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Methamphetamine (MA), a highly abused amphetamine-like psychostimulant, has surged in popularity worldwide in the last decade. Repeated MA exposure has been shown to affect the alternative splice variant expression of large conductance Ca2+-activated K+ (BK) channels. It remains unclear whether MA affects BK channel activity. The present study investigated the effects of MA on BK channels in NG108-15 mouse neuroblastoma × rat glioma hybrid cells using whole-cell and cell-attached patch clamp techniques. In whole-cell recordings, the macroscopic K+ outward currents were inhibited by MA with an EC50 of 146 μM, but not affected by dopamine (DA). It implies that DA is not involved in the effects of MA on K+ outward currents. In cell-attached patches, MA significantly decreased BK channel activity. Moreover, MA significantly decreased the BK channel opener NS1619-evoked whole-cell K+ outward currents and BK channel activity. Finally, the effect of MA on membrane potential was examined by current-clamp configuration. MA caused membrane depolarization and application of NS1619 returned the depolarized potential to resting value. These findings suggest that MA might act as an inhibitor of BK channels, and thereby increase the neuronal excitability and enhance neurotransmitter release.

Human genetic evidence has identified the voltage-gated sodium channel NaV1.7 as an attractive target for the treatment of pain. We initially identified naphthalene sulfonamide 3 as a potent and selective inhibitor of NaV1.7. Optimization to reduce biliary clearance by balancing hydrophilicity and hydrophobicity (Log D) while maintaining NaV1.7 potency led to the identification of quinazoline 16 (AM-2099). Compound 16 demonstrated a favorable pharmacokinetic profile in rat and dog and demonstrated dose-dependent reduction of histamine-induced scratching bouts in a mouse behavioral model following oral dosing.

1,3-Bis-[2-hydroxy-5-(trifluoromethyl)phenyl]urea (NS1643) is reported to be an activator of human ether-à-go-go-related gene current. However, it remains unknown whether it has any effects on other types of ion channels. The effects of NS1643 on ion currents and membrane potential were investigated in this study. NS1643 stimulated Ca(2+)-activated K(+) current [I(K(Ca))] in a concentration-dependent manner with an EC(50) value of 1.8 microM in pituitary tumor (GH(3)) cells. In inside-out recordings, this compound applied to the intracellular side of the detached channels stimulated large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels with no change in single-channel conductance. It shifted the activation curve of BK(Ca) channels to less depolarized voltages without altering the gating charge of the channels. NS1643-stimulated channel activity depended on intracellular Ca(2+), and mean closed time during exposure to NS1643 was reduced. NS1643 (3 microM) had little or no effect on peak amplitude of ether-à-go-go-related gene-mediated K(+) current evoked by membrane hyperpolarization, although it increased the amplitude of late-sustained components of K(+) inward current, which was suppressed by paxilline but not by azimilide. NS1643 (3 microM) had no effect on L-type Ca(2+) current. This compound reduced repetitive firing of action potentials, and further application of paxilline attenuated its decrease in firing rate. In addition, NS1643 enhanced BK(Ca)-channel activity in human embryonic kidney 293T cells expressing alpha-hSlo. In summary, we clearly show that NS1643 interacts directly with the BK(Ca) channel to increase the amplitude of I(K(Ca)) in pituitary tumor (GH(3)) cells. The alpha-subunit of the channel may be a target for the action of this small compound.

General anesthetic propofol (2,6-bis(isopropyl)-phenol) possess a chemical structure unrelated to other anesthetic drugs. It has been known to block a variety of ion currents. This study is designed to determine the effect of this drug on ion currents in differentiated H9c2 cardiac cells. The effects of propofol, an intravenous anesthetic agent with a distinct chemical structure, on ion currents of differentiated clonal cardiac (H9c2) cells were investigated in this study. Propofol (10-300 microM) suppressed the amplitude of delayed rectifier K(+) current (I(K(DR))) in a concentration-dependent manner with an IC(50) value of 36 microM. This compound reduced activation time constant and increased current inactivation, although no voltage dependency of propofol-induced block of I(K(DR)) can demonstrated. Neither diazoxide, pinacidil, nor caffeic acid phenethyl ester had any effect on propofol-induced block of I(K(DR)). Propofol (30 microM) had no effect on erg-mediated K(+) current in these cells; however, it suppressed L-type Ca(2+) current (I(Ca,L)) of cardiac and skeletal types to a similar extent. Intracellular dialysis with propofol (100 microM) had no effects on I(K(DR)) or I(Ca,L). Numerical simulations of I(K(DR)) based on a Markovian model reproduce the experimental results and show that propofol-induced blockade of I(K(DR)) is associated with an decrease in forward rate of the activation process and an increase in transitional rate into the inactivated state. Propofol can suppress I(K(DR)) in differentiated H9c2 cardiac cells in a concentration- and state-dependent manner. These effects can significantly contribute its action on functional activity of heart cells.

