Nuclear transport of the human aryl hydrocarbon receptor and subsequent gene induction relies on its residue histidine 291.

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor involved in the metabolism of physiological substances and xenobiotics, representing an interesting target in both toxicology and pharmacology. In this study, we investigated the ligand-dependent conjunction of nuclear import of the human AHR in living cells and target gene induction. Our findings strengthen the theory that the AHR triggers a precisely defined and rapid reaction upon binding to endogenous ligands, while the xenobiotic ß-naphthoflavone only induces rather unspecific and slow effects. To better illuminate the ligand-mediated responses of the human AHR, we applied site-directed mutagenesis and identified histidine 291 as key residue for AHR functionality, essential for both nuclear import and target gene induction. Contrary, replacing histidine at position 291 by alanine did not affect nucleo-cytoplasmic shuttling, showing that permanent endogenous import and ligand-induced import of the AHR into the nucleus are two independent and differently regulated processes. Combining these observations with our structural investigations using a homology model of the AHR-PAS B domain, we suggest a dual role of histidine 291: (1) a major role for shaping the ligand binding site including direct interactions with ligands and, (2) an essential role for the conformational dynamics of a PAS B loop, which most likely influences the association of the AHR with the AHR nuclear translocator through interference with their protein-protein interface.

Enhanced immunostimulatory activity of in silico discovered agonists of Toll-like receptor 2 (TLR2).

BACKGROUND: Emergent therapies in anticancer vaccination use Toll-like receptors (TLRs) agonists as dendritic cell (DC) vaccine adjuvants. DCs from the patient are isolated, stimulated with TLR agonists and tumor antigens ex vivo and then infused back into the patient. Although some TLR ligands have been tested in clinical trials, novel TLR agonists with improved immunomodulatory properties are essential to optimize treatment success. We report on the discovery of small-molecule TLR2 agonists, with favorable properties as synthetic adjuvants. METHODS: We performed a shape- and featured-based similarity virtual screening against a commercially available compound library. The selected virtual hits were experimentally tested in TLR2-reporter cells and their activity in phagocytes and DCs was characterized. A binding model of the compounds to TLR2 (docking studies) was proposed. RESULTS: Through a virtual screening approach against a library of three million compounds four virtual hits (AG1, AG2, AG3, AG4) were found to synergistically augment the NF-kB activation induced by the lipopeptide ligand Pam3CSK4 in luciferase reporter assays using HEK293-TLR2 cells. Biacore experiments indicated that AG1-AG4 are ago-allosteric modulators of TLR2 and AG2 bound TLR2 with high affinity (KD 0.8µM). The compounds induced TNF-α production in human peripheral blood mononuclear cells (PBMCs) and they activated DCs as indicated by IL-12 production and upregulation of CD83/CD86. CONCLUSIONS: Following a combined in silico/in vitro approach we have discovered TLR2-agonists (AG1-AG4) that activate human and mouse immune cells. GENERAL SIGNIFICANCE: We introduce four novel TLR2 ago-allosteric modulators that stimulate myeloid cell activity and constitute promising candidates as synthetic adjuvants.

Ecdysteroids: A novel class of anabolic agents?

Increasing numbers of dietary supplements with ecdysteroids are marketed as "natural anabolic agents". Results of recent studies suggested that their anabolic effect is mediated by estrogen receptor (ER) binding. Within this study the anabolic potency of ecdysterone was compared to well characterized anabolic substances. Effects on the fiber sizes of the soleus muscle in rats as well the diameter of C2C12 derived myotubes were used as biological readouts. Ecdysterone exhibited a strong hypertrophic effect on the fiber size of rat soleus muscle that was found even stronger compared to the test compounds metandienone (dianabol), estradienedione (trenbolox), and SARM S 1, all administered in the same dose (5 mg/kg body weight, for 21 days). In C2C12 myotubes ecdysterone (1 µM) induced a significant increase of the diameter comparable to dihydrotestosterone (1 µM) and IGF 1 (1.3 nM). Molecular docking experiments supported the ERß mediated action of ecdysterone. To clarify its status in sports, ecdysterone should be considered to be included in the class "S1.2 Other Anabolic Agents" of the list of prohibited substances of the World Anti-Doping Agency.

Binding characteristics of [3H]-JSM10292: a new cell membrane-permeant non-peptide bradykinin B2 receptor antagonist.

