Multitracer screening for in vivo element-element interrelations

Radioactive multitracer technique was applied to study the screening of in vivo interrelations between radioactive tracers (⁴⁶Sc, ⁵⁴Mn, ⁵⁸Co, ⁶⁵Zn, ⁷⁵Se, ⁸³Rb, ⁸⁵Sr and ⁸⁸Zr) and stable Zn species. Comparative uptake rates were examined in the blood, nine organs (thymus, lung, cardiac muscle, spleen, pancreas, kidney, liver, testes and bone) and eight brain regions (cerebral cortex, striatum, hippocampus, thalamus and hypothalamus, midbrain, cerebellum, pons and medulla, olfactory bulb) using 3-week-old mice fed by four kinds of Zn-deficient, -adequate and -excessive diets with Zn content from 0.7 to 3520 ppm. As a result, no significant difference between the dietary Zn-deficient state (Zn content: 3.6 ppm) and Zn-adequate state (Zn content: 36 ppm) was observed in the uptake rates of ⁶⁵Zn and other ⁴⁶Sc, ⁵⁴Mn, ⁵⁸Co and ⁷⁵Se, except for ⁸³Rb. In addition, significant differences among the organ and brain regional uptakes of ⁴⁶Sc, ⁵⁴Mn, ⁵⁸Co, ⁷⁵Se and ⁸³Rb were found in the dietary Zn-excessive state (Zn content: 3520 ppm). These results indicate that the organ and brain regional uptakes of tracers in Zn-deficient and excessive mice are strongly correlated with the blood uptakes and retentions of the tracers. Furthermore, the multitracer screening gives us new findings concerning the diet-related element-element interrelations in living bodies.     

Multitracer study on transport and distribution of metal ions in plants

Transport and distribution of metal ions in rice and soybean plants were studied using multitracers produced by irradiating an Au target by 135 MeV/nucleon¹²C,¹⁴N, or¹⁶O ions accelerated by RIKEN Ring Cyclotron. The multitracer consisted of radioisotopes of the following elements: Be, Na, Sc, Mn, Fe, Co, Zn, Se, Rb, Sr, Y, Zr, Nb, Ag, Te, Ba, Ce, Pm, Eu, Gd, Tb, Tm, Yb, Lu, Hf, Ir, and Pt. Rice and soybean plants were grown in a nutrient solution and also on soil containing a multitracer. -ray spectroscopy of different parts of the plants showed that all the elements were more or less taken up by roots, while appreciable transport to leaves and seeds was observed only for Mn, Zn, Se, Rb, Sr, and Ba.     

Multitracer study on the behavior of various elements in atmosphere plant-systems

The direct absorption of atmospheric elements via soybean leaves has been studied using a radioactive multitra r technique. Soybean was cultivated until it bore seeds in a box under no-rain conditions and with introduction of multitracer-adsorbed cellulose powders. The radioactive nuclides of 40 elements were produced from Au target irradiated with¹⁴N, and the nuclides with relatively long half-life of the elements Sc, Mn, Co, As, Se, Rb, Sr, Y, Eu, Gd, Yb, Re, and Ir were dominantly observed in each part of soybean plant.     

Multitracer study on distribution of carrier-free radioactive isotopes in organs of rats

Multitracer, a new tracer technique developed by us, can analyze metabolism and behavior of different elements in the living bodies simultaneously. We were able to reproduce completely the results on the behavior of various elements obtained by individual experiments of our precursors only by a single run of this technique. At the same time, we could also show the behavior and interaction of many other trace elements which past researchers did not pay attention to. For example, our results on the behavior of rare-earth elements suggested that there is a correlation between their uptakes and ion radii. We also discovered that affinities of platinum and iridium for their binding proteins were changed, the latter being increased, in the case of zinc deficiency. In this article, we discuss the usefulness of the multitracer technique in the studies of trace elements in living bodies.     

In vivo toxicity screening programs using metabonomics

Metabonomics is an emerging technology that enables rapid in vivo screening for toxicity, disease state, or drug efficacy. The technology combines the power of high-resolution nuclear magnetic resonance (NMR) techniques with statistical data analysis methods to rapidly evaluate the metabolic "status" of an animal. Complimentary to other profiling technologies like proteomics and genomics, metabonomics provides a fingerprint of the small-molecules contained in a given biofluid through the time course of a study. This article reviews the steps in implementing a metabonomics-based screening program from study design through data analysis. While metabonomics is still a relatively new technology in comparison to the other "omics", published results from metabonomics studies demonstrate its potential impact in the drug discovery process by enabling the incorporation of safety endpoints much earlier in the drug discovery process, reducing the likelihood (and cost) of later stage attrition.     

