Stability of the Transthyretin Molecule as a Key Factor in the Interaction with A-Beta Peptide - Relevance in Alzheimer's Disease

Transthyretin (TTR) protects against A-Beta toxicity by binding the peptide thus inhibiting its aggregation. Previous work showed different TTR mutations interact differently with A-Beta, with increasing affinities correlating with decreasing amyloidogenecity of the TTR mutant; this did not impact on the levels of inhibition of A-Beta aggregation, as assessed by transmission electron microscopy. Our work aimed at probing differences in binding to A-Beta by WT, T119M and L55P TTR using quantitative assays, and at identifying factors affecting this interaction. We addressed the impact of such factors in TTR ability to degrade A-Beta. Using a dot blot approach with the anti-oligomeric antibody A11, we showed that A-Beta formed oligomers transiently, indicating aggregation and fibril formation, whereas in the presence of WT and T119M TTR the oligomers persisted longer, indicative that these variants avoided further aggregation into fibrils. In contrast, L55PTTR was not able to inhibit oligomerization or to prevent evolution to aggregates and fibrils. Furthermore, apoptosis assessment showed WT and T119M TTR were able to protect against A-Beta toxicity. Because the amyloidogenic potential of TTR is inversely correlated with its stability, the use of drugs able to stabilize TTR tetrameric fold could result in increased TTR/A-Beta binding. Here we showed that iododiflunisal, 3-dinitrophenol, resveratrol, [2-(3,5-dichlorophenyl)amino] (DCPA) and [4-(3,5-difluorophenyl)] (DFPB) were able to increase TTR binding to A-Beta; however only DCPA and DFPB improved TTR proteolytic activity. Thyroxine, a TTR ligand, did not influence TTR/A-Beta interaction and A-Beta degradation by TTR, whereas RBP, another TTR ligand, not only obstructed the interaction but also inhibited TTR proteolytic activity. Our results showed differences between WT and T119M TTR, and L55PTTR mutant regarding their interaction with A-Beta and prompt the stability of TTR as a key factor in this interaction, which may be relevant in AD pathogenesis and for the design of therapeutic TTR-based therapies.     

Alpine vascular plant species richness: the importance of daily maximum temperature and pH

Species richness in the alpine zone varies dramatically when communities are compared. We explored (i) which stress and disturbance factors were highly correlated with species richness, (ii) whether the intermediate stress hypothesis (ISH) and the intermediate disturbance hypothesis (IDH) can be applied to alpine ecosystems, and (iii) whether standing crop can be used as an easily measurable surrogate for causal factors determining species richness in the alpine zone. Species numbers and standing crop were determined in 14 alpine plant communities in the Swiss Alps. To quantify the stress and disturbance factors in each community, air temperature, relative air humidity, wind speed, global radiation, UV-B radiation, length of the growing season, soil suction, pH, main soil nutrients, waterlogging, soil movement, number of avalanches, level of denudation, winter dieback, herbivory, wind damage, and days with frost were measured or observed. The present study revealed that 82% of the variance in␣vascular species richness among sites could be explained by just two abiotic factors, daily maximum temperature and soil pH. Daily maximum temperature and pH affect species richness both directly and via their effects on other environmental variables. Some stress and disturbance factors were related to species richness in a monotonic way, others in an unimodal way. Monotonic relationships suggest that the harsher the environment is, the fewer species can survive in such habitats. In cases of unimodal relationships (ISH and IDH) species richness decreases at both ends of the gradients due to the harsh environment and/or the interaction of other environmental factors. Competition and disturbance seemed only to play a secondary role in the form of fine-tuning species richness in specific communities. Thus, we concluded that neither the ISH nor the IDH can be considered useful conceptual models for the alpine zone. Furthermore, we found that standing crop can be used as an easily measurable surrogate for causal factors determining species richness in the alpine zone, even though there is no direct causality.     

A Systematic Approach towards Optimizing a Cohabitation Challenge Model for Infectious Pancreatic Necrosis Virus in Atlantic Salmon (Salmo salar L.)

