Evaluation of Z-(R,R)-IQNP for the potential imaging of m2 mAChR rich regions of the brain and heart

Alterations in the function or density of the m2 muscarinic (mAChR) subtype have been postulated to play an important role in various dementias such as Alzheimer's disease. The ability to image and quantify the m2 mAChR subtype is of importance for a better understanding of the m2 subtype function in various dementias. Z-(R)-1-Azabicyclo[2.2.2]oct-3-y (R)-alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate (Z-(R,R)-IQNP) has demonstrated significant uptake in cerebral regions that contain a high concentration of m2 mAChR subtype in addition to heart tissue. The present study was undertaken to determine if the uptake of Z-(R,R)-IQNP in these regions is a receptor mediated process and to identify the radiospecies responsible for binding at the receptor site. A blocking study demonstrated cerebral and cardiac levels of activity were significantly reduced by pretreatment (2-3 mg/kg) of (R)-3-quinuclidinyl benzilate, dexetimide and scopolamine, established muscarinic antagonists. A direct comparison of the cerebral and cardiac uptake of [I-125]-Z-(R,R)-IQNP and [I-131]-E-(R,R)-IQNP (high uptake in ml, m4 rich mAChR cerebral regions) demonstrated Z-(R,R)-IQNP localized to a higher degree in cerebral and cardiac regions containing a high concentration of the m2 mAChR subtype as directly compared to E-(R,R)-IQNP. In addition, a study utilizing [I-123]-Z-(R,R)-IQNP, [I-131]-iododexetimide and [I-125]-R-3-quinuclidinyl S-4-iodobenzilate, Z-(R,R)-IQNP demonstrated significantly higher uptake and longer residence time in those regions which contain a high concentration of the m2 receptor subtype. Folch extraction of global brain and heart tissue at various times post injection of [I-125]-Z-(R,R)-IQNP demonstrated that approximately 80% of the activity was extracted in the lipid soluble fraction and identified as the parent ligand by TLC and HPLC analysis. These results demonstrate Z-(R,R)-IQNP has significant uptake, long residence time and high stability in cerebral and cardiac tissues containing high levels of the m2 mAChR subtype. These combined results strongly suggest that Z-(R,R)-IQNP is an attractive ligand for the in vivo imaging and evaluation of m2 rich cerebral and cardiac regions by SPECT.     

Epidermal growth factor promotes oligodendrocyte process formation and regrowth after injury

Oligodendrocytes (OLs) form myelin within the central nervous system and are targets in numerous demyelinating diseases and injuries. OLs grown in culture maintain the developmental timetable which occurs in vivo and mature into cells with a relatively normal phenotype. In this study, cultured cells are used to test whether EGF can modulate process formation in OLs both before and after transection injury. EGF had no effect on the formation of new processes by OLs at any stage of development. To test the effect of EGF on process outgrowth after injury, mature OLs were selected and injured by laser transection of a single process, then imaged at 24-h intervals for 120 h. EGF promoted the recovery and regrowth of injured processes and also significantly increased outgrowth in uninjured processes. As well, it increased the number of new sprouts formed by OLs after injury. Results suggest that the effects of EGF on process outgrowth are a consequence of EGF interaction with a signaling pathway that is specifically activated within injured OLs. The potent effect of EGF on OL process formation after an injury suggests that modulation of the signaling pathways involved might provide a mechanism to promote remyelination.     

A powerful strategy to account for multiple testing in the context of haplotype analysis

Haplotypes--that is, linear arrangements of alleles on the same chromosome that were inherited as a unit--are expected to carry important information in the context of association fine mapping of complex diseases. In consideration of a set of tightly linked markers, there is an enormous number of different marker combinations that can be analyzed. Therefore, a severe multiple-testing problem is introduced. One method to deal with this problem is Bonferroni correction by the number of combinations that are considered. Bonferroni correction is appropriate for independent tests but will result in a loss of power in the presence of linkage disequilibrium in the region. A second method is to perform simulations. It is unfortunate that most methods of haplotype analysis already require simulations to obtain an uncorrected P value for a specific marker combination. Thus, it seems that nested simulations are necessary to obtain P values that are corrected for multiple testing, which, apparently, limits the applicability of this approach because of computer running-time restrictions. Here, an algorithm is described that avoids such nested simulations. We check the validity of our approach under two disease models for haplotype analysis of family data. The true type I error rate of our algorithm corresponds to the nominal significance level. Furthermore, we observe a strong gain in power with our method to obtain the global P value, compared with the Bonferroni procedure to calculate the global P value. The method described here has been implemented in the latest update of our program FAMHAP.     

