

Spectrin repeats are three-helix bundle structures which occur in a large number of diverse proteins, either as single copies or in tandem arrangements of multiple repeats. They can serve structural purposes, by coordination of cytoskeletal interactions with high spatial precision, as well as a 'switchboard' for interactions with multiple proteins with a more regulatory role. We describe the structure of the alpha-actinin spectrin repeats as a prototypical example, their assembly in a defined antiparallel dimer, and the interactions of spectrin repeats with multiple other proteins. The alpha-actinin rod domain shares several features common to other spectrin repeats. (1) The rod domain forms a rigid connection between two actin-binding domains positioned at the two ends of the alpha-actinin dimer. The exact distance and rigidity are important, for example, for organizing the muscle Z-line and maintaining its architecture during muscle contraction. (2) The spectrin repeats of alpha-actinin have evolved to make tight antiparallel homodimer contacts. (3) The spectrin repeats are important interaction sites for multiple structural and signalling proteins. The interactions of spectrin repeats are, however, diverse and defy any simple classification of their preferred interaction sites, which is possible for other domains (e.g. src-homology domains 3 or 2). Nevertheless, the binding properties of the repeats perform important roles in the biology of the proteins where they are found, and lead to the assembly of complex, multiprotein structures involved both in cytoskeletal architecture as well as in forming large signal transduction complexes.     

Cellular MR imaging

Cellular MR imaging is a young field that aims to visualize targeted cells in living organisms. In order to provide a different signal intensity of the targeted cell, they are either labeled with MR contrast agents in vivo or prelabeled in vitro. Either (ultrasmall) superparamagnetic iron oxide [(U)SPIO] particles or (polymeric) paramagnetic chelates can be used for this purpose. For in vivo cellular labeling, Gd3+- and Mn2+-chelates have mainly been used for targeted hepatobiliary imaging, and (U)SPIO-based cellular imaging has been focused on imaging of macrophage activity. Several of these magnetopharmaceuticals have been FDA-approved or are in late-phase clinical trials. As for prelabeling of cells in vitro, a challenge has been to induce a sufficient uptake of contrast agents into nonphagocytic cells, without affecting normal cellular function. It appears that this issue has now largely been resolved, leading to an active research on monitoring the cellular biodistribution in vivo following transplantation or transfusion of these cells, including cell migration and trafficking. New applications of cellular MR imaging will be directed, for instance, towards our understanding of hematopoietic (immune) cell trafficking and of novel guided (stem) cell-based therapies aimed to be translated to the clinic in the future.     

RNA POLYMERASE ACTIVITY IN VARIOUS CLASSES OF NUCLEI FROM DIFFERENT REGIONS OF RAT BRAIN DURING POSTNATAL DEVELOPMENT

—The changes in the wet weight and the numbers of cell nuclei recovered from the cerebral hemispheres, the cerebellum and the brain stem of rats from the period of 5–30 days after birth have been determined. In parallel a study has been made of the RNA polymerase activity, both in the unfractionated nuclei from these regions and in the nuclei separated by zonal centrifugation. In general there is a considerable decline in activity during this period, which occurs in all class of nuclei although not to the same extent. The most dense nuclei from the cerebellum retain relatively high activity at 20 days after birth, possibly due to the contribution of the microneuronal nuclei.     

Affinity parameters of amino acid derivative binding to molecularly imprinted nanospheres consisting of poly[(ethylene glycol dimethacrylate)-co-(methacrylic acid)

The binding of L-Boc-phenylalanine anilide (BFA) and L-Boc-phenylalanine (phe) to molecularly imprinted and non-imprinted polymer nanoparticles consisting of poly[(ethylene glycol dimethacrylate)-co-(methacrylic acid)] has been investigated by adsorption experiments and mathematical modeling. The experimental isotherms have been mathematically adapted following the models of Freundlich, Langmuir, Langmuir-Freundlich, Bi-Langmuir, and extended Langmuir. The extended Langmuir model differentiated between specific and nonspecific binding of the ligand to the receptor nanoparticles and rendered excellent fitting of the experimental data. It delivered a thermodynamic and kinetic parameter set on the experimental association curves of L-BFA by L-BFA-imprinted nanospheres in suspension experiments with the equilibrium constant KD= 4.09 +/- 0.69 micromol L(-1) and the kinetic association rate constant Ka= 5.60 mL micromol(-1) min(-1).     

Influence of age, sex, and sexual activity on trace element levels and antioxidant enzyme activities in field mice (Apodemus sylvaticus and Mus spretus)

The influence of age, gender and sexual activity on both hepatic levels of some trace elements (Fe, Cu, Zn, Mn, Se) and the activities of glutathione-S-transferase (GST) and superoxide dismutase (SOD) was investigated in Wood mice (Apodemus sylvaticus) and Algerian mice (Mus spretus). Animals were taken from a riverside community of an unpolluted area of central Portugal. Adult A. sylvaticus presented the highest hepatic mean concentrations of Cu and Mn, whereas adult M. spretus had the highest Fe concentration in the liver. Moreover, an influence of age on the contents of Fe, Zn, and Mn has been observed in A. sylvaticus, whereas in M. spretus an influence of gender and sexual activity was only detected on Zn levels. In contrast, enzyme activities were not influenced by the studied variables, despite a tendency for an increase in SOD activity in sexually active M. spretus. GST activity was species dependent, whereas SOD activity was similar between species. These findings were analyzed regarding the relationship of both essential trace elements and the two antioxidant enzymes with physiological and metabolic pathways related to life cycles in the two species of mice. Results enhanced the understanding of A. sylvaticus and M. spretus as biological models, allowing their future use as bioindicators of environmental toxicity.