BACKGROUND AND PURPOSE: A (3) H-labelled derivative of the novel small-molecule bradykinin (BK) B(2) receptor antagonist JSM10292 was used to directly study its binding properties to human and animal B(2) receptors in intact cells and to closely define its binding site. EXPERIMENTAL APPROACH: Equilibrium binding, dissociation and competition studies with various B(2) receptor ligands and [(3) H]-JSM10292 were performed at 4°C and 37°C. The experiments were carried out using HEK293 cells stably (over)expressing wild-type and mutant B(2) receptors of human and animal origin. KEY RESULTS: [(3) H]-JSM10292 bound to B(2) receptors at 4°C and at 37°C with the same high affinity. Its dissociation strongly depended on the temperature and increased when unlabelled B(2) receptor agonists or antagonists were added. [(3) H]-JSM10292 is cell membrane-permeant and thus also bound to intracellular, active B(2) receptors, as indicated by the different 'nonspecific' binding in the presence of unlabelled JSM10292 or of membrane-impermeant BK. Equilibrium binding curves with [(3) H]-JSM10292 and competition experiments with unlabelled JSM10292 and [(3) H]-BK showed a different affinity profile for the wild-type B(2) receptor in different species (man, cynomolgus, rabbit, mouse, rat, dog, pig, guinea pig). Characterization of B(2) receptor mutants and species orthologues combined with homology modelling, using the CXCR4 as template, suggests that the binding site of JSM10292 is different from that of BK but overlaps with that of MEN16132, another small non-peptide B(2) receptor ligand. CONCLUSIONS AND IMPLICATIONS: [(3) H]-JSM10292 is a novel, cell membrane-permeant, high-affinity B(2) receptor antagonist that allows direct in detail studies of active, surface and intracellularly located wild-type and mutant B(2) receptors.

Natural products in structure-assisted design of molecular cancer therapeutics.

Since the late 1990's, novel insights into molecular biology and carcinogenesis enabled the rational design of mechanism-based anticancer therapeutics. The large number of natural product (NP)-derived drugs currently under clinical evaluation and the recent approval of temsirolimus (Torisel) as a first mTOR protein kinase inhibitor indicate that NPs have to be considered not only as a seminal source of cytotoxic, but also as a source of molecularly targeted agents. Whereas molecular modeling is well established as an important and successful method to discover and rationalize bioactivities in medicinal chemistry research, its application has also proven to be also a powerful tool in the field of NPs. This review highlights the impact of computer-assisted approaches on NPs as molecularly targeted anticancer drugs. Examples of applications are provided focusing on innovative targets such as protein kinases, tumour vasculature, epigenetic modulators, heat shock protein (Hsp) 90, and direct apoptosis enhancers.

Enhancing drug discovery through in silico screening: strategies to increase true positives retrieval rates.

Computational chemistry software for lead discovery has become well established in pharmaceutical industry and has found its way to the desktop computers of medicinal chemists for different purposes, providing insight on the mode of action and binding properties, and creating new ideas for lead structure refinement. In this review we investigate the performance and reliability of recent state-of-the-art data modeling techniques, as well as ligand-based and structure-based modeling approaches for 3D virtual screening. We discuss and summarize recently published success stories and lately developed techniques. Parallel screening is one of these emerging approaches allowing for efficient activity in silico profiling of several compounds against different targets or anti-targets simultaneously. This is of special interest to medicinal chemists, as the approach allows revealing unknown binding modes ('target-fishing') as well as integrated ADME profiling or--more generally--the prediction of off-target effects.

Pharmacophore definition and 3D searches.

The most common pharmacophore building concepts based on either 3D structure of the target or ligand information are discussed together with the application of such models as queries for 3D database search. An overview of the key techniques available on the market is given and differences with respect to algorithms used and performance obtained are highlighted. Pharmacophore modelling and 3D database search are shown to be successful tools for enriching screening experiments aimed at the discovery of novel bio-active compounds.:

Dose-dependent differential effects of low and pulsed dose-rate brachytherapy in a radioresistant syngenic rat prostate tumour model.

PURPOSE: To study the response of the Dunning prostate carcinoma (R3327-AT1 subline) to continuous low dose-rate (CLDR) and pulsed dose-rate (PDR) brachytherapy. MATERIALS AND METHODS: After subcutaneous tumour transplantation into the thigh of the Copenhagen rat, doses of 0, 20, 30, 40 and 50 Gy were applied to the tumour surface (tumour diameter 9+/-1mm). Eight animals were irradiated per dose group and exposure condition. Interstitial PDR ((192)Ir source, 37 GBq) and CLDR ((192)Ir seed, 150 MBq) brachytherapy were carried out with 0.75 Gy/pulse h(-1) and a dose-rate of 0.75Gyh(-1), respectively. Treatment response was assessed in terms of growth delay expressed as the time (T(5)) required for each tumour to reach five times the initial tumour volume. RESULTS: The median T(5) times for the CLDR groups (in the order: control, 20, 30, 40, 50 Gy) were 12 (12), 54.5 (21), 64.5 (31), 85.5 (51), and 65 (47.5) days. Values after PDR brachytherapy are given in parentheses and resulted in a significantly impaired tumour growth delay (log-rank test) in the 20Gy (p =0.006) and 30 Gy (p =0.036) groups. No significant difference was found in the 40-50 Gy dose range. CONCLUSIONS: In contrast to previous results and predictions of biological models we observed dose-dependent differential effects of PDR and CLDR brachytherapy with reduced efficacy of PDR in the lower dose range.