Riluzole is known to be of therapeutic use in the management of amyotrophic lateral sclerosis. In this study, we investigated the effects of riluzole on ion currents in cultured differentiated human skeletal muscle cells (dHSkMCs). Western blotting revealed the protein expression of alpha-subunits for both large-conductance Ca2+-activated K+ (BK(Ca)) channel and Na+ channel (Na(v)1.5) in these cells. Riluzole could reduce the frequency of spontaneous beating in dHSkMCs. In whole-cell configuration, riluzole suppressed voltage-gated Na+ current (I(Na)) in a concentration-dependent manner with an IC50 value of 2.3 microM. Riluzole (10 microM) also effectively increased Ca2+-activated K+ current (I(K(Ca))) which could be reversed by iberiotoxin (200 nM) and paxilline (1 microM), but not by apamin (200 nM). In inside-out patches, when applied to the inside of the cell membrane, riluzole (10 microM) increased BK(Ca)-channel activity with a decrease in mean closed time. Simulation studies also unraveled that both decreased conductance of I(Na) and increased conductance of I(K(Ca)) utilized to mimic riluzole actions in skeletal muscle cells could combine to decrease the amplitude of action potentials and increase the repolarization of action potentials. Taken together, inhibition of I(Na) and stimulation of BK(Ca)-channel activity caused by this drug are partly, if not entirely, responsible for its muscle relaxant actions in clinical setting.

Cardiac fibroblasts are involved in the maintenance of myocardial tissue structure. However, little is known about ion currents in human cardiac fibroblasts. It has been recently reported that cardiac fibroblasts can interact electrically with cardiomyocytes through gap junctions. Ca(2+)-activated K(+) currents (I (K[Ca])) of cultured human cardiac fibroblasts were characterized in this study. In whole-cell configuration, depolarizing pulses evoked I (K(Ca)) in an outward rectification in these cells, the amplitude of which was suppressed by paxilline (1 microM: ) or iberiotoxin (200 nM: ). A large-conductance, Ca(2+)-activated K(+) (BK(Ca)) channel with single-channel conductance of 162 +/- 8 pS was also observed in human cardiac fibroblasts. Western blot analysis revealed the presence of alpha-subunit of BK(Ca) channels. The dynamic Luo-Rudy model was applied to predict cell behavior during direct electrical coupling of cardiomyocytes and cardiac fibroblasts. In the simulation, electrically coupled cardiac fibroblasts also exhibited action potential; however, they were electrically inert with no gap-junctional coupling. The simulation predicts that changes in gap junction coupling conductance can influence the configuration of cardiac action potential and cardiomyocyte excitability. I (k(Ca)) can be elicited by simulated action potential waveforms of cardiac fibroblasts when they are electrically coupled to cardiomyocytes. This study demonstrates that a BK(Ca) channel is functionally expressed in human cardiac fibroblasts. The activity of these BK(Ca) channels present in human cardiac fibroblasts may contribute to the functional activities of heart cells through transfer of electrical signals between these two cell types.

Introduction We have previously shown that a tag single nucleotide polymorphism (rs10235235), which maps to the CYP3A locus (7q22.1), was associated with a reduction in premenopausal urinary estrone glucuronide levels and a modest reduction in risk of breast cancer in women age ≤50 years. Methods We further investigated the association of rs10235235 with breast cancer risk in a large case control study of 47,346 cases and 47,570 controls from 52 studies participating in the Breast Cancer Association Consortium. Genotyping of rs10235235 was conducted using a custom Illumina Infinium array. Stratified analyses were conducted to determine whether this association was modified by age at diagnosis, ethnicity, age at menarche or tumor characteristics. Results We confirmed the association of rs10235235 with breast cancer risk for women of European ancestry but found no evidence that this association differed with age at diagnosis. Heterozygote and homozygote odds ratios (ORs) were OR = 0.98 (95% CI 0.94, 1.01; P = 0.2) and OR = 0.80 (95% CI 0.69, 0.93; P = 0.004), respectively (Ptrend = 0.02). There was no evidence of effect modification by tumor characteristics. rs10235235 was, however, associated with age at menarche in controls (Ptrend = 0.005) but not cases (Ptrend = 0.97). Consequently the association between rs10235235 and breast cancer risk differed according to age at menarche (Phet = 0.02); the rare allele of rs10235235 was associated with a reduction in breast cancer risk for women who had their menarche age ≥15 years (ORhet = 0.84, 95% CI 0.75, 0.94; ORhom = 0.81, 95% CI 0.51, 1.30; Ptrend = 0.002) but not for those who had their menarche age ≤11 years (ORhet = 1.06, 95% CI 0.95, 1.19, ORhom = 1.07, 95% CI 0.67, 1.72; Ptrend = 0.29). Conclusions To our knowledge rs10235235 is the first single nucleotide polymorphism to be associated with both breast cancer risk and age at menarche consistent with the well-documented association between later age at menarche and a reduction in breast cancer risk. These associations are likely mediated via an effect on circulating hormone levels.

