Dose-response Testing Research Articles

The Recovery Time of Myometrial Responsiveness After Oxytocin-Induced Desensitization in Human Myometrium In Vitro.

Postpartum hemorrhage secondary to uterine atony is a leading cause of maternal morbidity. Prolonged exposure to oxytocin for labor augmentation can result in the desensitization phenomenon, a decrease in the responsiveness of myometrium to further oxytocin. It is currently not known whether waiting for a specific time interval after the cessation of oxytocin allows the oxytocin receptors to resensitize and recover, thereby improving subsequent oxytocin-induced myometrial contractility. We aimed to investigate the effect of a rest period of 30, 60, and 90 minutes after oxytocin administration on the recovery of oxytocin-desensitized human myometrium in vitro. We hypothesized that the longer the rest period, the better the responsiveness and subsequent oxytocin-induced contractility of the myometrium. Myometrial tissue was obtained from women undergoing elective cesarean deliveries. The myometrial sample was dissected into 4 strips, and each strip was mounted in a single organ bath with physiological salt solution (PSS) under homeostatic conditions and then pretreated for 2 hours with oxytocin 10 M. After pretreatment, each strip was washed with PSS and allowed to rest in PSS solution for 30, 60, or 90 minutes. At the end of the rest period, dose-response testing to oxytocin 10 to 10 M was performed. A control group consisted of oxytocin dose-response testing without any oxytocin pretreatment. Contractile parameters were measured and compared among the groups after square root transformation. The primary outcome was motility index (frequency × amplitude), and secondary outcomes included frequency, amplitude, and area under the curve. Fifty-five experiments were conducted from samples obtained from 16 women. The mean motility index (√g·contractions/10 min) during the dose-response curve (oxytocin 10 to 10 M) in the control group was significantly greater than all the experimental groups; the mean estimated differences (95% confidence intervals) were -1.33 (-2.50 to -0.15, P = 0.02), -1.59 (-2.68 to -0.50, P = 0.004), and -1.88 (-2.97 to -0.80, P = 0.001) for the 30-, 60-, and 90-minute groups, respectively. When the experimental groups were compared, there were no significant differences in any of the contractility parameters; however, confidence intervals were wide. Our study shows that oxytocin pretreatment attenuates oxytocin-induced contractility in human myometrium despite a rest period of up to 90 minutes after oxytocin administration. However, we were unable to determine whether increasing the rest period from 30 to 90 minutes results in improvement in myometrial contractility because of our small sample size relative to the variability in the contractile parameters. Further laboratory and clinical in vivo studies are necessary to determine whether a rest period up to 90 minutes results in improvement in myometrial contractility. In addition, further experimental studies are necessary to determine the key mechanisms of oxytocin receptor resensitization.

The Effect of Extracellular Calcium on Oxytocin-Induced Contractility in Naive and Oxytocin-Pretreated Human Myometrium In Vitro

Prolonged exposure to oxytocin during augmentation of labor is a significant risk factor for uterine atony, resulting in the desensitization phenomenon, a decrease in the responsiveness of myometrium to further oxytocin. The importance of extracellular calcium is well established in spontaneous myometrial contractility; however, its significance is unknown in the context of desensitized myometrium. We aimed to investigate the effect of low, normal, and high extracellular calcium concentration on oxytocin-induced contractility in oxytocin-pretreated human myometrium in vitro. We hypothesized that extracellular normocalcemia would provide superior oxytocin-induced contractility in both naive and oxytocin-pretreated myometrium compared with hypocalcemia and hypercalcemia. Myometrial tissue was obtained from women undergoing elective cesarean deliveries and was dissected into longitudinal strips. Each strip was mounted in a single organ bath with physiological salt solution (PSS) under homeostatic conditions and then pretreated for 2 hours with either oxytocin 10 M or PSS (control). The tissue was then washed with PSS, and calcium concentrations were altered to reflect low (1.25 mM), normal (2.5 mM), or high (3.75 mM) levels, thereby providing 6 study groups. After equilibration in the desired calcium concentration, a dose-response testing to oxytocin 10 M to 10 M was performed. Contractile parameters were measured and compared among groups after square root transformation. The primary outcome was motility index (frequency × amplitude), and secondary outcomes included frequency, amplitude, and area under the curve. One hundred seventy-four experiments were conducted from samples obtained from 36 women. In the control group, the mean motility index (√g·contractions/10 min) was significantly lower in the hypocalcemic group than in the normocalcemic group (estimated difference, -0.43; 95% confidence interval [CI], -0.82 to -0.04; P = 0.03). In addition, the mean frequency of contractions (√contractions/10 min) was significantly lower in the hypocalcemic (estimated difference, -0.27; 95% CI, -0.46 to -0.09; P = 0.002) and hypercalcemic groups (estimated difference, -0.18; 95% CI, -0.34 to -0.02; P = 0.03) compared with the normocalcemic group. In the oxytocin-pretreated group, there were no significant differences in the values of any of the contractility parameters of the hypocalcemic or hypercalcemic groups compared with the normocalcemic group (mean motility index [√g·contractions/10 min] estimated difference, 0.10; 95% CI, -0.23 to 0.43; P = 0.74 and -0.39; 95% CI, -1.10 to 0.32; P = 0.39, respectively). In oxytocin-naive myometrium, normocalcemia provides superior oxytocin-induced contractility compared with hypocalcemic and hypercalcemic conditions. We were unable to draw conclusions regarding oxytocin-pretreated myometrium because of the small sample size relative to the large variability of the data. These observations warrant further investigations in laboratory and clinical settings.