A comparison of gamma and neutron irradiation on Raji cells: effects on DNA damage, repair, cell cycle distribution and lethality.

The Comet assay (microgel electrophoresis) was used to study DNA damage in Raji cells, a B-lymphoblastoid cell line, after treatment with different doses of neutrons (0.5 to 16 Gy) or gamma rays (1.4 to 44.8 Gy). A better growth recovery was observed in cells after gamma-ray treatments compared with neutron treatments. The relative biological effectiveness (RBE) of neutron in cell killing was determined to be 2.5. Initially, the number of damaged cells per unit dose was approximately the same after neutron and gamma-ray irradiation. One hour after treatment, however, the number of normal cells per unit dose was much lower for neutrons than for gamma rays, suggesting a more efficient initial repair for gamma rays. Twenty-four hours after treatment, the numbers of damaged cells per unit dose of neutrons or gamma rays were again at comparable level. Cell cycle kinetic studies showed a strong G2/M arrest at equivalent unit dose (neutrons up to 8 Gy; gamma rays up to 5.6 Gy), suggesting a period in cell cycle for DNA repair. However, only cells treated with low doses (up to 2 Gy) seemed to be capable of returning into normal cell cycle within 4 days. For the highest dose of neutrons, decline in the number of normal cells seen at already 3 days after treatment was deeper compared with equivalent unit doses of gamma rays. Our present results support different mechanisms of action by these two irradiations and suggest the generation of locally multiply damaged sites (LMDS) for high linear energy transfer (LET) radiation which are known to be repaired at lower efficiency.

Morphometric analysis of bromodeoxyuridine distribution and cell density in the rat Dunning prostate tumor R3327-AT1 following treatment with radiation and/or hyperthermia.

To monitor cellular response to single doses of radiation (RT) and/or local tumor hyperthermia (LTH) proliferation kinetics were determined in the anaplastic prostate adenocarcinoma R3327-AT1 grown in Copenhagen rats. Tumor-bearing animals were injected i.v. with a bolus of bromodeoxyuridine (BrdUrd), and at defined times after treatment the tumors were surgically removed, fixed and embedded in paraffin. BrdUrd incorporated into the DNA of S-Phase nuclei was detected on 4-6 microns-thick tissue sections using a monoclonal anti-BrdUrd antibody followed by streptavidin-biotin and alkaline phosphatase as a reporter system. Cell nuclei were stained with the fluorescence dye DAPI (Diaminophenylindole). Morphometric analysis was performed using a computer-assisted Leitz-TAS/plus system. Depending on tumor size, up to 18,000 nuclei were routinely analyzed. Untreated tumors of standardized size (8-10 mm) exhibited a BrdUrd-labeling index (LI) of (6.9 +/- 1.6)%. In general, the LI was higher in the periphery than in the center, being more pronounced in larger tumors. After 6 Gy gamma-rays, the mean LI decreased to 1.8% (24 h) and rose afterwards to 5.4% by 168 h. Following LTH (43.5 degrees C, 35 min water bath), the mean LI rapidly decreased to 2% (8 h), rose to 9.8% (48 h), and plateaued at 6% after 168 h. A combined treatment consisting of irradiation (6 Gy) followed by LTH yielded smallest LI (2.4 +/- 0.18%) and lowest cell density (111 +/- 0.6 nuclei per field) by 168 h. The morphometric procedure was reliable and reproducible and can be used to characterize and compare the effects of different therapies on cell kinetics. Of particular value is that these analyses are done on an intact tissue architecture and hence enable a better interpretation of flow cytometric results of treatment-induced alterations within different topohistological regions in solid tumors. Moreover, the technique provides the basis for 3D reconstruction of the cellular activity and heterogeneity of experimental neoplasms.

Differential sensitivity of three sublines of the rat Dunning prostate tumor system R3327 to radiation and/or local tumor hyperthermia.