The photocycloaddition of methacrylonitrile to 5-substituted adamantan-2-ones (1-X) produces two geometrically isomeric oxetanes in which the oxygen atom and the 5-substituent are in anti or syn positions. The substituent was varied from fluoro, chloro, bromo, phenyl to SiMe3 and the product ratios, except for SiMe3, were similar (ca. 56:44) in all instances. Structure determination of the anti and syn oxetanes were carried out by 1H and 13C NMR and mass spectroscopy. The structure of syn-5-bromospiro[adamantane-2,2′-oxetane](syn-5-Br) was further supported by single-crystal X-ray crystallography. The product formation bias resulting from the attack on the zu-face is discussed in terms of transition-state hyperconjugation, but new results for photocycloaddition reactions of 5-trimethylsilyl-substituted adamantan-2-one (1-SiMe3), with both fumaronitrile and methacrylonitrile, show an unprecedented σ-electron-withdrawing characteristic with this substituent.

Diosgenin (3β-hydroxy-5-spirostene, 1), a plant-derived sapogenin, is used as a dietary supplement. However, the biological effects of 1 related to viral replication remain unexplored. In this study, the effects of 1 on hepatitis C virus (HCV) replication were evaluated. Based on a reporter-based HCV subgenomic replicon system, 1 was found to inhibit HCV replication at low micromolar concentrations. The EC(50) (concentration at which 50% of HCV replication is inhibited) of 1 was 3.8 μM. No cellular toxicity was observed at this concentration. Diosgenin (1) also significantly reduced the levels of viral RNA and viral proteins as evaluated by quantitative real-time reverse transcriptase PCR and Western blot analysis, respectively. In addition, in an alternative HCV antiviral system more closely aligned to all steps involved in the HCV infection and life cycle, 1 totally abolished HCV replication at 20 μM. Moreover, 1 reduced the phosphorylation of signal transducer and activator of transcription 3. A combination of 1 and interferon-α exerted an additive effect on the resultant anti-HCV activity.

Aconitine (ACO), a highly toxic diterpenoid alkaloid, is recognized to have effects on cardiac voltage-gated Na(+) channels. However, it remains unknown whether it has any effects on K(+) currents. The effects of ACO on ion currents in differentiated clonal cardiac (H9c2) cells and in cultured neonatal rat ventricular myocytes were investigated in this study. In H9c2 cells, ACO suppressed ultrarapid-delayed rectifier K(+) current (I(Kur)) in a time- and concentration-dependent fashion. The IC(50) value for ACO-induced inhibition of I(Kur) was 1.4 microM. ACO could accelerate the inactivation of I(Kur) with no change in the activation time constant of this current. Steady-state inactivation curve of I(Kur) during exposure to ACO could be demonstrated. Recovery from block by ACO was fitted by a single-exponential function. The inhibition of I(Kur) by ACO could still be observed in H9c2 cells preincubated with ruthenium red (30 microM). Intracellular dialysis with ACO (30 microM) had no effects on I(Kur). I(Kur) elicited by simulated action potential (AP) waveforms was sensitive to block by ACO. Single-cell Ca(2+) imaging revealed that ACO (10 microM) alone did not affect intracellular Ca(2+) in H9c2 cells. In cultured neonatal rat ventricular myocytes, ACO also blocked I(Kur) and prolonged AP along with appearance of early afterdepolarizations. Multielectrode recordings on neonatal rat ventricular tissues also suggested that ACO-induced electrocardiographic changes could be associated with inhibition of I(Kur). This study provides the evidence that ACO can produce a depressant action on I(Kur) in cardiac myocytes.