A cohabitation challenge model was developed for use in evaluating the efficacy of vaccines developed against infectious pancreatic necrosis virus (IPNV) in Atlantic salmon (Salmo salar L) using a stepwise approach. The study involved identifying a set of input variables that were optimized before inclusion in the model. Input variables identified included the highly virulent Norwegian Sp strain NVI015-TA encoding the T₂₁₇A₂₂₁ motif having the ability to cause >90% mortality and a hazard risk ratio of 490.18 (p<0.000) for use as challenge virus. The challenge dose was estimated at 1x10⁷ TCID₅₀/mL per fish while the proportion of virus shedders was estimated at 12.5% of the total number of fish per tank. The model was designed based on a three parallel tank system in which the Cox hazard proportional regression model was used to estimate the minimum number of fish required to show significant differences between the vaccinated and control fish in each tank. All input variables were optimized to generate mortality >75% in the unvaccinated fish in order to attain a high discriminatory capacity (DC) between the vaccinated and control fish as a measure of vaccine efficacy. The model shows the importance of using highly susceptible fish to IPNV in the optimization of challenge models by showing that highly susceptible fish had a better DC of differentiating vaccine protected fish from the unvaccinated control fish than the less susceptible fish. Once all input variables were optimized, the model was tested for its reproducibility by generating similar results from three independent cohabitation challenge trials using the same input variables. Overall, data presented here show that the cohabitation challenge model developed in this study is reproducible and that it can reliably be used to evaluate the efficacy of vaccines developed against IPNV in Atlantic salmon. We envision that the approach used here will open new avenues for developing optimal challenge models for use in evaluating the efficacy of different vaccines used in aquaculture.     

Calmodulin-binding proteins also have a calmodulin-like binding site within their structure. The flip-flop model.

The flip-flop model is a mechanistic model proposed to describe how calmodulin activates enzymes. One prediction based upon this model is that calmodulin-activated enzymes would contain a calmodulin-like binding site which, among other attributes, would bind the peptide melittin. Five purified calmodulin-activated enzymes, namely calcineurin, myosin light chain kinase, phosphorylase b kinase, phosphodiesterase, and NAD kinase, were all found to bind biotinylated melittin and to also bind an antimelittin antibody and biotinylated calmodulins. Using gel blots of crude tissue extracts (rat brain and Arabidopsis), most proteins did not bind any of the probes and thus do not have these characteristics. However, among those which bind any of these probes, a strong correlation was found between those proteins which bind biotinylated calmodulins and those which bind melittin and antimelittin. Gel blots of phosphorylase b kinase demonstrate that the alpha, beta, and gamma subunits all bind calmodulin and melittin. A putative calmodulin-like binding site sequence was identified in eight enzymes or subunits which may play an important role in both melittin binding and calmodulin-dependent regulation of these enzymes.     

The effects of maturation on self-induced dynamic body sway frequencies of girls performing acrobatics or classical dance

We investigated the effects of maturation on the dynamic body sways of healthy girls. Prepubertal and postpubertal girls practising professional physical activities requiring a good ability to maintain equilibrium (acrobats and dancers) were asked to stand on a free seesaw platform and the results compared to those for untrained age-matched girls. This platform (stabilometer) allows self-induced body sways. Stabilograms were obtained by a double integration of the angular acceleration from the recordings of the platform sways made with an accelerometer. Fast Fourier transform processing of stabilograms allowed spectral frequency analysis. The total spectrum energy and the energies of three frequency bands (0–0.5 Hz, 0.5–2 Hz, 2–20 Hz) were determined. ANOVA showed that, for all groups of different equilibrium activity and independent of visual input, prepubertal girls had higher energy values than postpubertal girls in the 0- to 0.5-Hz band whereas the opposite was true for 0.5- to 2-Hz band. Ballet dancers were more dependent than acrobats on visual inputs for the regulation of their postural control but were less dependent than untrained girls at both ages. Maturation seemed to shift body sways towards higher frequencies and the utilization of the cues of postural control was different according to the type of equilibrium activity practised by the subjects. Accepted: 7 February 1997     

Chemo-enzymatic synthesis of the Galili epitope Gal(alpha)(1-->3)Galbeta(1-->4)GlcNAc on a homogeneously soluble PEG polymer by a multi-enzyme system.

