Scopoletin from Cirsium setidens Increases Melanin Synthesis via CREB Phosphorylation in B16F10 Cells.

In this study, we isolated scopoletin from Cirsium setidens Nakai (Compositae) and tested its effects on melanogenesis. Scopoletin was not toxic to cells at concentrations less than 50 µM and increased melanin synthesis in a dose-dependent manner. As melanin synthesis increased, scopoletin stimulated the total tyrosinase activity, the rate-limiting enzyme of melanogenesis. In a cell-free system, however, scopoletin did not increase tyrosinase activity, indicating that scopoletin is not a direct activator of tyrosinase. Furthermore, Western blot analysis showed that scopoletin stimulated the production of microphthalmia-associated transcription factor (MITF) and tyrosinase expression via cAMP response element-binding protein (CREB) phosphorylation in a dose-dependent manner. Based on these results, preclinical and clinical studies are needed to assess the use of scopoletin for the treatment of vitiligo.

Peripherally restricted transthyretin-based delivery system for probes and therapeutics avoiding opioid-related side effects.

Several investigations into the sites of action of opioid analgesics have utilized peripherally acting mu-opioid receptor antagonists (PAMORAs), which have been incorrectly assumed to possess limited permeability across the blood-brain barrier. Unfortunately, the poor pharmacokinetic properties of current PAMORAs have resulted in misunderstandings of the role of central nervous system and gastrointestinal tract in precipitating side effects such as opioid-induced constipation. Here, we develop a drug delivery approach for restricting the passage of small molecules across the blood-brain barrier. This allows us to develop naloxone- and oxycodone-based conjugates that display superior potency, peripheral selectivity, pharmacokinetics, and efficacy in rats compared to other clinically used PAMORAs. These probes allow us to demonstrate that the mu-opioid receptors in the central nervous system have a fundamental role in precipitating opioid-induced constipation. Therefore, our conjugates have immediate use as pharmacological probes and potential therapeutic agents for treating constipation and other opioid-related side effects.

Potential role of the Virchow Robin space in the pathogenesis of bacterial meningitis.

Meningitis is an infectious disease commonly arising from a bacterial etiology. The rapid progression of morbidity and mortality due to bacterial meningitis requires critical and imminent time-dependent clinical intervention. Although it is unambiguously clear that bacteria must infiltrate the cerebrospinal fluid, the sequence of events in the pathogenesis of bacterial meningitis has not been fully elucidated. Most reviews of the pathogenesis of bacterial meningitis do not specify the anatomical location of bacteria following BBB traversal. We propose an additional hypothesis focusing on the Virchow-Robin space (VRS). The VRS consists of a small, but identifiable perivascular space formed by a sheath of cells derived from the pia mater. The VRS has been described as an immunological space and possibly having a role in several neuropathological diseases. Solute exchange between cerebrospinal fluid and extracellular fluid occurs at the VRS, with subsequent drainage into the subarachnoid space. Because the VRS is continuous with the subpial space, a more direct route to the meninges is facilitated. The involvement of the VRS may have profound implications on the pathogenesis and therapeutic strategies: (1) nasopharyngeal colonization; (2) penetration into the blood stream after crossing the mucosal and epithelial membranes; (3) proliferation in the bloodstream; (4) extravasations through the endothelium of the post-capillary venules to the perivascular VRS; (5) migration from VRS to subpial space; (6) traversal through pia mater, entering the CSF in the subarachnoid space; (7) invasion of the meninges. The implication of the VRS in the pathogenesis of bacterial meningitis would be twofold. First, the VRS could provide an additional route of entry of bacteria into the brain. Second, the VRS could provide an area for bacterial proliferation, and thereby serve as a bacterial reservoir in relatively close proximity to the meninges. The clinical consequences of this hypothesis are: 1) clinical interpretation of laboratory findings, and 2) effective antibiotic delivery into the VRS. If the role of the VRS is established as part of bacterial meningitis pathogenesis, antibiotic pharmacokinetics and pharmacodynamics in the VRS need to be determined. This may result in developing novel antibiotic delivery and clinical strategies to improve morbidity and mortality.

Reduction in warfarin adverse events requiring patient hospitalization after implementation of a pharmacist-managed anticoagulation service.