The carbapenem antibiotic ertapenem has been shown to be safe, well tolerated and effective in treating adults with complicated urinary tract infection, skin and soft-tissue infection and community-acquired pneumonia. In this study, we evaluated ertapenem for treating these infections in children in a randomised, double-blind, active-controlled clinical trial. The primary outcome was the incidence of clinical and laboratory drug-related serious adverse events (AEs). Children were randomised in a 3:1 ratio (ertapenem:ceftriaxone) stratified by index infection and age to receive ertapenem or ceftriaxone; 303 children received ertapenem and 100 children received ceftriaxone. The median duration of parenteral therapy was 4 days for both treatments. The most commonly reported drug-related clinical AEs during parenteral therapy were diarrhoea (5.9% ertapenem, 10% ceftriaxone), infusion site erythema (3% ertapenem, 2% ceftriaxone) and infusion site pain (5% ertapenem, 1% ceftriaxone). One child in each group reported a serious drug-related clinical AE. No serious drug-related laboratory AEs were reported. In children aged 3 months to 17 years, ertapenem was well tolerated and had a comparable safety profile to that of ceftriaxone.

Microscopic findings associated with paclitaxel (Taxol) chemotherapy toxicity were described years ago but whether they are specific for toxicity remains unclear. Further, epithelial changes associated with taxanes can mimic high-grade dysplasia (HGD) in non-neoplastic gastrointestinal (GI) tract mucosa. Similar changes associated with colchicine are only seen in patients with toxicity.GI tract specimens were reviewed (221 total; 93 esophageal, 55 gastric cardiac, 48 oxyntic, 7 antral, 8 small bowel, 6 colonic, 3 appendiceal, 1 anal) from 71 patients (63M, 8F), 38 to 84 years (median, 55 y) undergoing chemotherapy for esophageal, breast, or lung cancer who had all received taxanes at some time [either paclitaxel (Taxol, 55 patients) or docetaxel (Taxotere, 16 patients)]. Epithelial changes (mitotic arrest/ring mitoses, apoptosis) associated with taxanes were graded on a scale of 0 to 3 (0=no mitotic arrest; 1=rare arrest; 2=scattered arrest; 3=striking mitotic arrest and apoptosis). Samples from the patients taken before administration of taxanes were also reviewed; all samples were reviewed without knowledge of the interval between drug doses and the biopsy/resection.Five samples had striking mitotic arrest mimicking HGD in non-neoplastic mucosa and were from the esophagus, gastric cardia, gastric body, gastric antrum, and appendix of 5 separate patients. All 5 had GI tract samples obtained 1 to 3 days after taxane administration. These patients had all taken Taxol (rather than Taxotere). On follow-up, in 3/5 patients with samples 1 day posttreatment, 1 had acute appendicitis (died 180 d postappendectomy), 1 died a day later of metastases, and 1 was asymptomatic (alive with metastatic disease at 126 d postbiopsy). The remaining 2 died of metastases at 90 and 210 days postbiopsy with no signs of drug toxicity at any time.In contrast to colchicine-associated changes in non-neoplastic mucosa, the mitotic arrest mimicking HGD seen in GI tract specimens after taxane administration is not specific for toxicity, but may also reflect taxane effect. It can be encountered in asymptomatic patients who have recently had medication. If these findings are seen histologically, they merit correlation with the clinical impression, and should not be interpreted as toxicity in isolation.

The effects of aconitine (ACO), a highly toxic alkaloid, on ion currents in differentiated NG108-15 neuronal cells were investigated in this study. ACO (0.3-30 microM) suppressed the amplitude of delayed rectifier K+ current (I K(DR)) in a concentration-dependent manner with an IC50 value of 3.1 microM. The presence of ACO enhanced the rate and extent of I K(DR) inactivation, although it had no effect on the initial activation phase of I K(DR). It could shift the inactivation curve of I K(DR) to a hyperpolarized potential with no change in the slope factor. Cumulative inactivation for I K(DR) was also enhanced by ACO. Orphenadrine (30 microM) or methyllycaconitine (30 microM) slightly suppressed I K(DR) without modifying current decay. ACO (10 microM) had an inhibitory effect on voltage-dependent Na+ current (I Na). Under current-clamp recordings, ACO increased the firing and widening of action potentials in these cells. With the aid of the minimal binding scheme, the ACO actions on I K(DR) was quantitatively provided with a dissociation constant of 0.6 microM. A modeled cell was designed to duplicate its inhibitory effect on spontaneous pacemaking. ACO also blocked I K(DR) in neuroblastoma SH-SY5Y cells. Taken together, the experimental data and simulations show that ACO can block delayed rectifier K+ channels of neurons in a concentration- and state-dependent manner. Changes in action potentials induced by ACO in neurons in vivo can be explained mainly by its blocking actions on I K(DR) and I Na.