To better understand the relationship of the growth characteristics of tumor tissues and their response to ionizing radiation alone and in combination with local tumor hyperthermia, we compared three different tumor sublines of the Dunning rat prostate carcinoma R3327. This report includes results obtained with the anaplastic AT1 subline (volume doubling time 5.2 days), the moderately differentiated mucin-secreting HI subline (volume doubling time about 9 days) and the well-differentiated, hormone-dependent H subline (volume doubling time about 17 days). The effects of single doses of photons (10 to 40 Gy) with and without local tumor hyperthermia (35 min immersion at 43.5 degrees C) were quantified by growth delay. The time to reach five times the volume at the time of treatment after 30 Gy alone was found to be 56.0, 134.9 and 184.0 days for the R3327-AT1, HI and H tumors, respectively. The R3327-H tumor was more radiosensitive than the AT1 or HI subline. Five of nine R3327-H tumors were controlled locally with a single dose of photons (40 Gy). Local tumor hyperthermia alone induced growth delay in both differentiated tumors, while the anaplastic tumor subline did not respond. Combined treatment modalities with heat applied directly after irradiation revealed isoeffective thermal enhancement ratios for 30 Gy which decreased from 1.59 for the AT1 tumor and 1.42 for the HI tumor to 1.23 in the well-differentiated subline R3327-H.

Improved therapeutic response by distinct timing of multiple heat treatments during interstitial radiation in the Dunning R3327 prostate tumor model.

PURPOSE: As a continuation of a previous study showing the efficacy of a single local tumor heat treatment (LTH) in combination with interstitial radiation (IR) in the Dunning tumor system R3327 (subline AT1), we evaluated higher doses and/or lower dose rates with an extended time course of IR treatment, which allowed greater flexibility for LTH applications. METHODS: IR was carried out by the insertion of one removable 192Ir seed into the center of a R3327-AT1 tumor, transplanted s.c. into the distal thigh of Copenhagen rats. LTH (43.5 degrees C, for 35 min) varied from one treatment just before IR to multiple applications beginning at 0 h and repeated every 48 h or 72 h. RESULTS: The Dunning subline R3327-AT1 is a very thermoresistant tumor, which did not reveal any thermal response when heated up to 44.5 degrees C for 35 min. IR alone produced a delay in tumor growth, related to dose and dose rates of 18-53 cGy/h. During longer treatment times, a single LTH just before the IR was no more effective than IR alone. Thermoradiotherapy with multiple LTH treatments given at intervals of between 48 h and 72 h resulted in a clear increase in growth delay. Radiosensitization was highest in all dose-rate groups where LTH was applied every 72 h during a complete course of IR. CONCLUSIONS: These results demonstrate the importance of administering a sequence of multiple applications of LTH during continuous low-dose-rate irradiation and they substantiate our earlier findings, with shorter exposure times, where one LTH given every 72 h appeared to be most efficient in the combined treatment of the Dunning rat prostate tumor R3327-AT1.

A new fast-neutron source for radiobiological research.

A biomedical cyclotron facility primarily dedicated to radionuclide production has been extended by the addition of an experimental fast-neutron source for radiobiological and biophysical studies. Several beams of fast-neutrons with different average energies and LET distributions can now be provided. The neutrons are produced by bombarding berryllium targets with 18-32 McV protons or 8.7-15 MeV deuterons from our K = 32 negative-ion cyclotron. Average neutron energies range from approximately 4 to 15 MeV. Doses at maximum build-up vary from 0.24 to 1.85 cGy microA-1 min-1 at 1 m SSD, i.e. approximately 55 cGy min-1 at 30 microA of proton current at maximum energy. The design of the facility and some dosimetric results are presented.

Interstitial radiation and hyperthermia in the Dunning R3327 prostate tumour model: therapeutic efficacy depends on radiation dose-rate, sequence and frequency of heating.

