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.

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.

Response of LNCaP spheroids after treatment with an alpha-particle emitter (213Bi)-labeled anti-prostate-specific membrane antigen antibody (J591).

A theoretical drawback to alpha-particle therapy with 213Bi is the short range of the particle track coupled with the short half-life of the radionuclide, thereby potentially limiting effective cytotoxicity to rapidly accessible, disseminated individual tumor cells (e.g., as in leukemia). In this work, a prostate carcinoma spheroid model was used to evaluate the feasibility of targeting micrometastatic clusters of tumor cells using 213Bi-labeled anti-prostate-specific membrane antigen (PSMA) antibody, J591. In prostate cancer, vascular dissemination of tumor cells or tumor cell clusters to the marrow constitutes an important step in the progression of this disease to widespread skeletal involvement, an incurable state. Such prevascularized clusters are ideal targets for radiolabeled antibodies because the barriers to antibody penetration that are associated with the capillary basal lamina have not yet formed. Beta- and gamma-emitting radionuclides such as 131I, which are widely used in radioimmunotherapy, are not expected to be effective when targeting single cells or small cell clusters. This is because the range of the emissions is one to two orders of magnitude greater than the target size, and the energy deposited per traversal is insufficient to produce any significant radiobiological effect. Spheroids of the prostate cancer cell line, LNCaP-LN3, were used as a model of prevascularized micrometastases; their response to an anti-PSMA antibody, J591, radiolabeled with the alpha-particle emitter 213Bi (T(1/2), 45.6 min.) has been measured. The time course of spheroid volume reductions was found to be sensitive to the initial spheroid volume. J591 labeled with 0.9 MBq/ml 213Bi resulted in a 3-log reduction in spheroid volume on day 33, relative to control, for spheroids with an initial diameter of 130 microm; 1.8 MBq/ml were required to achieve a similar response for spheroids with an initial diameter of 180 microm. Equivalent spheroid responses were observed after 12 Gy of acute external beam photon irradiation. Monte Carlo-based microdosimetric analyses of the 213Bi decay distribution in individual spheroids of 130-microm diameter yielded an average alpha-particle dose of 3.7 Gy to the spheroids, resulting in a relative biological effectiveness factor of 3.2 over photon irradiation. The activity concentrations used in the experiments were clinically relevant, and this work supports the possibility of using 213Bi-labeled antibodies not only for disseminated single tumor cells, as found in patients with leukemia, but also for micrometastatic tumor deposits up to 180 microm in diameter (1200 cells).

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.

Estimation of local cerebral glucose utilization by positron emission tomography: comparison of [18F]2-fluoro-2-deoxy-D-glucose and [18F]2-fluoro-2-deoxy-D-mannose in patients with focal brain lesions.

A comparative PET study of [18F]2-fluoro-2-deoxy-D-glucose (FDG) and [18F]2-fluoro-2-deoxy-D-mannose (FDM) uptake was performed in 13 patients with focal brain lesions. Differences between FDG and FDM with respect to model rate constants, lumped constant, and estimated metabolic rate for glucose were determined on a regional basis. Across whole brain, the transport rate constant K1* was almost unchanged, whereas k2*, describing the transport back from tissue to plasma, was 6% higher, and the phosphorylation rate constant k3* was 9% lower for FDM compared to FDG. This implies a 20% lower lumped constant for FDM. No significant regional variability of this differential tracer behavior was observed in normal or in lesioned brain tissue. Thus, results from previous FDG studies, where the radiotracer was not 100% pure FDG but contained varying amounts of FDM, can easily be corrected by adjustment of the lumped constant employed in metabolic quantitation.

Efficient stereospecific synthesis of no-carrier-added 2-[18F]-fluoro-2-deoxy-D-glucose using aminopolyether supported nucleophilic substitution.

