Statistical Evaluation of HTS Assays for Enzymatic Hydrolysis of ß-Keto Esters.

ß-keto esters are used as precursors for the synthesis of ß-amino acids, which are building blocks for some classes of pharmaceuticals. Here we describe the comparison of screening procedures for hydrolases to be used for the hydrolysis of ß-keto esters, the first step in the preparation of ß-amino acids. Two of the tested high throughput screening (HTS) assays depend on coupled enzymatic reactions which detect the alcohol released during ester hydrolysis by luminescence or absorption. The third assay detects the pH shift due to acid formation using an indicator dye. To choose the most efficient approach for screening, we assessed these assays with different statistical methods-namely, the classical Z'-factor, standardized mean difference (SSMD), the Kolmogorov-Smirnov-test, and t-statistics. This revealed that all three assays are suitable for HTS, the pH assay performing best. Based on our data we discuss the explanatory power of different statistical measures. Finally, we successfully employed the pH assay to identify a very fast hydrolase in an enzyme-substrate screening.

Tectonics of a K⁺ channel: The importance of the N-terminus for channel gating.

The small K⁺ channel Kcv represents the pore module of complex potassium channels. It was found that its gating can be modified by sensor domains, which are N-terminally coupled to the pore. This implies that the short N-terminus of the channel can transmit conformational changes from upstream sensors to the channel gates. To understand the functional role of the N-terminus in the context of the entire channel protein, we apply combinatorial screening of the mechanical coupling and long-range interactions in the Kcv potassium channel by reduced molecular models. The dynamics and mechanical connections in the channel complex show that the N-terminus is indeed mechanically connected to the pore domain. This includes a long rang coupling to the pore and the inner and outer transmembrane domains. Since the latter domains host the two gates of the channel, the data support the hypothesis that mechanical perturbation of the N-terminus can be transmitted to the channel gates. This effect is solely determined by the topology of the channel; sequence details only have an implicit effect on the coarse-grained dynamics via the fold and not through biochemical details at a smaller scale. This observation has important implications for engineering of synthetic channels on the basis of a K⁺ channel pore.

Dynamical regimes due to technological change in a microeconomical model of production.

We develop a microeconomical model to investigate the impact of technological change onto production decisions of suppliers-modeling an effective feedback mechanism of the market. An important property-the time horizon of production planning-is related to the Kolmogorov entropy of the one-dimensional maps describing price dynamics. We simulate this price dynamics in an ensemble representing the whole macroeconomy. We show how this model can be used to support ongoing research in economic growth and incorporate the obtained microeconomic findings into the discussion about appropriate macroeconomic quantities such as the production function-thus effectively underpinning macroeconomics with microeconomical dynamics. From there we can show that the model exhibits different dynamical regimes (suggesting "phase transitions") with respect to an order parameter. The non-linear feedback under technological change was found to be the crucial mechanism. The implications of the obtained regimes are finally discussed.

Granuloma annulare of the palms.

Granuloma annulare (GA) is a common, benign skin condition, which was first described over a century ago, but still remains an enigma with respect to etiology, associated systemic diseases, and treatment. A number of clinical variants have been classified. We report an atypical presentation of GA localized to the palms.

Relating sequence evolution of HIV1-protease to its underlying molecular mechanics.

We investigate the connection between sequence evolution of the human immunodeficiency virus (HIV) type 1 protease under neutral selection or selective pressure induced by protease inhibitors and the functional and molecular-stability characteristics of the molecule in the physical domain. To this end we analyze sequence data on more than 45,000 patients with bioinformatical tools, namely mutual information between residue pairings. In addition we perform extensive computations on the molecular mechanics of the molecule subject to artificial mutations. The changes in the mechanics and dynamics of the molecule in three-dimensional space upon perturbation are then related to the sequence stability as described by the mutual information. We distinguish physical interactions by their evolutionary background and give hints for potential new drug targets. In addition we discuss how such targets can be efficiently chosen to give the HI virus less opportunity to develop resistance towards such drugs while maintaining the protease function at the same time. The interactions between residue no. 28 and 23' in different chains as well as the interaction between residue no. 92 and 94 within one chain were identified as particular crucial. In addition we find interactions in the beta-sheet-dimerization interface to be important for conserving the protein function and stability while these are at the same time evolutionary conserved - implications of and comparisons to experimental results are finally discussed.

Information theoretical measures to analyze trajectories in rational molecular design.

We develop a new methodology to analyze molecular dynamics trajectories and other time series data from simulation runs. This methodology is based on an information measure of the difference between distributions of various data extract from such simulations. The method is fast as it only involves the numerical integration/summation of the distributions in one dimension while avoiding sampling issues at the same time. The method is most suitable for applications in which different scenarios are to be compared, e.g. to guide rational molecular design. We show the power of the proposed method in an application of rational drug design by reduced model computations on the BH3 motif in the apoptosis inducing BCL(2) protein family.