To determine the most effective means by which to apply the combined treatments of local tumour hyperthermia (LTH) with interstitial low dose-rate irradiation (IRT) we examined the significance of such factors as dose-rate of radiation, and the sequence and frequency of hyperthermia applications in the anaplastic Dunning prostate tumour subline R3327-AT1. IRT was carried out by the insertion of a single Ir-192 seed into the center of the tumour. For LTH treatments, the tumour-bearing leg was positioned in a circulating constant temperature water bath (43.5 +/- 0.1 degrees C) for 35 min. Neither LTH treatment alone nor the insertion of a dummy seed produced any change in tumour growth compared with sham-treated controls. With regard to the sequence of heating and IRT our results showed that during a 72-h treatment time (30 Gy, 40 cGy/h) a single heat treatment given just before the start of IRT was more efficient (TER = 1.47) in terms of growth delay than LTH given in the middle or the end of radiation treatment (TER approximately 1.0). The growth delay for both the 20 and 40 cGy/h groups appear to be linear with increasing dose for the IRT as well as the IRT + LTH groups. The higher dose-rate was more effective especially with respect to long-term delay in tumour growth in some of the animals. As TER at 40 cGy/h decreased subsequently with increasing treatment time from 1.47 to 1.25 at 60 Gy, we conclude that for treatment times > 72 h, one LTH just before IRT might not be sufficient. If multiple heat treatments are applied during a comparable time course, two LTH treatments one just before the start, the other at the end yielded the greatest local tumour control. In contrast, three LTH given daily were not more effective than the one LTH given just before the start of IRT. These data indicate a clear thermal enhancement of low dose-rate irradiation, with maximal sensitization when hyperthermia was given just before IRT. For multiple heatings a better understanding of the underlying mechanisms of sequencing and timing hopefully provides guidelines how to apply optimally both modalities in the treatment of cancer.

Neutron dosimetry in french nuclear power plants. Problems and their solutions in 1995.

Personal neutron dosimetry in nuclear power plants is one of the hardest to achieve because there is no nuclear reaction that is both efficient and which ensures a suitable discrimination between 50 keV to 2 MeV fast neutrons and gamma photons. Neutron doses being low, the method used until now has been based on ambient dosimetry and the spent time in exposed areas. The advent of bubble detectors meets our immediate requirements until the direct reading electronic neutron dosimeter, which is still at the feasability research stage, can be utilized.

A variety of fast neutron beams for radiobiological research.

The design and construction of a new fast neutron facility and first dosimetric results obtained from seven neutron beams are presented. The neutrons are produced by bombarding beryllium targets with protons and deuterons from our K = 32 negative ion cyclotron. The dose rate in air 1 m distance from the thick target within a 13 x 13 cm2 field amounts to about 50 cGy/min at 30 microA of 32 MeV protons.

[The relative biological effectiveness (RBE) of fast neutrons on the Dunning rat prostate carcinoma R3327-HI]. / Relative biologische Wirksamkeit (RBW) von schnellen Neutronen auf das Dunning-Rattenprostatakarzinom R3327-HI.

Human prostate tumors are known to be good candidates for neutron therapy. The Dunning rat prostate tumor system R3327 was found in many studies to be an excellent model for human prostate tumors. There is still a paucity of studies on the response of the Dunning tumors to fast neutrons. Tumors of the R3327-HI subline are moderately well differentiated and mucin producing. They show one euploid cell population, a bromodeoxyuridine labelling index of 5%, a potential doubling time of 8.9 days, a volume doubling time of about ten days and a cell loss rate of 10%. Tumors were transplanted s.c. in the distal thigh of Copenhagen rats and treated with 60Co-photons (10, 20, 30, 40 Gy, 45 cGy/min) and 14-MeV-neutrons (8, 10, 12 Gy, 7 to 11 cGy/min). Tumor volumes were measured twice weekly. Growth delay was defined as time in days until the tumors reached twice their treatment volume. Linear regressions on the median growth delays of the different treatment groups were calculated. The ratio of the neutron- and photon-slopes yielded an RBE of 3.1 +/- 0.3. Additionally isoeffect-RBE values between 2.3 and 2.6 were graphically estimated.

Informed decision making in persons acquitted not guilty by reason of insanity.

Deciding to raise an insanity defense carries serious consequences. This is especially true for persons charged with minor offenses, for whom an acquittal not guilty by reason of insanity (NGRI) might lead to a longer period of incarceration than would conviction. Before raising an insanity defense, a defendant should be provided with information necessary to make an informed decision and should be competent to understand the consequences of the verdict. This study attempted, through retrospective review and concurrent evaluation, to determine the degree to which trial courts in Virginia attended to these important aspects of informed decision making before finding defendants charged with misdemeanors NGRI. The study also attempted to assess the degree to which defendants were competent and informed at the time of adjudication. In most instances, trial courts did not consider defendants' competence to make decisions regarding the insanity defense and did not consider whether defendants were informed about the consequences of a successful insanity defense at the time of adjudication. The average length of stay for these patients was (at least) 21 months; most would have been released earlier had they been committed civilly rather than committed as a result of insanity pleas. We stress the need to educate judges, attorneys and forensic evaluators to the importance of considering defendants competence to plead insanity and of providing information about the consequences of a successful plea. We also propose that laws be changed to recognize the importance of these elements in the decision making process regarding pleas of insanity.