Soil-Residual Protoporphyrinogen Oxidase–Inhibiting Herbicides Influence the Frequency of Associated Resistance in Waterhemp (Amaranthus tuberculatus)

The extensive use of foliar-applied protoporphyrinogen oxidase (PPO)-inhibiting herbicides indisputably contributes to the continued selection of waterhemp resistant to PPO-inhibiting herbicides (PPO-R). However, the role of soil-residual applications of PPO-inhibiting herbicides in the selection of PPO-R has been clouded by the efficacy that these herbicides have on PPO-R waterhemp. The aim of the present study was to understand if soil-residual PPO-inhibiting herbicides have the potential to influence the proportion of resistant waterhemp in emerging plants as herbicide concentrations diminish in the soil. Greenhouse and field experiments were conducted in a PRE dose-response experiment testing fomesafen or fomesafen pluss-metolachlor in the presence of mixed seed populations of PPO-R and -susceptible (S) waterhemp. The first 20 (greenhouse) or 10 (field) waterhemp plants that survived the residual herbicide treatment were sampled for genotypic analysis to detect the presence of the allele responsible for PPO-R in waterhemp (ΔG210). Relative to the nontreated control, the highest rate of fomesafen increased the frequency of resistance (FOR) by 70% in the greenhouse experiments and 20% in the field experiments. The addition ofs-metolachlor did not reduce the fomesafen-induced increase in the FOR in the surviving plants from field or greenhouse experiments. However, the additional herbicide from an alternate site of action (s-metolachlor) substantially improved soil-residual herbicide efficacy over fomesafen alone, which limited the number of waterhemp plants surviving the herbicide application. Thus, the selection for resistance can be delayed with the addition of a herbicide from an alternative site of action by postponing and/or reducing waterhemp emergence. These data strongly suggest that soil-residual PPO-inhibiting herbicides can influence the FOR in a field population, placing even greater importance on the implementation of best management practices such as full herbicide use rates and using herbicides from multiple sites of action to mitigate the risk of selecting for herbicide-resistant weed biotypes.

Nicotine and ethanol co-use in Long-Evans rats: Stimulatory effects of perinatal exposure to a fat-rich diet

Clinical studies demonstrate frequent co-existence of nicotine and alcohol abuse and suggest that this may result, in part, from the ready access to and intake of fat-rich diets. Whereas animal studies show that high-fat diet intake in adults can enhance the consumption of either nicotine or ethanol and that maternal consumption of a fat-rich diet during pregnancy increases operant responding for nicotine in offspring, little is known about the impact of dietary fat on the co-abuse of these two drugs. The goal of this study was to test in Long-Evans rats the effects of perinatal exposure to fat on the co-use of nicotine and ethanol, using a novel paradigm that involves simultaneous intravenous (IV) self-administration of these two drugs. Fat- vs. chow-exposed offspring were characterized and compared, first in terms of their nicotine self-administration behavior, then in terms of their nicotine/ethanol self-administration behavior, and lastly in terms of their self-administration of ethanol in the absence of nicotine. The results demonstrate that maternal consumption of fat compared to low-fat chow during gestation and lactation significantly stimulates nicotine self-administration during fixed-ratio testing. It also increases nicotine/ethanol self-administration during fixed-ratio and dose-response testing, with BEC elevated to 120 mg/dL, and causes an increase in breakpoint during progressive ratio testing. Of particular note is the finding that rats perinatally exposed to fat self-administer significantly more of the nicotine/ethanol mixture as compared to nicotine alone, an effect not evident in the chow-control rats. After removal of nicotine from the nicotine/ethanol mixture, this difference between the fat- and chow-exposed rats was lost, with both groups failing to acquire the self-administration of ethanol alone. Together, these findings suggest that perinatal exposure to a fat-rich diet, in addition to stimulating self-administration of nicotine, causes an even greater vulnerability to the excessive co-use of nicotine and ethanol.