STUDY OBJECTIVE: To compare adverse events related to anticoagulation in patients assigned to a pharmacist-managed anticoagulation service versus those receiving usual care. DESIGN: Retrospective cohort analysis. SETTING: Three hundred-bed community hospital. PATIENTS: Four hundred twenty patients referred for anticoagulation management. MEASUREMENTS AND MAIN RESULTS: Primary outcomes were the number of adverse events requiring patient hospitalization and the number of patients experiencing such events. Secondary outcomes were the median length of hospital stay/admission and the total number of hospital days. The total numbers of adverse events requiring hospitalization were three for the pharmacist-managed group and 14 for the usual care group (p=0.0153). The number of patients experiencing an adverse event requiring hospitalization was also lower for the pharmacist-managed group than for the usual care group (3 vs 10, p = 0.0962). The median length of hospital stay associated with each adverse event was not significantly different between the two groups; however, the total number of hospital days accrued was higher in the usual care group. CONCLUSION: At 6 months after discontinuation of the pharmacist-managed anticoagulation service, the frequency of adverse events increased significantly, resulting in both an increased number of hospitalizations and an increased number of hospital days accrued. This coordinated anticoagulation program using a pharmacist reduced warfarin-related complications.

Differential regulation of calcium homeostasis in adenocarcinoma cell line A549 and its Taxol-resistant subclone.

Drug resistance is a fundamental problem in cancer chemotherapy. Intracellular calcium concentration ([Ca2+](i)) may play a role in the development of chemoresistance. We investigated the regulatory role of [Ca2+](i) in Taxol resistance in the non-small-cell lung cancer cell line A549 and its chemoresistant subclone A549-T24. Measurement of cytosolic calcium ([Ca2+](c)) in single cells and cell populations revealed similar levels of basal calcium in the two cell lines. However, a reduced response to thapsigargin (a sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor) in A549-T24 cells compared to the parent cell line suggested a lower ER Ca2+ content in these cells. mRNA expression of SERCA2b and SERCA3, major Ca2+ pumps involved in ER Ca2+ homeostasis, did not significantly differ between the two cell lines, as revealed by RT-PCR. An altered calcium influx pathway in the Taxol-resistant cell line was observed. Modulation of the ER calcium pools using CMC (4-chloro-m-cresol) and ATP revealed lower ryanodine receptor (RyR) and IP(3) receptor (IP(3)R)-sensitive Ca2+ stores in the chemoresistant cell line. Western blot and RT-PCR studies suggested that A549-T24 cells expressed higher levels of the antiapoptotic protein Bcl-2 and the calcium-binding protein sorcin, respectively, in comparison to the parent cell line. Both of these proteins have been previously implicated in chemoresistance, in part, due to their ability to modulate[Ca2+](i). These results suggest that altered intracellular calcium homeostasis may contribute to the Taxol-resistant phenotype.

Incorporation of Bloom's taxonomy into multiple-choice examination questions for a pharmacotherapeutics course.

OBJECTIVE: To incorporate Bloom's taxonomy into multiple-choice examination questions in a pharmacotherapeutics course and assess its effectiveness in detecting areas of improvement in learning. DESIGN: Bloom's taxonomy was incorporated into examination questions through a multi-step process: Sample questions representing each learning domain within Bloom's taxonomy (knowledge, comprehension, application, analysis, synthesis, and evaluation) were introduced to students during lecture presentations and discussions. Quiz and examination containing questions categorized according to Bloom's taxonomy were administered to students. During review sessions following each quiz or examination, the categorization of each question was provided to students and feedback from students was gathered. ASSESSMENT: The effect of the 5 types of test questions on the correct response fraction and discrimination index was determined after combining synthesis and evaluation. Correct response fractions for knowledge, comprehension, and application questions were significantly higher than those for analysis and synthesis/evaluation questions (p<0.05). However, discrimination index for application and synthesis/evaluation questions were significantly higher than those for knowledge and comprehension questions (p<0.05). In interviews with students who had requested learning assistance, the majority realized the importance of critical-thinking skills in the learning process. CONCLUSION: Well-designed multiple-choice questions incorporating different learning domains of Bloom's taxonomy may be a potential method of assessing critical-thinking skills in large classes of students.

p23 co-chaperone protects the aryl hydrocarbon receptor from degradation in mouse and human cell lines.