An aminopolyether mediated synthesis of fluorine-18 (18F) 2-fluoro-2-deoxy-D-glucose (FDG) has been developed. The nucleophilic fluorination with accelerator-produced [18F]fluoride works at the no-carrier-added level and gives epimerically pure 2-18FDG with an uncorrected radiochemical yield of a maximum 50% in a synthesis time of approximately 50 min from EOB.

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.

A cell-culture reactor for the on-line evaluation of radiopharmaceuticals: evaluation of the lumped constant of FDG in human glioma cells.

UNLABELLED: A fluidized-bed cell-culture reactor with on-line radioactivity detection was developed for the in vitro evaluation of radiopharmaceuticals. The technique was applied to measure the dependency of the lumped constant (LC) of FDG on the glucose concentration in the culture medium in a human glioma cell line. METHODS: Human glioblastoma cells (86HG39) immobilized in open porous microcarriers were cultivated in a continuously operating fluidized-bed bioreactor. At different glucose concentrations in the culture medium, step inputs (0.1 MBq/mL) of FDG were performed and the cellular uptake of FDG was measured on-line and compared with analyzed samples. From these results, the LC of FDG and its dependency on the glucose concentration were calculated. RESULTS: This fluidized-bed technique enabled precise and reproducible adjustment of all relevant experimental parameters, including radiotracer time-concentration course, medium composition, pH, dissolved oxygen and temperature under steady-state conditions, and an on-line determination of the intracellular radiotracer uptake. The immobilized glioma cells formed stable, 3-dimensional, tumor-like spheroids and were continuously proliferating, as proven by an S-phase portion of 25%-40%. For further examination of the cells, an enzymatic method for detachment from the carriers without cellular destruction was introduced. In the FDG experiments, a significant dependency of the LC on the glucose level was found. For normoglycemic glucose concentrations, the LC was determined to be in the range of 0.7+/-0.1, whereas in hypoglycemia LC increased progressively up to a value of 1.22+/-0.01 at a glucose concentration of 3 mmol/L. CONCLUSION: The bioreactor represents an improved in vitro model for the on-line evaluation of radiotracers and combines a wide range of experimental setups and 3-dimensional, tissue-like cell cultivation with a technique for on-line radioactivity detection.

Noninvasive assessment of regional cardiac adenosine using positron emission tomography.

One of the early metabolic changes associated with myocardial ischemia is the breakdown of adenine nucleotides resulting in the enhanced production of adenosine. In order to image regional cardiac adenosine by positron emission tomography (PET) the enzymatic conversion of adenosine into [11C]-S-adenosylhomocysteine ([11C]SAH) was used in the presence of 11C-labeled homocysteine thiolactone (adenosine + [11C] - homocysteine-->[11C] - SAH + H2O). Following production of an experimental coronary constriction in anesthetized dogs carrier added 1-[11C]-D,L-homocysteine thiolactone (5-27 mCi, 30 mg/kg) was infused over 1 min. This intervention, while hemodynamically ineffective, increased the plasma homocysteine concentration from 2.5 to 306 microM, which thereafter declined with a T1/2 of 28 min to 97 microM after 60 min. During the first minutes following infusion of [11C] homocysteine, the radioactivity concentration in the blood pool, the nonischemic and the ischemic myocardium were similar. Between 20 and 60 min, however, the regional radioactivity concentration was highest in the perfusion area of the stenosed vessel: 6.6% compared to 5.2 and 5.2% of the injected dose per 1 I tissue. The elevated radioactivity concentration was strictly confined to the perfusion area of the occluded artery. Using [35S]-L-homocysteine (20 microCi; 30 mg/kg) chromatographic separation of SAH in tissue extracts confirmed that the radioactivity accumulation was due to trapping of adenosine in the cellular SAH-pool. These experiments provide first evidence that 1-[11C]homocysteine thiolactone can be successfully used to assess regional adenosine formation in the heart with PET via measurement of [11C] SAH accumulation.

Parametric images of antibody pharmacokinetics in Bi213-HuM195 therapy of leukemia.