Oxytocin Pretreatment Attenuates Oxytocin-induced Contractions in Human Myometrium In Vitro

Oxytocin receptor desensitization has been shown to occur in humans at biomolecular level and in isolated rat myometrium; however, its effect on human myometrial contractility has not been demonstrated. The objective of this in vitro study was to investigate the contractile response of human pregnant myometrium to oxytocin after pretreatment with different concentrations of oxytocin for variable durations. Myometrial samples were obtained from 62 women undergoing elective cesarean deliveries under regional anesthesia. The strips were pretreated with oxytocin 10, 10, 10 M, or physiological salt solution (control) for 2, 4, 6, or 12 h, followed by a dose-response testing with oxytocin 10 to 10 M. Amplitude and frequency of contractions, motility index, and area under the curve during the dose-response period were recorded, analyzed with linear regression models, and compared among groups. Pretreatment with oxytocin 10 and 10 M significantly reduced motility index (estimate [standard error]: -0.771 [0.270] square root units, P = 0.005 and -0.697 [0.293], P = 0.02, respectively) and area under the curve (-3.947 [1.909], P = 0.04 and -4.241 [2.189], P = 0.05, respectively) compared with control group, whereas pretreatment with oxytocin 10 M did not significantly attenuate contractions. Increase in duration of oxytocin pretreatment from 2 to 12 h significantly decreased amplitude (type 3 generalized estimating equation analysis: chi-square = 14.0; df = 3; P = 0.003), motility index (chi-square = 9.3; df = 3; P = 0.03), and area under the curve (chi-square = 10.5; df = 3; P = 0.02), but not the frequency of oxytocin-induced contractions. Pretreatment with oxytocin decreases oxytocin-induced myometrial contractions in a concentration and time-dependent manner, likely as a function of the oxytocin receptor desensitization phenomenon.

Stimulation of nicotine reward and central cholinergic activity in Sprague-Dawley rats exposed perinatally to a fat-rich diet.

While clinical studies show maternal consumption of palatable fat-rich diets during pregnancy to negatively impact the children's behaviors and increase their vulnerability to drug abuse, the precise behavioral and neurochemical mechanisms mediating these phenomena have yet to be examined. The study examined in rats whether gestational exposure to a high-fat diet (HFD) can increase the offspring's propensity to use nicotine and whether disturbances in central nicotinic cholinergic signaling accompany this behavioral effect. Rat offspring exposed perinatally to a HFD or chow diet were characterized in terms of their nicotine self-administration behavior in a series of operant response experiments and the activity of acetylcholinesterase (AChE) and density of nicotinic ACh receptors (nAChRs) in different brain areas. Perinatal HFD compared to chow exposure increased nicotine-self administration behavior during fixed ratio and dose-response testing and caused an increase in breakpoint using progressive ratio testing, while nicotine seeking in response to nicotine prime-induced reinstatement was reduced. This behavioral change induced by the HFD was associated with a significant reduction in activity of AChE in the midbrain, hypothalamus, and striatum and increased density of β2-nAChRs in the ventral tegmental area and substantia nigra and of α7-nAChRs in the lateral and ventromedial hypothalamus. Perinatal exposure to a HFD increases the vulnerability of the offspring to excessive nicotine use by enhancing its reward potential, and these behavioral changes are accompanied by a stimulation of nicotinic cholinergic signaling in mesostriatal and hypothalamic brain areas important for reinforcement and consummatory behavior.