Multitracer study on permeation of rare earth elements through a supported liquid membrane

Separation by means of supported liquid membranes is a useful method for the preparation and preconcentration of radioactive nuclides. The permeation of rare earth elements through a bis(2-ethylhexyl) hydrogen phosphate-decalin membrane supported on a microporous polytetrafluoroethylene sheet was studied using a multitracer containing radioactive nuclides of Sc, Zr, Nb, Hf, Ce, Pm, Gd, Yb, and Lu. Permeation rates of these elements from feed solutions of various acidity to receiving solutions of 0.5 mol·dm⁻³ HCl were determined simultaneously. The feed solution at pH 1.4 gave the highest permeation rate for Ce, Pm, and Gd, amounting to about 95% of permeation for Ce and Pm, 80% for Gd, and 10% for Yb in 21 h. Scandium, Zr, Nb, Hf, and Lu were not transported at all from the feed solution. Permeation rates of Yb and Lu from the feed solution at pH 1.4 to receiving solutions of 0.75, 1.0, 2.3, and 4.0 mol·dm⁻³ HCl increased with the concentration. The results obtained indicate that the light rare earth elements can be separated from the heavy ones by this method.     

Streamlined in vivo selection and screening of human prostate carcinoma avid phage particles for development of peptide based in vivo tumor imaging agents

Bacteriophage (phage) display has been exploited for the purpose of discovering new cancer specific targeting peptides. However, this approach has resulted in only a small number of tumor targeting peptides useful as in vivo imaging agents. We hypothesize that in vivo screening for tumor uptake of fluorescently tagged phage particles displaying multiple copies of an in vivo selected tumor targeting peptide will expedite the development of peptide based imaging agents. In this study, both in vivo selection and in vivo screening of phage displaying foreign peptides were utilized to best predict peptides with the pharmacokinetic properties necessary for translation into efficacious in vivo imaging agents. An in vivo selection of phage display libraries was performed in SCID mice bearing human PC-3 prostate carcinoma tumors. Eight randomly selected phage clones and four control phage clones were fluorescently labeled with AlexaFluor 680 for subsequent in vivo screening and analyses. The corresponding peptides of six of these phage clones were tested as 111In-labeled peptide conjugates for single photon emission computed tomography (SPECT) imaging of PC-3 prostate carcinomas. Two peptide sequences, G1 and H5, were successful as in vivo imaging agents. The affinities of G1 and H5 peptides for cultured PC-3 cells were then analyzed via cell flow cytometry resulting in Kd values of 1.8 μM and 2.2 μM, respectively. The peptides bound preferentially to prostate tumor cell lines compared to that of other carcinoma and normal cell lines, and H5 appeared to possess cytotoxic properties. This study demonstrates the value of in vivo screening of fluorescently labeled phage for the prediction of the efficacy of the corresponding 111In-labeled synthetic peptide as an in vivo SPECT tumor imaging agent.     

Bond length interrelations in benzenoid hydrocarbons and their heteroatom analogues

The geometries of some benzenoid hydrocarbons and their analogues with XY (XY = BB, BC, BN, CN, and NN) bonds have been determined at the B3LYP/6-311++G^∗∗ level of theory. It is shown that the lengths of peripheral XY bonds are strictly correlated with the lengths of their CC counterparts in native hydrocarbons. No correlation of this type is observed for the XY bonds located inside the rings.     

Multitracer studies on the permeation of various elements through a supported liquid membrane containing TBP

The permeation of various elements through a tributyl phosphate (TBP)-decalin membrane supported on a microporous polytetrafluoroethylene sheet was studied using a multitracer. Permeation rates of elements from feed solutions to distilled water as a receiving agent were determined by changing the HCl concentration in feed solutions. An increase in the transport was observed for most of the elements studied with increasing HCl concentration in the feed solutions. Among them, Fe, Zn, Se, Zr, Nb, Te, Hf and Re gave relatively high percentages of permeation from 4-12 mol^.dm^-3 HCl feed solutions. The permeability coefficients of these elements were determined.     

New aldehyde tag sequences identified by screening formylglycine generating enzymes in vitro and in vivo

Formylglycine generating enzyme (FGE) performs a critical posttranslational modification of type I sulfatases, converting cysteine within the motif CxPxR to the aldehyde-bearing residue formylglycine (FGly). This concise motif can be installed within heterologous proteins as a genetically encoded "aldehyde tag" for site-specific labeling with aminooxy- or hydrazide-functionalized probes. In this report, we screened FGEs from M. tuberculosis and S. coelicolor against synthetic peptide libraries and identified new substrate sequences that diverge from the canonical motif. We found that E. coli's FGE-like activity is similarly promiscuous, enabling the use of novel aldehyde tag sequences for in vivo modification of recombinant proteins.     