No-carrier-added nucleophilic 18F-labelling in an electrochemical cell exemplified by the routine production of [18F]altanserin.

A new type of electrochemical cell with anodic deposition of no-carrier-added [(18)F]fluoride and variable reaction volume has been developed. The reactor is designed for small reaction volumes and non-thermal drying of [(18)F]fluoride. The implementation of this reactor into a complete remotely controlled synthesis device is described for the routine production of [(18)F]altanserin. A radiochemical yield of 23+/-5% was obtained via cryptate-mediated nucleophilic (18)F-fluorination. Batches of up to 6 GBq [(18)F]altanserin, suitable for human application, with a molar activity of >500 GBq/micromol were obtained within 75 min.

A minimal model for stabilization of biomolecules by hydrocarbon cross-linking.

Programmed cell death regulating protein motifs play an essential role in the development of an organism, its immune response, and disease-related cellular mechanisms. Among those motifs the BH3 domain of the BCL-2 family is found to be of crucial importance. Recent experiments showed how the isolated, otherwise unstructured BH3 peptide can be modified by a hydrocarbon linkage to regain function. We parametrized a reduced, dynamic model for the stability effects of such covalent cross-linking and confirmed that the model reproduces the reinforcement of the structural stability of the BH3 motif by cross-linking. We show that an analytically solvable model for thermostability around the native state is not capable of reproducing the stabilization effect. This points to the crucial importance of the peptide dynamics and the fluctuations neglected in the analytic model for the cross-linking system to function properly. This conclusion is supported by a thorough analysis of a simulated Go model. The resulting model is suitable for rational design of generic cross-linking systems in silicio.

Computing the Amino Acid Specificity of Fluctuations in Biomolecular Systems.

We developed a new amino acid specific method for the computation of spatial fluctuations of proteins around their native structures. We show the consistency with experimental values and the increased performance in comparison to an established model, based on statistical estimates for a set of test proteins. We apply the new method to HIV-1 protease in its wild-type form and to a V82F-I84V mutant that shows resistance to protease inhibitors. We further show how the method can be successfully used to explain the molecular biophysics of drug resistance of the mutant.

O-(2-[18F]fluorethyl)-L-tyrosine PET in the clinical evaluation of primary brain tumours.

PURPOSE: The aim of this study was to evaluate the differential uptake of O-(2-[18F]fluorethyl)-L-tyrosine (FET) in suspected primary brain tumours. METHODS: Positron emission tomography (PET) was performed in 44 patients referred for the evaluation of a suspected brain tumour. Acquisition consisted of four 10-min frames starting upon i.v. injection of FET. Tumour uptake was calculated as the ratio of maximal tumour intensity to mean activity within a reference region (FETmax). RESULTS: FET uptake above the cortical level was observed in 35/44 lesions. All histologically confirmed gliomas and many other lesions showed FET uptake to a variable extent. No uptake was observed in nine lesions (one inflammatory lesion, one dysembryoplastic neuroepithelial tumour, one mature teratoma, six lesions without histological confirmation). An analysis of uptake dynamics was done in the patients with increased FET uptake (22 gliomas, three lymphomas, three non-neoplastic lesions, three lesions with unknown histology and four other primaries). Upon classification of tumours into low (i.e. WHO I and II) and high grade (i.e. WHO III and IV), a significant difference in FETmax between the two categories was observed only in the first image frame (0-10 min p.i.), with FETmax=2.0 in low-grade and 3.2 in high-grade tumours (p<0.05); no significant differences were found in frame 4 (30-40 min p.i.), with FETmax=2.4 vs 2.7. Similar results were obtained when the analysis was applied only to astrocytic tumours (2.0 vs 3.1 in the first frame; 2.4 vs 2.6 in the fourth frame). CONCLUSION: These initial results indicate that FET PET is a useful method to identify malignant brain lesions. It appears that high- and low-grade brain tumours exhibit a different uptake kinetics of FET. A kinetic analysis of FET PET may provide additional information in the differentiation of suspected brain lesions.

Efficient routine production of the 18F-labelled amino acid O-2-18F fluoroethyl-L-tyrosine.

A convenient remotely controlled no-carrier-added synthesis of enantiomerically pure O-(2-[18F]fluoroethyl)-L-tyrosine (FET) is described. This allows the distribution of the radiotracer to other laboratories according to the satellite concept. The radiochemical yield obtained within 80 min is about 60%. The FET containing HPLC fraction can be used immediately (after adding sodium chloride) for human application.