Development of a High-Throughput Cell-Based Assay for Identification of IL-17 Inhibitors

Human interleukin 17 (IL-17) is a proinflammatory cytokine derived mainly from activated T cells. Extensive evidence points to a significant role of IL-17 in many autoimmune and infectious diseases, as well as tumorigenesis and transplant rejection, and suggests that targeting IL-17 could be a promising therapeutic strategy. Robust cell-based assays would thus be essential for lead identification and the optimization of therapeutic candidates. Herein, we report a well-characterized two-step assay, consisting of (a) in vitro activation and stimulation of CD4+ T lymphocytes by a defined complex of antibodies and cytokines, leading to T helper 17 (Th17) cell differentiation and IL-17 production, and (b) IL-17 quantification in cell supernatants using a homogeneous time-resolved fluorescence (HTRF) assay. The system was optimized for and shown to be reliable in high-throughput compatible 96- and 384-well plate formats. The assay is robust (Z′ > 0.5) and simple to perform, yields a stable response, and allows for sufficient discrimination of positive (IL-17–producing cells) and negative controls (uninduced cells). The assay was validated by performing dose-response testing of rapamycin and cyclosporine A, which had previously been reported to inhibit IL-17, and determining, for the first time, their in vitro potencies (IC50s of 80 ± 23 pM and 223 ± 52 nM, respectively). Also, IKK 16, a selective small-molecule inhibitor of IκB kinase, was found to inhibit IL-17 production, with an IC50 of 315 ± 79 nM.

Contractile Efficacy of Various Prostaglandins in Pregnant Rat Myometrium Pretreated With Oxytocin

Oxytocin pretreatment of pregnant rat myometrium has been shown to reduce the contractions produced by further administration of oxytocin, as a function of the desensitization phenomenon. It is unclear whether this phenomenon affects the contractions produced by various prostaglandins that are used in the management of postpartum hemorrhage. The objective of this study was to investigate the contractile effects of various prostaglandins after oxytocin pretreatment and to compare their relative efficacies in vitro on pregnant rat myometrial strips. Myometrial samples from 29 pregnant Wistar rats at term were isolated and pretreated with oxytocin (10(-8) mol/L, experimental group) or physiological salt solution (control group) for 1 hour. They were then subjected to dose-response testing with oxytocin (n = 32), PGF2α (n = 16), dinoprostone (n = 14), alprostadil (n = 14), or misoprostol (n = 15) with cumulative increases in the organ bath concentrations from 10(-10) to 10(-5) mol/L. The contractile efficacies of various prostaglandins and oxytocin during the dose response were analyzed using mixed linear modeling and compared between the groups. There was no significant difference in the amplitude, frequency, motility index (amplitude × frequency), or area under the curve of all prostaglandins between the groups pretreated with oxytocin and the control group. However, there was a significant decrease in the frequency (P = .02) and motility index (P = .05) in the dose-response curves of oxytocin in the groups pretreated with oxytocin compared with the control groups. Overall, oxytocin produced superior contractions compared with all other prostaglandins, while dinoprostone and misoprostol produced the weakest contractions. The uterotonic effects of various prostaglandins are not affected by oxytocin desensitization; and despite desensitization, oxytocin provides superior contractions compared with the prostaglandins.

Cocaine self-administration behaviors in ClockΔ19 mice

A key role has been identified for the circadian locomotor output cycles kaput (Clock) gene in the regulation of drug reward. Mice bearing a dominant negative mutation in the Clock gene (ClockΔ19 mice) exhibit increased cocaine-induced conditioned place preference, reduced anxiety- and depression-like behavior, increased sensitivity to intracranial self-stimulation, and increased dopaminergic cell activity in the ventral tegmental area. We sought to determine if this hyperhedonic phenotype extends to cocaine self-administration and measures of motivation. Two separate serial testing procedures were carried out (n = 7-10/genotype/schedule). Testing began with acquisition of sucrose pellet self-administration, implantation of intravenous catheter, acquisition of cocaine self-administration, and dose-response testing (fixed ratio or progressive ratio). To evaluate diurnal variations in acquisition behavior, these sessions occurred at Zeitgeber 2 (ZT2) or ZT14. WT and ClockΔ19 mice exhibited similar learning and readily acquired food self-administration at both ZT2 and ZT14. However, only ClockΔ19 mice acquired cocaine self-administration at ZT2. A greater percentage of ClockΔ19 mice reached acquisition criteria at ZT2 and ZT14. ClockΔ19 mice self-administered more cocaine than WT mice. Using fixed ratio and progressive ratio schedules of reinforcement dose-response paradigms, we found that cocaine is a more efficacious reinforcer in ClockΔ19 mice than in WT mice. Our results demonstrate that the Clock gene plays an important role in cocaine reinforcement and that decreased CLOCK function increases vulnerability for cocaine use.