Activation analysis and radiochemistry,comments to their interrelations

It is the aim of this paper, to demonstrate the manyfold interrelation between radiochemistry and activation analysis. One of the main problems of both fields is the behaviour of submicro concentrations of elements and of their compounds in solids, in solution and in the gas phase. The radiochemists were the first, who have accumulated much experience, investigating the properties of the natural radionuclides. HEVESY has, somewhat later, by his proposal of activation analysis, pointed out a special and powerful way for the determination of submicro amouts of substances. In the further development till to the present day, both fields have stimulated each other. Their exponents are specialists in understanding and handling submicro concentrations, i.e. in a section of science and technology, growing up to an outstanding importance.     

Design of a compound screening collection for use in high throughput screening

In this paper we introduce a quantitative model that relates chemical structural similarity to biological activity, and in particular to the activity of lead series of compounds in high-throughput assays. From this model we derive the optimal screening collection make up for a given fixed size of screening collection, and identify the conditions under which a diverse collection of compounds or a collection focusing on particular regions of chemical space are appropriate strategies. We derive from the model a diversity function that may be used to assess compounds for acquisition or libraries for combinatorial synthesis by their ability to complement an existing screening collection. The diversity function is linked directly through the model to the goal of more frequent discovery of lead series from high-throughput screening. We show how the model may also be used to derive relationships between collection size and probabilities of lead discovery in high-throughput screening, and to guide the judicious application of structural filters.     

Structure-based discovery of novel non-nucleosidic DNA alkyltransferase inhibitors: virtual screening and in vitro and in vivo activities

The human DNA-repair O (6)-alkylguanine DNA alkyltransferase (MGMT or hAGT) protein protects DNA from environmental alkylating agents and also plays an important role in tumor resistance to chemotherapy treatment. Available inhibitors, based on pseudosubstrate analogs, have been shown to induce substantial bone marrow toxicity in vivo. These deficiencies and the important role of MGMT as a resistance mechanism in the treatment of some tumors with dismal prognosis like glioblastoma multiforme, the most common and lethal primary malignant brain tumor, are increasing the attention toward the development of improved MGMT inhibitors. Here, we report the identification for the first time of novel non-nucleosidic MGMT inhibitors by using docking and virtual screening techniques. The discovered compounds are shown to be active in both in vitro and in vivo cellular assays, with activities in the low to medium micromolar range. The chemical structures of these new compounds can be classified into two families according to their chemical architecture. The first family corresponds to quinolinone derivatives, while the second is formed by alkylphenyl-triazolo-pyrimidine derivatives. The predicted inhibitor protein interactions suggest that the inhibitor binding mode mimics the complex between the excised, flipped out damaged base and MGMT. This study opens the door to the development of a new generation of MGMT inhibitors.     

Biosensor-guided screening for macrolides

Macrolides are complex polyketides of microbial origin that possess an extraordinary variety of pharmacological properties, paired with an impressive structural diversity. Bioassays for specific detection of such compounds will be of advantage for a class-specific drug screening. The current paper describes a cell-based microbial biosensor, assigning a luminescence response to natural or chemically modified macrolides, independent from their biological activity. This biosensor is based on the coupling of the structural luciferase genes of Vibrio fischeri to the regulatory control mechanism of a bacterial erythromycin resistance operon. The bioassays is easy to handle and can be applied to various screening formats. The feasibility of the test system for natural products screening is exemplified by the isolation and characterization of picromycin from a Streptomyces species. Biosensor-guided screening for macrolides is based on macrolide-promoted expression of lux genes and induction of luminescence (independent of macrolide antibiotic activity).     

Two-photon Lysotrackers for in vivo imaging

We report two-photon Lysotrackers (CLT-blue and CLT-yellow) that can be excited by 750-840 nm femtosecond laser pulses and emit at 470 and 550 nm, respectively. They can be easily loaded into cells and tissue slices for visualization of lysosomes in live cells and tissues for a long period of time through two-photon microscopy. When combined with appropriate two-photon probes for other biological targets, these novel probes would greatly facilitate the two-photon microscopy colocalization experiments.     

ROCS-derived features for virtual screening

Rapid overlay of chemical structures (ROCS) is a standard tool for the calculation of 3D shape and chemical (“color”) similarity. ROCS uses unweighted sums to combine many aspects of similarity, yielding parameter-free models for virtual screening. In this report, we decompose the ROCS color force field into color components and color atom overlaps, novel color similarity features that can be weighted in a system-specific manner by machine learning algorithms. In cross-validation experiments, these additional features significantly improve virtual screening performance relative to standard ROCS.