Distribution of 5HT2A receptors in the human brain: comparison of data in vivo and post mortem.

AIM: The study presented here firstly compares the distribution of the binding potential of the serotonin-5HT2A receptor as measured in vivo with data of receptor density taken from literature. Secondly, the sensitivity of the method to detect gradual differences in receptor densities is evaluated. METHODS: Positron emission tomography (PET) studies were carried out in 6 healthy volunteers using the selective serotonin-5HT2A ligand 18F-altanserin. The binding potential was quantified in 12 regions using Logan's graphical method and the equilibrium method. These data were compared to the distribution of receptor density as taken from literature. RESULTS: The binding data in vivo correlated to autoradiography data (post mortem) with r = 0.83 (Pearson regression coefficient; p < 0.0001). A difference in the receptor density between two regions could be detected with p < 0.05 when it amounted at least to 18%. CONCLUSION: This study demonstrates a good agreement between in vivo data obtained with 18F-altanserin and PET in healthy volunteers and the true autoradiographically determined distribution of 5HT2A receptors in human brains. The in vivo method seems to be sensitive enough to detect changes in receptor density of more than 18%.

Disorder induced quantum phase transition in random-exchange spin-1/2 chains.

We investigate the effect of quenched bond disorder on the anisotropic antiferromagnetic spin-1/2 (XXZ) chain as a model for disorder-induced quantum phase transitions. We find nonuniversal behavior of the average correlation functions for weak disorder, followed by a quantum phase transition into a strongly disordered phase with only short-range xy correlations. We find no evidence for the universal strong-disorder fixed point predicted by the real-space renormalization group, suggesting a qualitatively different view of the relationship between quantum fluctuations and disorder.

Uptake of cis-4-[18 F]fluoro-L-proline in scar formation: a marker of collagen synthesis?

Excessive scar formation is accompanied by abnormal collagen synthesis. The feasibility of monitoring collagen synthesis in vivo with no-carrier-added cis-4[18F]fluoro-L-proline (cis-FPro) was evaluated in an animal model with scar formation induced by implanted meshes. The abdominal wall of rats was replaced by alloplastic meshes. At days 3, 7, 14, 21 and 90 after implantation, the uptake of cis-FPro at 4 h post-injection was determined for resected samples of the mesh and normal tissues. The highest uptake was found in the kidneys (1.73+/-0.47%ID/g) followed by the liver (0.59+/-0.19%ID/g). The meshes showed the maximum uptake at day 3 (0.20+/-0.07%ID/g) with a decrease to 0.10+/-0.03%ID/g at day 90 (P<0.001). After 3 days no connective tissue was shown by histopathological morphometric analysis. The maximum partial volume (PV%) of connective tissue was 43+/-14 PV% 90 days after implantation. The maximum levels of granulocytes and inflammatory infiltrate were found at day 3 with minimal levels at day 90, paralleling the course of cis-FPro uptake. In conclusion, the uptake of cis-FPro at 4 h post-injection is not related to the content of connective tissue. Cis-FPro radiolabelled with 18F appears not to be a suitable radiopharmaceutical for in vivo monitoring of collagen synthesis in scar formation.

Electrochemical cell for separation of [18F]fluoride from irradiated 18O-water and subsequent no carrier added nucleophilic fluorination.

An electrochemical cell was designed allowing the anodic deposition of n.c.a. [18F]fluoride solubilized in an 18O-water target and subsequent n.c.a. nucleophilic 18F-fluorination. The recovery of the deposited [18F]fluoride can be achieved in the presence of an aprotic solvent containing a phase-transfer catalyst (PTC). The radioisotope adsorbed electrochemically at the cylindrical surface of a glassy carbon electrode can be dried easily by washing the cell twice with dry aprotic solvents while maintaining a low electric field. This simple washing step makes an azeotropic drying process obsolete. Accordingly, less basic cryptates like [K within 2.2.2.]oxalate or triflate can be used for nucleophilic 18F-fluorination because loss of activity as a consequence of azeotropic drying under conditions of low basicity does not occur. The usefulness of this technique is exemplified for the n.c.a. synthesis of various 18F-labelled compounds. The radiotracers were synthesized with higher radiochemical yields and under much easier conditions than the conventional 18F-fluorination procedure which includes an additional drying step of the PTC.

Theoretical estimation of absorbed dose to organs in radioimmunotherapy using radionuclides with multiple unstable daughters.