Dose-response testing of Australian populations of onion thrips Thrips tabaci Lindeman (Thysanoptera: Thripidae) further refines baseline data and detects methidathion and likely imidacloprid resistance

Field strains of Thrips tabaci Lindeman were collected from bulb onion in New South Wales, Queensland and South Australia and returned to the laboratory for culturing and subsequent bioassay to determine chemical specific responses. Chemicals tested included diazinon, dimethoate, λ-cyhalothrin and methidathion with imidacloprid and spinetoram evaluated to establish reliable susceptible baseline responses. Reference (susceptible) responses were compared back with those previously published and for all insecticides more susceptible strains were found for resistance comparison. Results for methidathion in particular were found to be significantly different from those reported previously. These differences influenced resistance factor calculation considerably. A high 49-fold methidathion resistance was detected, a result consistent with anecdotal grower concerns of poor product performance against T. tabaci. Resistance to diazinon remains generally low with all strains tested showing <6-fold at the LC50 level. LC50 dimethoate resistance peaked at 17-fold. Maximum (56-fold) λ-cyhalothrin resistance was significantly less than previously found but responses were very heterogeneous implying resistance could very quickly increase. Worryingly, the difference in imidacloprid responses between the least and most tolerant was 41-fold although the chemical is neither used nor registered for use against T. tabaci. We consider such a high and significant difference in response to imidacloprid is most likely caused by an unknown cross-resistance that may compromise any future development of imidacloprid for use against T. tabaci. Thrips remain susceptible to spinetoram.

Wet-Weather Piloting Toward the Largest Compressible Media Filter on the Planet

Onsite pilot testing was recently completed to confirm performance and design criteria for the 380-ML/d (100-mgd) excess flow high-rate treatment facilities that are being designed for the City of Springfield’s wastewater treatment plant. The performance of the compressible media filter pilot demonstrated consistent compliance with all regulatory requirements for CSO treatment and produced average effluent TSS concentrations below 30 mg/L while treating wetweather flows. Furthermore, dose response testing demonstrated that the effluent from the compressible media filter was consistently amenable to hypochlorite disinfection, requiring doses and contact times equivalent to those typically used for conventional secondary effluents. The new high-rate treatment facilities will consist of new structures and equipment for influent excess flow interception, screening, compressible media filtration, hypochlorite disinfection, bisulfite dechlorination and effluent pumping. Construction of these upgrades is scheduled to be completed in 2014.

Novel Chemical Suppressors of Long QT Syndrome Identified by an In Vivo Functional Screen

Genetic long QT (LQT) syndrome is a life-threatening disorder caused by mutations that result in prolongation of cardiac repolarization. Recent work has demonstrated that a zebrafish model of LQT syndrome faithfully recapitulates several features of human disease, including prolongation of ventricular action potential duration, spontaneous early afterdepolarizations, and 2:1 atrioventricular block in early stages of development. Because of their transparency, small size, and absorption of small molecules from their environment, zebrafish are amenable to high-throughput chemical screens. We describe a small-molecule screen using the zebrafish KCNH2 mutant breakdance to identify compounds that can rescue the LQT type 2 phenotype. Zebrafish breakdance embryos were exposed to test compounds at 48 hours of development and scored for rescue of 2:1 atrioventricular block at 72 hours in a 96-well format. Only compounds that suppressed the LQT phenotype in 3 of 3 fish were considered hits. Screen compounds were obtained from commercially available small-molecule libraries (Prestwick and Chembridge). Initial hits were confirmed with dose-response testing and time-course studies. Optical mapping with the voltage-sensitive dye di-4 ANEPPS was performed to measure compound effects on cardiac action potential durations. Screening of 1200 small molecules resulted in the identification of flurandrenolide and 2-methoxy-N-(4-methylphenyl) benzamide (2-MMB) as compounds that reproducibly suppressed the LQT phenotype. Optical mapping confirmed that treatment with each compound caused shortening of ventricular action potential durations. Structure activity studies and steroid receptor knockdown suggest that flurandrenolide functions via the glucocorticoid signaling pathway. Using a zebrafish model of LQT type 2 syndrome in a high-throughput chemical screen, we have identified 2 compounds, flurandrenolide and the novel compound 2-MMB, as small molecules that rescue the zebrafish LQT type 2 syndrome by shortening the ventricular action potential duration. We provide evidence that flurandrenolide functions via the glucocorticoid receptor-mediated pathway. These 2 molecules and future discoveries from this screen should yield novel tools for the study of cardiac electrophysiology and may lead to novel therapeutics for human LQT patients.