UNLABELLED: Kinetic analysis of gamma camera patient images can provide time-dependent information about antibody behavior. Current region-of-interest-based techniques for the kinetic analysis of these images rely on user selection and drawing of regions to be analyzed. Such analyses do not reveal unexpected kinetic activity outside of the selected regions of interest and do not provide a whole-image assessment regarding the pharmacokinetics of an agent. At Memorial Sloan-Kettering Cancer Center, a method for generating images in which the pixel value represents a kinetic parameter has been developed. This work extends the method into a new application in which whole-body parametric images are used to examine the kinetics of Bi213-HuM195 in patients with leukemia. METHODS: Bi213-HuM195 is typically administered in multiple injections over 2-4 d, yielding a progressive increase in the amount of antibody administered. Patients are injected with individual doses while positioned in a gamma camera, and imaging is initiated at the start of the injection. The acquisition is performed in dynamic mode with images collected at several time intervals over 1 h. Using software developed in-house, images are corrected for patient movement through iterative alignments, decay corrected, and summed to yield a series of images over regular time intervals. Parametric rate images are obtained by fitting a linear expression to the counts in each pixel. In this study, rate images from a patient's first injection were compared with rate images from the last injection. RESULTS: The conventional planar images of antibody distribution showed significant uptake in liver, spleen, and marrow, whereas the generated rate images displayed different patterns, sometimes with negative values in liver and spleen and positive values in marrow, reflecting clearance and uptake rates rather than total accumulation. The impact of the progressive increase in antibody administration was observed by comparing the first with the last rate images. Interpatient comparisons were also made and showed that rate image patterns varied depending on patient-specific conditions such as the amount of disease and previous therapies undergone by the patient. CONCLUSION: Rate images make it possible to succinctly display kinetic information about an agent's behavior over the entire acquired image.

Uptake of cis-4-[18F]fluoro-L-proline in urologic tumors.

UNLABELLED: Tumor uptake of the amino acid cis-4-[18F]fluoro-L-proline (cis-FPro) was studied with PET in eight patients with urologic tumors. METHODS: Three patients had primary renal cell carcinomas (RCCs), one had a local recurrence of RCC, one had squamous RCC, one had an adrenal hemangioma, one had inguinal metastases of penile squamous carcinoma, and one had suspected metastatic disease from prostate cancer. PET scans of the trunk were acquired at 1 and 3-5 h after intravenous injection of 400 MBq cis-FPro and compared with 18F-FDG PET scans and CT. RESULTS: None of the tumors or metastases showed significant uptake of cis-FPro. FDG uptake was seen in one of the three primary RCCs, in the local recurrence of RCC, in the squamous RCC, and in the metastases of penile cancer. CONCLUSION: Cis-FPro appears not to be a promising PET tracer in oncology.

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.

A schema for estimating absorbed dose to organs following the administration of radionuclides with multiple unstable daughters: a matrix approach.

The dosimetry of alpha particle emitters requires that all decays, including those of unstable intermediates be included in the calculation. Such calculations are complicated by the potential differential biologic distribution of each of the intermediates. In this work we present a formalism which will account for the known biodistribution factors of the daughters and the resulting effective biodistribution which will depend upon the site at which the parent radionuclide decays. The number of decays or cumulated activity of a daughter radionuclide present in a particular tissue is estimated using a probability matrix which describes the likelihood of daughter decay in a particular tissue as a function of the decay site of the parent. An example of three initial compartments is provided to illustrate the use of this formalism. Such modeling may be used to evaluate the feasibility of using radionuclides whose decay includes alpha-emitting intermediates. Model validation and refinement will require an assessment of the fate of free, alpha-emitting intermediates in various biological milieus.

A comparative study of N.C.A. fluorine-18 labeling of proteins via acylation and photochemical conjugation.