The long acidic tail of high mobility group box 1 (HMGB1) protein forms an extended and flexible structure that interacts with specific residues within and between the HMG boxes

HMGB1 (high mobility group B1) is a conserved chromosomal protein composed of two similar DNA binding domains (HMG box A and box B) linked by a short basic stretch to an acidic C-terminal tail of 30 residues. The acidic tail modulates the DNA binding properties of HMGB1, and its length differentiates the various HMGB family members. We synthesized a peptide that corresponds to the acidic tail in HMGB1 (T-peptide) and studied its binding to the single boxes and to the fragment corresponding to tailless HMGB1 (designated as AB(bt) fragment). CD spectroscopy showed that T-peptide stabilizes significantly the AB(bt) fragment and that the complex has an identical thermal stability as full-length HMGB1. Calorimetric and NMR data showed that T-peptide binds with a dissociation constant of 9 microM to box A and much more weakly to box B. (1)H-(15)N HSQC spectra of full-length HMGB1 and of the AB(bt) fragment are very similar; the small chemical shift differences that exist correspond to those residues of the AB(bt) fragment that were affected by the addition of the T-peptide. We conclude that the T-peptide mimics closely the acidic tail and that the basic stretch and the acidic tail form an extended and flexible segment. The tail interacts with specific residues in the boxes and shields them from other interactions.     

Steroid hormone profiles and relative body condition of calling and satellite toads: implications for proximate regulation of behavior in anurans

Males of most anuran species (frogs and toads) vocalize to attractmates. However, individuals of many vocal species may also adoptalternative noncalling "satellite" tactics. Satellite malescharacteristically remain in close proximity to calling conspecificsand attempt to intercept incoming females attracted to advertisingmales. Emerson proposed that alternation between calling andnoncalling behavior in anurans is mediated by a reciprocal interactionbetween circulating levels of corticosterone and androgens thatis driven by depletion of energy reserves during vocalization.We tested this hypothesis by examining steroid hormone profilesand the relative body condition of calling and satellite Woodhouse'stoads (Bufo woodhousii) and Great Plains toads (B. cognatus).Consistent with Emerson's hypothesis, callers had significantlyhigher circulating corticosterone levels and were in bettercondition than satellites. However, levels of testosterone anddihydrotestosterone did not differ significantly between satellitesand callers, and we found no evidence that high levels of corticosteronehad an inhibitory effect on androgen production in either species.These data thus support a relationship between corticosteronelevels and depletion of energy reserves during bouts of vocalizationbut suggest that alternation between calling and satellite behaviormay be associated with direct effects of corticosterone on brainvocal control centers. We propose a model that incorporatesrelationships among energy reserves, androgens, corticosterone,and arginine vasotocin-producing neurons in the telencephalonto explain transitions between calling and satellite tacticsin toads.     

Phylogenetic relationships in Nicotiana (Solanaceae) inferred from multiple plastid DNA regions

For Nicotiana, with 75 naturally occurring species (40 diploids and 35 allopolyploids), we produced 4656bp of plastid DNA sequence for 87 accessions and various outgroups. The loci sequenced were trnL intron and trnL-F spacer, trnS-G spacer and two genes, ndhF and matK. Parsimony and Bayesian analyses yielded identical relationships for the diploids, and these are consistent with other data, producing the best-supported phylogenetic assessment currently available for the genus. For the allopolyploids, the line of maternal inheritance is traced via the plastid tree. Nicotiana and the Australian endemic tribe Anthocercideae form a sister pair. Symonanthus is sister to the rest of Anthocercideae. Nicotiana sect. Tomentosae is sister to the rest of the genus. The maternal parent of the allopolyploid species of N. sect. Polydicliae were ancestors of the same species, but the allopolyploids were produced at different times, thus making such sections paraphyletic to their extant diploid relatives. Nicotiana is likely to have evolved in southern South America east of the Andes and later dispersed to Africa, Australia, and southwestern North America.     