The effects of decorin and HGF-primed vocal fold fibroblasts in vitro and ex vivo in a porcine model of vocal fold scarring

Vocal fold injury can be irreversible, leading to vocal fold scarring, with permanent functional effects and no optimal treatment. A porcine model of vocal fold scarring was used to test effects of decorin and primed vocal fold fibroblasts in vitro using a cell migration assay and immunoblotting, and by using functional measurements of porcine larynges and excised porcine vocal folds. In vitro: primary pig vocal fold fibroblasts (PVFFs) were subjected to cell migration assays (scratch) and treated with decorin 20 μg/mL, hepatocyte growth factor (HGF) 200 ng/mL, epidermal growth factor (EGF) 1 nM, or transforming growth factor-β1 10 ng/mL. Cells also underwent decorin dose response testing. Scratch assays were analyzed in MetaMorph® Imaging; cell lysates were processed for MMP-8 and type I collagen content. Eleven pigs underwent unilateral vocal fold stripping procedures. At day 3 postoperatively, subjects underwent superficial injection into the affected vocal fold either with decorin 20 μg/mL or 1 × 10(6) HGF-primed fibroblasts. Larynges were harvested and either used for ex vivo laryngeal testing or for rheological testing. Scratch assay indicated significantly reduced cell migration in PVFFs treated with decorin or HGF. MMP-8 production was increased (P <0.01) and collagen was decreased in cells treated with decorin at increasing doses. Viscoelastic measurements suggested somewhat increased stiffness for decorin treated samples. Ex vivo aerodynamic testing suggested improved vocal efficiency scores in decorin-treated larynges. Decorin has a noticeable effect on PVFF migration in vitro and appears to increase vocal fold stiffness but either does not change or slightly increases vocal efficiency.

The Use of Animal Models for Cancer Chemoprevention Drug Development

Animal models currently are used to assess the efficacy of potential chemopreventive agents, including synthetic chemicals, chemical agents obtained from natural products, and natural product mixtures. The observations made in these models as well as other data are then used to prioritize agents to determine which are qualified to progress to clinical chemoprevention trials. Organ-specific animal models are employed to determine which agents or classes of agents are likely to be the most effective at nontoxic doses to prevent organ-specific forms of cancer. These results are then used to target specific organs in high-risk populations in clinical trials. The animal models used are either carcinogen-induced with carcinogens specific for particular organ sites or they are transgenic/mutant animals with insertions, deletions, or mutations at targeted gene sites known to enhance cancers in a specific organ. Animal tumor models with characteristics favorable to chemoprevention studies are available for cancers of the lung, colon, skin, bladder, mammary, prostate, head and neck, esophagus, ovary, and pancreas. In addition to single-agent dose-response testing, such models are frequently used for testing combinations of agents, testing different routes of administration, evaluating surrogate endpoint biomarkers, and generating initial pharmacokinetics and toxicology data. For some of the more standard animal models there is significant correlation with human chemopreventive trial results. There are a growing number of positive human chemoprevention trials that have used agents or combinations that were positive in animal testing. There have been fewer negative human clinical trials, but their results again correlate with negative animal results. Clearly the validation of animal models to predict the efficacy of agents in human clinical trials will await further human data on positive and negative outcomes with chemopreventive agents. Whether validated or not, animal efficacy data remain central to the clinical trial decision-making process.

Discovery of PDK1 Kinase Inhibitors with a Novel Mechanism of Action by Ultrahigh Throughput Screening