Di-8-ANEPPS (4-{2-[6-(dibutylamino)-2-naphthalenyl]-ethenyl}-1-(3-sulfopropyl)pyridinium inner salt) has been used as a fast-response voltage-sensitive styrylpyridinium probe. However, little is known regarding the mechanism of di-8-ANEPPS actions on ion currents. In this study, the effects of this dye on ion currents were investigated in pituitary GH(3) cells. In whole-cell configuration, di-8-ANEPPS (10 microM) reversibly increased the amplitude of Ca(2+)-activated K(+) current. In inside-out configuration, di-8-ANEPPS (10 microM) applied to the intracellular surface of the membrane caused no change in single-channel conductance; however, it did enhance the activity of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels with an EC(50) value of 7.5 microM. This compound caused a left shift in the activation curve of BK(Ca) channels with no change in the gating charge of these channels. A decrease in mean closed time of the channels was seen in the presence of this dye. In the cell-attached mode, di-8-ANEPPS applied on the extracellular side of the membrane also activated BK(Ca) channels. However, neither voltage-gated K(+) nor ether-à-go-go-related gene (erg)-mediated K(+) currents in GH(3) cells were affected by di-8-APPNES. Under current-clamp configuration, di-8-ANEPPS (10 microM) decreased the firing of action potentials in GH(3) cells. In pancreatic betaTC-6 cells, di-8-APPNES (10 microM) also increased BK(Ca)-channel activity. Taken together, this study suggests that during the exposure to di-8-ANEPPS, the stimulatory effects on BK(Ca) channels could be one of potential mechanisms through which it may affect cell excitability.

The effects of rottlerin, a known inhibitor of protein kinase C-δ activation, on ion currents were investigated in pituitary tumor (GH3) cells. Rottlerin (0.3–100 µM) increased the amplitude of Ca2+-activated K+ current (IK(Ca)) in a concentration-dependent manner with an EC50 value of 1.7 µM. In intracellular perfusion with rottlerin (1 µM) or staurosporine (10 µM), phorbol 12-myristate 13-acetate-induced inhibition of IK(Ca) in these cells was abolished. In cell-attached mode, rottlerin applied on the extracellular side of the membrane caused activation of large-conductance Ca2+-activated K+ (BKCa) channels, and a further application of BAPTA-AM (10 µM) to the bath had no effect on rottlerin-stimulated channel activity. When cells were exposed to rottlerin, the activation curve of these channels was shifted to less positive potential with no change in the slope factor. Rottlerin increased BKCa-channel activity in outside-out patches. Its change in kinetic behavior of BKCa channels is primarily due to an increase in mean open time. With the aid of minimal kinetic scheme, a quantitative description of rottlerin stimulation on BKCa channels in GH3 cells was also provided. Under current-clamp configuration, rottlerin (1 µM) decreased the firing of action potentials. IK(Ca) elicited by simulated action potential waveforms was enhanced by this compound. In human cortical HCN-1A cells, rottlerin (1 µM) could also interact with the BKCa channel to stimulate IK(Ca). Therefore, rottlerin may directly activate BKCa channels in neurons or endocrine cells. J. Cell. Physiol. 210: 655–666, 2007. © 2006 Wiley-Liss, Inc.

We present the fabrication and characterization of a nano-scale sensor made of amorphous silicon (a-Si), functionalized for the detection of pH and DNA hybridization. The detector is composed of a nano-scale a-Si semicircle with a surface functionalized by receptor molecules containing single-strand DNA. Target complimentary DNA molecules bind the receptor molecules and their charge accumulating near the surface of the nanoscale semicircle causes in turn a change in the charge carrier density under the surface. This change of charge carrier density results in an effective change of conductance that can be monitored electronically. The sensor structure allows for direct conversion of molecular recognition and binding events to electronic signals.