Ligand-linked structural transitions in crystals of a cooperative dimeric hemoglobin

Cooperative ligand binding in the dimeric hemoglobin (HbI) from the blood clam Scapharca inaequivalvis is mediated primarily by tertiary structural changes, but with a small quaternary rearrangement (approximately 3 degrees), based on analysis of distinct crystal forms for ligated and unligated molecules. We report here ligand transition structures in both crystal forms. Binding CO to unligated HbI crystals results in a structure that approaches, but does not attain, the full allosteric transition. In contrast, removing CO from the HbI-CO crystals results in a structure that possesses all the key low affinity attributes previously identified from analysis of HbI crystals grown in the unligated state. Subsequent binding of CO shows the reversibility of this process. The observed structural changes include the quaternary rearrangement even under the constraints of lattice interactions, demonstrating that subunit rotation is an integral component of the ligand-linked structural transition in HbI. Analysis of both crystal forms, along with data from HbI mutants, suggests that the quaternary structural change is linked to the movement of the heme group, supporting a hypothesis that the heme movement is the central event that triggers cooperative ligand binding in this hemoglobin dimer. These results show both the effects of a crystal lattice in limiting quaternary structural transitions and provide the first example of complete allosteric transitions within another crystal lattice.     

Do enzymes change the nature of transition states? Mapping the transition state for general acid-base catalysis of a serine protease

The properties of the transition state for serine protease-catalyzed hydrolysis of an amide bond were determined for a series of subtilisin variants from Bacillus lentus. There is no significant change in the structure of the enzyme upon introduction of charged mutations S156E/S166D, suggesting that changes in catalytic activity reflect global properties of the enzyme. The effect of charged mutations on the pK(a) of the active site histidine-64 N(epsilon)(2)-H was correlated with changes in the second-order rate constant k(cat)/K(m) for hydrolysis of tetrapeptide anilides at low ionic strength with a Br?nsted slope alpha = 1.1. The solvent isotope effect (D)2(O)(k(cat)/K(m))(1) = 1.4 +/- 0.2. These results are consistent with a rate-limiting breakdown of the tetrahedral intermediate in the acylation step with hydrogen bond stabilization of the departing amine leaving group. There is an increase in the ratio of hydrolysis of succinyl-Ala-Ala-Pro-Phe-anilides for p-nitroaniline versus aniline leaving groups with variants with more basic active site histidines that can be described by the interaction coefficient p(xy) = delta beta(lg)/delta pK(a) (H64) = 0.15. This is attributed to increased hydrogen bonding of the active site imidazolium N-H to the more basic amine leaving group as well as electrostatic destabilization of the transition state. A qualitative characterization of the transition state is presented in terms of a reaction coordinate diagram that is defined by the structure-reactivity parameters.     

Tuning heme redox potentials in the cytochrome C subunit of photosynthetic reaction centers

The photosynthetic reaction center (RC) from Rhodopseudomonas viridis contains four cytochrome c hemes. They establish the initial part of the electron transfer (ET) chain through the RC. Despite their chemical identity, their midpoint potentials cover an interval of 440 mV. The individual heme midpoint potentials determine the ET kinetics and are therefore tuned by specific interactions with the protein environment. Here, we use an electrostatic approach based on the solution of the linearized Poisson-Boltzmann equation to evaluate the determinants of individual heme redox potentials. Our calculated redox potentials agree within 25 meV with the experimentally measured values. The heme redox potentials are mainly governed by solvent accessibility of the hemes and propionic acids, by neutralization of the negative charges at the propionates through either protonation or formation of salt bridges, by interactions with other hemes, and to a lesser extent, with other titratable protein side chains. In contrast to earlier computations on this system, we used quantum chemically derived atomic charges, considered an equilibrium-distributed protonation pattern, and accounted for interdependencies of site-site interactions. We provide values for the working potentials of all hemes as a function of the solution redox potential, which are crucial for calculations of ET rates. We identify residues whose site-directed mutation might significantly influence ET processes in the cytochrome c part of the RC. Redox potentials measured on a previously generated mutant could be reproduced by calculations based on a model structure of the mutant generated from the wild type RC.