The phosphoinositide 3-kinase/AKT signaling pathway plays a key role in cancer cell growth, survival, and angiogenesis. Phosphoinositide-dependent protein kinase-1 (PDK1) acts at a focal point in this pathway immediately downstream of phosphoinositide 3-kinase and PTEN, where it phosphorylates numerous AGC kinases. The PDK1 kinase domain has at least three ligand-binding sites: the ATP-binding pocket, the peptide substrate-binding site, and a groove in the N-terminal lobe that binds the C-terminal hydrophobic motif of its kinase substrates. Based on the unique PDK1 substrate recognition system, ultrahigh throughput TR-FRET and Alphascreen screening assays were developed using a biotinylated version of the PDK1-tide substrate containing the activation loop of AKT fused to a pseudo-activated hydrophobic motif peptide. Using full-length PDK1, K(m) values were determined as 5.6 mum for ATP and 40 nm for the fusion peptide, revealing 50-fold higher affinity compared with the classical AKT(Thr-308)-tide. Kinetic and biophysical studies confirmed the PDK1 catalytic mechanism as a rapid equilibrium random bireactant reaction. Following an ultrahigh throughput screen of a large library, 2,000 compounds were selected from the reconfirmed hits by computational analysis with a focus on novel scaffolds. ATP-competitive hits were deconvoluted by dose-response studies at 1x and 10x K(m) concentrations of ATP, and specificity of binding was assessed in thermal shift assay. Inhibition studies using fusion PDK1-tide1 substrate versus AKT(Thr-308)-tide and kinase selectivity profiling revealed a novel selective alkaloid scaffold that evidently binds to the PDK1-interacting fragment pocket. Molecular modeling suggests a structural paradigm for the design of inhibitory versus activating allosteric ligands of PDK1.

The effects of response operandum and prior food training on intravenous nicotine self-administration in rats

Nicotine intravenous self-administration (IVSA) in rats has been conducted using a variety of methodological procedures with equally variable results. Here, we addressed the importance of the type of response operandum and prior instrumental training with a natural reinforcer on nicotine IVSA and reinstatement. Rats were tested for spontaneous acquisition of IVSA using either nose poke (NP) or lever press (LVR) operandum. A dose-response test was then conducted, followed by extinction and cue- and nicotine-induced reinstatement. The use of the NP operandum resulted in markedly higher levels of IVSA across acquisition and across dose-response testing compared with the LVR group. Whereas both groups reinstated following a nicotine prime, only the LVR group demonstrated cue-induced reinstatement. As a positive control, the experiment was repeated with cocaine as the reinforcer: equivalent levels of IVSA were observed across all tests, irrespective of operandum. When rats self-administering nicotine received instrumental training with a sucrose reinforcer prior to IVSA, a facilitated acquisition of IVSA was observed in both LVR and NP groups to a similar extent (the effect of operandum remained), but had little effect on responding thereafter. During reinstatement testing, both groups now displayed cue- and nicotine-induced reinstatement, but this was also evident in saline control animals that had never received nicotine. These results suggest that, unlike cocaine, an increased physical response requirement can decrease nicotine intake. It also indicates that operandum and prior sucrose training may influence the role that visual cues play in nicotine dependence.

Discovery of Chemical Modulators of a Conserved Translational Control Pathway by Parallel Screening in Yeast

Eukaryotic initiation factor 2 (eIF2) B is a guanine nucleotide exchange factor that plays a central role in translation initiation and its control, especially in response to diverse cellular stresses. In addition, inherited mutations in human eIF2B subunits cause a fatal brain disorder commonly called childhood ataxia with central nervous system hypomyelination or leukoencephalopathy with vanishing white matter. In yeast, inhibiting activity of eIF2B up-regulates expression of the transcriptional activator general control nondepressible (GCN) 4. We report here evaluation of high-throughput screening (HTS) using a yeast-based reporter gene assay, in which strains containing either wild-type or a mutant eIF2B were screened in parallel to identify compounds modifying eIF2B-dependent responses. The goals of the HTS were twofold: first, to discover compounds that restore normal function to mutant eIF2B, which may have therapeutic utility for the fatal human disease; and second, to identify compounds that activate a GCN4 response, which might be useful experimental tools. The HTS assay measured cell growth by absorbance, and activation of gene expression via a beta-galactosidase reporter gene fusion. Because mutant eIF2B activates GCN4 in the absence of stress inducers, the mutant strain was screened for reduction in GCN4 activation. HTS revealed apparent mutant-selective inhibitors but did not reliably predict selectivity as these hits affected both wild-type and mutant strains equally on dose-response confirmation. Wild-type strain results from the HTS identified two GCN4 response activators, both of which were confirmed to be wild-type selective in dose-response testing, suggesting that these compounds may activate GCN4 by a mechanism that down-regulates eIF2B activity.