The alpha-Gal trisaccharide Gal(alpha)(1-->3)Galbeta(1-->4)GlcNAc 11 was synthesized on a homogeneously soluble polymeric support (polyethylene glycol, PEG) by use of a multi-enzyme system consisting of beta-1,4-galactosyltransferase (EC 2.4.1.38), alpha-1,3-galactosyltransferase (EC 2.4.1.151), sucrose synthase (EC 2.4.1.13) and UDP-glucose-4-epimerase (EC 5.1.3.2). In addition workup was simplified by use of dia-ultrafiltration. Thus the advantages of classic chemistry/enzymology and solid-phase synthesis could be united in one. Subsequent hydrogenolytic cleavage afforded the free alpha-Gal trisaccharide.     

Conformational analysis of the 3'-5'-cyclic dinucleotide d less than pApA greater than by means of molecular mechanics

The conformational properties of the cyclic dinucleotide d less than pApA greater than were studied by means of molecular mechanics calculations in which a multiconformation analysis was combined with minimum energy calculations. In this approach models of possible conformers are built by varying the torsion angles of the molecule systematically. These models are then subjected to energy minimization; in the present investigation use was made of the AMBER Force field. It followed that the lowest energy conformer has a pseudo-two-fold axis of symmetry. In this conformer the deoxyribose sugars adopt a N-type conformation. The conformation of the sugar-phosphate backbone is determined by the following torsion angles: alpha +, beta t, gamma +, epsilon t and zeta +. The conformation of this ringsystem corresponds to the structure derived earlier by means of NMR spectroscopy and X-ray diffraction. The observation of a preference for N-type sugar conformations in this molecule can be explained by the steric hindrance induced between opposite H3' atoms when one sugar is switched from N- to S-type puckers. The sugars can in principle switch from N- to S-type conformations, but this requires at least the transition of gamma + to gamma -. In this process the molecule obtains an extended shape in which the bases switch from a pseudo-axial to a pseudo-equatorial position. The calculations demonstrate that, apart from the results obtained for the lowest energy conformation, the 180 degrees change in the propagation direction of the phosphate backbone can be achieved by several different combinations of the backbone torsion angles. It appeared that in the low energy conformers five higher order correlations are found. The combination of torsion angles which are involved in changes in the propagation direction of the sugar-phosphate backbone in DNA-hairpin loops and in tRNA, are found in the dataset obtained for cyclic d less than pApA greater than. It turns out, that in the available examples, 180 degrees changes in the backbone direction are localized between two adjacent nucleotides.     

Sampling Atriplex bladders: a comparison of methods

Abstract. A major objection to the convenient sampling of Chenopodiacean bladders by brushing in aqueous solutions is the suspected leakage of ions from the leaf blade. To overcome this problem, a new method of sampling bladder hair is described, which also achieves rapid and quantitative separation and, at the same time, yields bladder samples of undoubtedly high purity. Leakage is stopped by the aid of liquid nitrogen. Comparison of this method to removal of bladders by brushing in aquenous solutions effectively confirm the validity of the customary method beyond dispute.     

Lung microsomal p-nitrophenol hydroxylase -- characterization and reconstitution of its activity

1. Formation of catechols from benzene and nitrobenzene have been implicated in the carcinogenic activity of these chemicals. In liver, p-nitrophenol, an intermediate of p-nitrobenzene is enzymatically converted to 4-nitrocatechol. 2. For the first time in this study, the presence of a highly active enzyme catalyzing the formation of 4-nitrocatechol from p-nitrophenol was detected in lung microsomes. The average specific activity of lung p-nitrophenol hydroxylase was found to be 0.494 nmol 4-nitrocatechol formed mg prot-1 min-1. 3. The optimum conditions for sheep lung microsomal p-nitrophenol hydroxylase were established. The maximal activity was noted at pH 6.8. The rate of p-nitrophenol hydroxylation was linear up to 2 mg prot/ml of incubation mixture. The maximal rate of 4-nitrocatechol formation was observed with 0.25 mM p-nitrophenol. 4. The Lineweaver-Burk and Eadie-Hofstee plots were found to be curve-linear. Two different Km values were calculated as 11.6 and 71.4 microM from the Lineweaver-Burk plot and as 10.7 and 74.5 microM from the Eadie-Hofstee plot. This suggested that there were either two forms of enzyme or two different enzymes participating in ortho hydroxylation of p-nitrophenol in lung microsomes. 5. Lung microsomal p-nitrophenol hydroxylase activity of sheep was reconstituted in the presence of purified lung microsomal cytochrome P-450, NADPH dependent cytochrome P-450 reductase and synthetic lipid, phosphatidylcholine dilauroyl.