Three methods for 18F-labeling of proteins were evaluated with respect to conjugation yields, suitability for remote-controlled routine synthesis, and in vivo stability of the conjugates-i.e., photochemical conjugation (PCC) using 4-azidophenacyl-[18F]fluoride ([18F]APF) as well as classical conjugation using 4-nitrophenyl 2-[18F]fluoropropionate ([18F]NPFP) and N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB). For this purpose, [18F]APF was synthesized in one step with a radiochemical yield (RCY) of up to 70% within about 15 min. The 18F-labeling was performed by photogeneration of the corresponding [18F]arylnitrene by irradiating [18F]APF with UV light in presence of the protein in aqueous buffered solution. Using this procedure, human serum albumin (HSA), transferrin, IgG, and avidin were labeled. The [18F]NPFP was synthesized according to a recently published method. Preparation of [18F]SFB was achieved within 35 min with radiochemical yields of 55 +/- 10% by an improved method using O-(N-succinimidyl)-N-N,N',N'-tetramethyluronium tetrafluoroborate (TSTU) as activating reagent. Compared to [18F]APF, protein labeling with [18F]NPFP and [18F]SFB gave rise to considerably higher RCY, of up to 90%. Labeling studies showed that conjugation yields using [18F]NPFP depend on the lysine, tyrosine, and histidine content of the proteins used, whereas conjugation with [18F]APF and [18F]SFB predominantly depends on the Lys content. Owing to competing O-acylation of Tyr residues, [18F]fluoropropionylated HSA was partially unstable under slightly basic conditions. Biodistribution studies with 18F-labeled HSA in NMRI mice revealed the highest in vivo stability for the [18F]SFB conjugate. Based on these results, [18F]SFB seems to be the most suitable 18F-labeling agent for proteins, particularly for the labeling of antibodies.

Preclinical evaluation of 4-[18F]fluoroprolines: diastereomeric effect on metabolism and uptake in mice.

The aim of this study was to evaluate the diastereomeric effect on uptake and metabolic behavior of (2S,4R)-4-[18F]fluoro-L-proline (trans-[18F]FPro) and (2S,4S)-4-[18F]fluoro-L-proline (cis-[18F]FPro) in view of their potential suitability as tracers for abnormal collagen synthesis. No-carrier-added 4-[18F]fluoro-L-prolines were prepared according to the literature in about 150 min (50-60% radiochemical yield). Both compounds exhibited high in vivo stability. The tumor uptake of cis-[18F]FPro in osteosarcomas of mice was high and at 240 min postinjection reached 11.8 +/- 2.2 %ID/g compared with 7.07 +/- 1.68 %ID/g for trans-[18F]FPro. In contrat to trans-[18F]FPro, which showed fast and complete renal clearance, the cis isomer was accumulated in the pancreas, and showed hepatic clearance and renal reuptake. Speciation studies on tissue homogenates revealed protein incorporation only for cis-[18F]FPro. However, due to the relatively slow protein incorporation rate of cis-[18F]FPro, the tumor uptake of both compounds in colon carcinomas, mammary carcinomas, and osteosarcomas 1 h postinjection predominantly reflect amino acid transport.

Whole-body kinetics and dosimetry of cis-4-[(18)F]fluoro-L-proline.

The whole-body distribution of 4-cis[(18)F]fluoro-L-proline (cis-FPro) was studied in six patients with urological tumors by PET. Based on the IMEDOSE and MIRDOSE procedures radiation absorbed doses were estimated from whole-body PET scans acquired at 1 and 3-5 h after i.v. injection of 400 MBq cis-FPro. Cis-FPro showed high retention in the renal cortex and a slight uptake in liver and pancreas. Urinary excretion ranged from 12 to 19% at 5 h p.i. Highest absorbed doses were found for the urinary bladder wall and the kidneys (44.1/44.0 microGy/mbq). The effective dose according to ICRP 60 was 15.1 microSv/mbq for adults. This leads to an effective dose of 6.0 mSv in a PET study using 400 MBq cis-FPro.