Toward a molecular equivalent dose: Use of the medaka model in comparative risk assessment

Recent changes in the risk assessment landscape underscore the need to be able to compare the results of toxicity and dose–response testing between a growing list of animal models and, quite possibly, an array of in vitro screening assays. How do we compare test results for a given compound between vastly different species? For example, what dose level in the ambient water of a small fish model would be equivalent to 10 ppm of a given compound in the rat's drinking water? Where do we begin? To initially address these questions, and in order to compare dose–response tests in a standard rodent model with a fish model, we used the concept of molecular dose. Assays that quantify types of DNA damage that are directly relevant to carcinogenesis integrate the factors such as chemical exposure, uptake, distribution, metabolism, etc. that tend to vary so widely between different phyletic levels. We performed parallel exposures in F344 rats and Japanese medaka ( Oryzias latipes) to the alkylating hepatocarcinogen, dimethylnitrosamine (DMN). In both models, we measured the DNA adducts 8-hydroxyguanine, N 7-methylguanine and O 6-methylguanine in the liver; mutation frequency using λ cII transgenic medaka and λ cII transgenic (Big Blue®) rats; and early morphological changes in the livers of both models using histopathology and immunohistochemistry. Pulse dose levels in fish were 0, 10, 25, 50, or 100 ppm DMN in the ambient water for 14 days. Since rats are reported to be especially sensitive to DMN, they received 0, 0.1, 1, 5, 10, or 25 ppm DMN in the drinking water for the same time period. While liver DNA adduct concentrations were similar in magnitude, mutant frequencies in the DMN-exposed medaka were up to 20 times higher than in the Big Blue rats. Future work with other compounds will generate a more complete picture of comparative dose response between different phyletic levels and will help guide risk assessors using “alternative” models.

Effect of age on kainate-induced seizure severity and cell death

While the onset and extent of epilepsy increases in the aged population, the reasons for this increased incidence remain unexplored. The present study used two inbred strains of mice (C57BL/6J and FVB/NJ) to address the genetic control of age-dependent neurodegeneration by building upon previous experiments that have identified phenotypic differences in susceptibility to hippocampal seizure-induced cell death. We determined if seizure induction and seizure-induced cell death are affected differentially in young adult, mature, and aged male C57BL/6J and FVB/NJ mice administered the excitotoxin, kainic acid. Dose response testing was performed in three to four groups of male mice from each strain. Following kainate injections, mice were scored for seizure activity and brains from mice in each age group were processed for light microscopic histopathologic evaluation 7 days following kainate administration to evaluate the severity of seizure-induced brain damage. Irrespective of the dose of kainate administered or the age group examined, resistant strains of mice (C57BL/6J) continued to be resistant to seizure-induced cell death. In contrast, aged animals of the FVB/NJ strain were more vulnerable to the induction of behavioral seizures and associated neuropathology after systemic injection of kainic acid than young or middle-aged mice. Results from these studies suggest that the age-related increased susceptibility to the neurotoxic effects of seizure induction and seizure-induced injury is regulated in a strain-dependent manner, similar to previous observations in young adult mice.

An implantable carotid sinus stimulator for drug-resistant hypertension: Surgical technique and short-term outcome from the multicenter phase II Rheos feasibility trial

A large number of patients have hypertension that is resistant to currently available pharmacologic therapy. Electrical stimulation of the carotid sinus baroreflex system has been shown to produce significant chronic blood pressure decreases in animals. The phase II Rheos Feasibility Trial was performed to assess the response of patients with multidrug-resistant hypertension to such stimulation. The system consists of an implantable pulse generator with bilateral perivascular carotid sinus leads. Implantation is performed bilaterally with patients under narcotic anesthesia (to preserve the reflex for assessment of optimal lead placement). Dose-response testing at 0 to 6 V is assessed before discharge and at monthly intervals thereafter; the device is activated after 1 month's recovery time. This was a Food and Drug Administration-monitored phase II trial performed at five centers in the United States. Ten patients with resistant hypertension (taking a median of six antihypertensive medications) underwent implantation. All 10 were successful, with no significant morbidity. The mean procedure time was 198 minutes. There were no adverse events attributable to the device. Predischarge dose-response testing revealed consistent (r = .88) reductions in systolic blood pressure of 41 mm Hg (mean fall is from 180-139 mm Hg), with a peak response at 4.8 V (P < .001) and without significant bradycardia or bothersome symptoms. A surgically implantable device for electrical stimulation of the carotid baroreflex system can be placed safely and produces a significant acute decrease in blood pressure without significant side effects.