The aryl hydrocarbon receptor (AhR) is a ligand-sensitive transcription factor which is responsible for most 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicities. Without ligand, the AhR complex is cytoplasmic and contains p23. Our objective was to investigate whether the wild type p23 levels are important for the AhR function. We generated eight p23-specific knockdown stable cell lines via either electroporation or lentiviral infection. Five of these stable cell lines were generated from a mouse hepatoma cell line (Hepa1c1c7) and three were from human hepatoma and cervical cell lines (Hep3B and HeLa). All of them expressed lower AhR protein levels, leading to reduced ligand-induced, DRE-driven downstream activity. The AhR protein levels in p23-specific knockdown stable cells were reversed back to wild type levels after exogenous p23 was introduced. Reduction of the AhR protein levels in these stable cells was caused by a decrease in the AhR message levels and an increase of the AhR protein degradation in the absence of ligand. This ligand-independent degradation of AhR was not reversed by MG132, suggesting that the 26S proteasome was not responsible for the degradation. In addition, MG132 could not protect AhR from the ligand-induced degradation in both mouse and human p23-knockdown stable cells.

The effect of 17 beta-estradiol on intracellular calcium homeostasis in human endothelial cells.

The cardiovascular effects of estrogen are mediated in part by augmenting the function of endothelial nitric oxide synthase. Endothelial nitric oxide synthase activity is dependent on many cofactors including Ca(2+). Hence, we investigated the effect of chronic 17 beta-estradiol treatment on the intracellular Ca(2+) concentration and endothelial nitric oxide synthase protein expression in the human endothelial cell line, EA.hy926, using spectrofluorometry and Western blot, respectively. Inhibiting the sarco(endo)plasmic reticulum Ca(2+) ATPase with thapsigargin caused an increase in the intracellular Ca(2+) concentration, which was higher in chronically 17 beta-estradiol-treated (1muM, 24h) cells loaded with Fura-2-acetoxymethyl ester compared to vehicle-treated cells, suggesting a higher endoplasmic reticulum Ca(2+) content in 17 beta-estradiol-treated cells. An enhanced Ca(2+) influx pathway in chronically 17 beta-estradiol-treated cells was also observed. In addition, 17 beta-estradiol-treated cells expressed higher levels of endothelial nitric oxide synthase protein in comparison to vehicle-treated cells. The chronic effect of 17 beta-estradiol on Ca(2+) homeostasis and endothelial nitric oxide synthase expression was attenuated with the nonselective estrogen receptor inhibitor, ICI 182,780 (10muM, 7alpha, 17beta-[9-[(4,4,5,5,5-Pentafluoropentyl)sulfinyl]nonyl] estra-1,3,5(10)-triene-3,17-diol). Furthermore, analysis of the thapsigargin-evoked Ca(2+) response in chronically 17 beta-estradiol-treated estrogen receptor alpha-knockdown cells showed no significant difference in Ca(2+) response compared to vehicle-treated estrogen receptor alpha-knockdown cells, indicating that the regulation of Ca(2+) homeostasis by 17 beta-estradiol is mediated through an estrogen receptor alpha-dependent pathway. These data revealed an estrogen receptor alpha-dependent modulation of Ca(2+) homeostasis accompanying the enhancement of endothelial nitric oxide synthase expression in 17 beta-estradiol-treated human endothelial cells.

P-glycoprotein induction and tumor cell-kill dynamics in response to differential doxorubicin dosing strategies: a theoretical pharmacodynamic model.

PURPOSE: The objectives of this work were 1) to develop a theoretical pharmacodynamic model that captures dynamic changes resulting from drug/therapy mediated P-glycoprotein (P-gp) induction and 2) to compare the pharmacodynamic outcomes of several doxorubicin (DOX) dosing schemes through simulations. METHODS: We developed a theoretical model that included a pharmacokinetic (PK) model for intracellular DOX-mediated P-gp induction and a pharmacodynamic (PD) model using a threshold trigger function for tumor cell-kill. In this model, both the level of P-gp induction and rate of tumor cell death were modulated by intracellular DOX concentration. Most model parameters were obtained from literature sources, and a few were either fixed or reasonably estimated. RESULTS: Comparative dosing simulations showed that a 10-week constant infusion in which a tumor cell population was continuously exposed to the drug did not produce the best PD profile. On the other hand, dosing schemes where the cell population was initially challenged with a high dose, followed by intermittent dosing, generated the best PD profile. The favorable outcome of the latter dosing schemes was correlated with the lowest expression of P-gp in terms of area under the curve (AUC) during treatment period. CONCLUSIONS: The simulations led us to conclude that drug resistance, particularly resistance caused by P-gp overexpression, induced during chemotherapy may, in part, be circumvented by designing optimal dosing strategies that minimize P-gp induction.