The toxicity and clinical utility of long-lived alpha emitters such as Ac-225 and Ra-223 will depend upon the fate of alpha-particle emitting unstable intermediates generated after decay of the conjugated parent. For example, decay of Ac-225 to a stable element yields four alpha particles and seven radionuclides. Each of these progeny has its own free-state biodistribution and characteristic half-life. Therefore, their inclusion for a more accurate prediction of absorbed dose and potential toxicity requires a formalism that takes these factors into consideration as well. To facilitate the incorporation of such intermediates into the dose calculation, a previously developed methodology (model 1) has been extended. Two new models (models 2 and 3) for allocation of daughter products are introduced and are compared with the previously developed model. Model 1 restricts the transport to a function that yields either the place of origin or the place(s) of biodistribution depending on the half-life of the parent radionuclide. Model 2 includes the transient time within the bloodstream and model 3 incorporates additional binding at or within the tumor. This means that model 2 also allows for radionuclide decay and further daughter production while moving from one location to the next and that model 3 relaxes the constraint that the residence time within the tumor is solely based on the half-life of the parent. The models are used to estimate normal organ absorbed doses for the following parent radionuclides: Ac-225, Pb-212, At-211, Ra-223, and Bi-213. Model simulations are for a 0.1 g rapidly accessible tumor and a 10 g solid tumor. Additionally, the effects of varying radiolabled carrier molecule purity and amount of carrier molecules, as well as tumor cell antigen saturation are examined. The results indicate that there is a distinct advantage in using parent radionuclides such as Ac-225 or Ra-223, each having a half-life of more than 10 days and yielding four alpha particles per parent decay, in that lower doses to normal organs result for a given tumor dose in comparison to those radionuclides yielding fewer alpha particles. In model 2, which accounts for transit time through the blood, a dose of 20 Gy to a rapidly accessible 0.1 g tumor will result in a liver and kidney dose of 1.7 and 0.9 Gy, respectively from Ac-225. An equivalent dose to tumor from Ra-223 would yield a maximum normal organ dose of 0.4 and 0.3 Gy to bone and small intestines, respectively; the corresponding absorbed dose to small intestines from Pb-212 and Bi-213 is 2.2 and 3.0 Gy, respectively.

Disturbance of serotonin 5HT2 receptors in remitted patients suffering from hereditary depressive disorder.

AIM: The characteristics of 5HT2 receptor binding were investigated in major depression in vivo using positron emission tomography and the radioligand F-18-altanserin. METHODS: Twelve patients from families with high loading of depression living in a geographically restricted region were examined and compared with normal control subjects. At the time of the PET measurement all patients were remitted; in some of them remission was sustained by antidepressive medication. Binding potential was assessed by Logan's graphical analysis method. RESULTS: The binding of F-18-altanserin was about 38% lower in patients than in healthy controls (p < 0.001). A multiple regression analysis revealed that this difference was mainly induced by depression rather than by medication. CONCLUSIONS: The data suggest that 5HT2 receptors are altered in depression. We present evidence for a reduction of the receptor density, which might be usable as trait marker of subjects susceptible for depressive illness.

Cellular dose conversion factors for alpha-particle--emitting radionuclides of interest in radionuclide therapy.

UNLABELLED: alpha-Particle--emitting radionuclides are of increasing interest in radionuclide therapy. The decay scheme of alpha-emitting radionuclides typically includes a chain of unstable progeny. It is generally assumed that alpha-particle emission by the parent radionuclide will break the chemical bond with its carrier molecule and that the resulting daughter atom will no longer be associated with the carrier molecule. If the daughter is very short lived, it will not have enough time to be carried any significant distance from the site of parent decay and a cellular, absorbed dose estimate must consider the energy deposited by the daughter as well as the parent. Depending on the site of parent decay and the expected removal rate of daughter atoms from this site, the contribution of emissions from longer-lived daughters may also be warranted. In this study, dose conversion factors (DCFs) for cellular dimensions that incorporate the fate of daughter radionuclides were derived for (225)Ac, (213)Bi, (211)At, and (223)Ra, the alpha-particle--emitting radionuclides of interest in radionuclide therapy. METHODS: The dose contribution of daughter radionuclides at the site of parent decay was made dependent on a cutoff time parameter, which was used to estimate the fraction of daughter decays expected at the site of parent decay. Previously tabulated S values (cell-surface to nucleus and cell-surface to cell) for each daughter in the decay scheme were scaled by this fraction and a sum over all daughters was performed to yield a cutoff time--dependent set of corresponding DCF values for each radionuclide. RESULTS: DCF values for the absorbed dose to the nuclear or cellular volume from cell-surface decays are presented as a function of the cutoff time for 4 different cellular and nuclear dimensions. CONCLUSION: In contrast to the cellular S values that account only for parent decay, the DCF values provided in this study make it possible to easily include the contribution of daughter decays in cellular alpha-particle emitter dose calculations.