Brain serotonin (5-hydroxytryptamine, 5-HT) has been suggested to be involved in central fatigue during prolonged exercise. Changes in the ratio of plasma free tryptophan (free Trp) to branched-chain amino acids (BCAA) are associated with altered brain 5-HT synthesis. The purposes of this study were to describe systematically the effects of prolonged exercise on changes in plasma free Trp and BCAA and to examine the effects of carbohydrate (CHO) feedings on these same variables. Eight well-trained men [VO2max = 57.8 (SE 4.1) ml kg-1 min-1] cycled for up to 255 min at a power output corresponding to VO2 at lactate threshold (approximately 68% VO2max) on three occasions separated by at least 1 week. Subjects drank 5 ml kg-1 body wt-1 of either a water placebo, or a liquid beverage containing a moderate (6% CHO) or high (12% CHO) concentration of carbohydrate beginning at min 14 of exercise and every 30 min thereafter. Exercise time to fatigue was shorter in subjects receiving placebo [190 (SE 4) min] as compared to 6% CHO [235 (SE 10) min] and 12% CHO [234 (SE 9) min] (P < 0.05). Glucose and insulin decreased in the placebo group, and free Trp, free-Trp/BCAA, and free fatty acids increased approximately five- to sevenfold (P < 0.05). These changes were attenuated in a dose-related manner by the carbohydrate drinks.(ABSTRACT TRUNCATED AT 250 WORDS)

The Tousled-like kinases (TLKs) are involved in chromatin assembly, DNA repair, and transcription. Two TLK genes exist in humans, and their expression is often dysregulated in cancer. TLKs phosphorylate Asf1 and Rad9, regulating double-strand break (DSB) repair and the DNA damage response (DDR). TLKs maintain genomic stability and are important therapeutic intervention targets. We identified specific inhibitors of TLKs from several compound libraries, some of which belong to the family of phenothiazine antipsychotics. The inhibitors prevented the TLK-mediated phosphorylation of Rad9(S328) and impaired checkpoint recovery and DSB repair. The inhibitor thioridazine (THD) potentiated tumor killing with chemotherapy and also had activity alone. Staining for γ-H2AX revealed few positive cells in untreated tumors, but large numbers in mice treated with low doxorubicin or THD alone, possibly the result of the accumulation of DSBs that are not promptly repaired as they may occur in the harsh tumor growth environment.

Type I phosphatidylinositol 4-phosphate 5-kinase (PI4P5K) catalyzes the phosphorylation of phosphatidylinositol 4 phosphate [PI(4)P] at carbon 5, producing phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2]. Phosphatidic acid (PA) activates PI4P5K in vitro and plays a central role in the activation of PIP5K pathways in vivo. This report demonstrates that actin fiber formation in murine fibroblasts involves PA activation of PIP5Ks and defines biochemical interactions between PA and the PIP5Ks. Inhibition of phospholipase D production of PA results in the loss of actin fibers. Overexpression of the beta isoform of the type I murine phosphatidylinositol 4-phosphate 5-kinase (mPIP5K-Ib) maintains actin fiber structure in the face of phospholipase D inhibition. PA activates mPIP5K-Ib by direct binding to mPIP5K-Ib through both electrostatic and hydrophobic interactions, with the fatty acid acyl chain length and degree of saturation acting as critical determinants of binding and activation. Furthermore, kinetic analysis suggests that phosphorylation of the PI(4)P substrate does not follow classical Michaelis-Menten kinetics. Instead, the kinetic data are consistent with a model in which mPIP5K-Ib initially binds to the lipid micelle and subsequently binds the PI(4)P substrate. In addition, the kinetics indicate substrate inhibition, suggesting that mPIP5K-Ib contains an inhibitory PI(4)P-binding site. These results suggest a model in which mPIP5K-Ib is surrounded by PI(4)P, but is unable to catalyze its conversion to PI(4,5)P2 unless PA is bound.

To probe the mechanism of chaperone substrate selection, we have investigated the kinetics of complex formation and dissociation between the molecular chaperone DnaK and a short peptide (Cro, representing amino acids 1-12 of the cro repressor protein). The Cro protein was N-terminally labeled with the environmentally sensitive fluorophore dansyl chloride (Cro*), and steady-state and stopped-flow fluorescence spectroscopies and fluorescence-detected high-performance size exclusion chromatography (HPSEC) were used to monitor complex formation and dissociation over a range of temperatures in the absence of ATP. The results are summarized as follows: (i) Cro* binds to DnaK with a second-order rate constant, k(on), which varies from 8 to 200 M-1 s-1 between 15 and 37 degrees C. The slow on-rate is a consequence of a large activation energy barrier. The activation enthalpy (delta H*) and the prefactor [omega exp delta S*/R)] are 26 kcal mol-1 and 7 x 10(20) M-1 s-1, respectively. (ii) Once formed, DnaK-Cro* complexes are long-lived, especially at low temperatures (T < 15 degrees C). The off-rate is unusually temperature-sensitive, for example, there is a 478-fold increase in k(off) from 2.3 x 10(6) to 1.1 x 10(-3) s-1 over a range of only 30 degrees C (5-35 degrees C). The steep temperature-dependence of the off-rate is a consequence of a very large activation energy barrier to DnaK-Cro* complex dissociation [delta H* = 34.6 kcal mol-1 and omega exp (delta S*/R) = 2 x 10(21) s-1]. The relatively low affinity of the Cro* peptide for DnaK is due to a large kinetic barrier to binding. We discuss possible causes for these large kinetic barriers.

Lysosome trafficking plays a significant role in tumor invasion, a key event for the development of metastasis. Previous studies from our laboratory have demonstrated that the anterograde (outward) movement of lysosomes to the cell surface in response to certain tumor microenvironment stimulus, such as hepatocyte growth factor (HGF) or acidic extracellular pH (pHe), increases cathepsin B secretion and tumor cell invasion. Anterograde lysosome trafficking depends on sodium-proton exchanger activity and can be reversed by blocking these ion pumps with Troglitazone or EIPA. Since these drugs cannot be advanced into the clinic due to toxicity, we have designed a high-content assay to discover drugs that block peripheral lysosome trafficking with the goal of identifying novel drugs that inhibit tumor cell invasion. An automated high-content imaging system (Cellomics) was used to measure the position of lysosomes relative to the nucleus. Among a total of 2210 repurposed and natural product drugs screened, 18 “hits” were identified. One of the compounds identified as an anterograde lysosome trafficking inhibitor was niclosamide, a marketed human anti-helminthic drug. Further studies revealed that niclosamide blocked acidic pHe, HGF, and epidermal growth factor (EGF)-induced anterograde lysosome redistribution, protease secretion, motility, and invasion of DU145 castrate resistant prostate cancer cells at clinically relevant concentrations. In an effort to identify the mechanism by which niclosamide prevented anterograde lysosome movement, we found that this drug exhibited no significant effect on the level of ATP, microtubules or actin filaments, and had minimal effect on the PI3K and MAPK pathways. Niclosamide collapsed intralysosomal pH without disruption of the lysosome membrane, while bafilomycin, an agent that impairs lysosome acidification, was also found to induce JLA in our model. Taken together, these data suggest that niclosamide promotes juxtanuclear lysosome aggregation (JLA) via modulation of pathways involved in lysosome acidification. In conclusion, we have designed a validated reproducible high-content assay to screen for drugs that inhibit lysosome trafficking and reduce tumor invasion and we summarize the action of one of these drugs.