The effects of diosgenin (3beta-hydroxy-5-spirostene), a plant-derived sapogenin, on ion currents in human cortical neurons (HCN-1A) were investigated. In the whole-cell configuration, diosgenin (0. -30 microM) increased the amplitude of K+ outward current (I(K)). Diosgenin-stimulated I(K) was sensitive to inhibition by paxilline (1 microM), but not by apamin (200 nM) or glibenclamide (10 muM). In the cell-attached configuration, diosgenin applied to the bath increased the activity of large-conductance Ca2+-activated K+ (BK(Ca)) channels without altering single-channel conductance. Diosgenin enhanced BK(Ca)-channel activity with an EC50 value of 25 microM. However, in inside-out patches, diosgenin applied to the intracellular surface had no effect on BK(Ca)-channel activity, while cilostazol or caffeic acid phenethyl ester increased it. As shown with the aid of intracellular Ca2+ measurements, diosgenin elevated intracellular Ca2+ in HCN-1A cells. Western blotting also revealed the presence of the alpha-subunit of BK (Ca) channels in these cells. The sustained stimulation of I(K) arises primarily from the diosgenin-induced Ca2+ influx across the cell membrane. The effect of diosgenin on these channels may affect the functional activity of cortical neurons. Abbreviations. I(K):K+ outward current I(K(Ca)):Ca2+-activated K+ current BK(Ca) channel:Large-conductance Ca2+-activated K+ channel CAPE:caffeic acid phenethyl ester [Ca2+] (i):intracellular Ca2+ concentration I/V relationship:current/voltage relationship K (ATP) channel:ATP-sensitive K+ channel K(Ca) channel:Ca2+-activated K+ channel.

We demonstrate the design and fabrication of optically pumped sub-diffraction nanophotonic waveguides using a self assembly process to attach two different types of quantum dots on a silicon dioxide substrate, thus enabling low crosstalk.

Background: Androgen receptor (AR) is a ligand-activated transcription factor that plays critical roles in lethal and incurable castration-resistant prostate cancer (CRPC). EPI-002 is a novel small molecule that specifically targets the AR N-terminal domain (NTD), while conventional therapies target the ligand-binding domain (LBD). Our previous work suggests that EPI-002 may have clinical potential to treat CRPC. Here, the objective was to evaluate EPI-002 against several clinically relevant conditions such as elevated expression of AR co-activators, variable lengths of the poly-glutamine tract (CAG-repeats) within AR NTD, and constitutively active AR splice variants lacking the LBD. Methods: To evaluate the effect of EPI-002 on AR transcriptional activity against overexpressed co-activators such as SRC1-3 and p300, luciferase reporter assays were performed using LNCaP cells transfected with an AR-driven reporter and various amounts of the co-activators. AR-negative COS1 cells were employed for reporter assays to examine if the length of CAG-repeats (varying from 0 to 49) affects EPI-002 inhibition. The effect of EPI-002 on constitutively active AR splice variants was studied in LNCaP95 cells, which express endogenous AR-V7 variant. The expression of several genes regulated by AR-V7 was analyzed by QPCR in LNCaP95 cells treated with antiandrogen enzalutamide or EPI-002. The proliferation of LNCaP95 cells treated with EPI-002 was measured using BrdU incorporation. The efficacy of EPI-002 on LNCaP95 xenografts was studied in castrated male mice by oral delivery and compared to control and enzalutamide. Results: EPI-002 effectively inhibited AR transcriptional activity in spite of overexpressed co-activators SRC1, SRC2, SRC3, or p300. The variable lengths of CAG-repeats within the NTD had no effect on the inhibition of AR by EPI-002. In LNCaP95 cells, EPI-002 significantly blocked the transcriptional activity of AR-V7 indicated by decreased levels of mRNA of AR-V7 regulated genes such as UBE2C. In contrast, enzalutamide had no effect on expression of these genes. EPI-002 significantly inhibited the androgen-independent and antiandrogen-resistant proliferation of LNCaP95 cells. In castrated male mice, EPI-002 attenuated the growth of LNCaP95 xenografts, whereas enzalutamide had no significant effect. By the end of the 28-day study, animals treated with EPI-002 had xenografts that were half of the size of those from the control arm. Conclusions: AR NTD inhibitor EPI-002 was effective against several clinically relevant conditions, including overexpressed co-activators, variable lengths of CAG-repeats, and constitutively active AR-V7. These findings support EPI-002 as a promising therapeutic agent to treat CRPC, particularly against castration-resistant tumors driven by constitutively active AR splice variants that are resistant to LBD-targeting therapies. Citation Format: Yu Chi (Kevin) Yang, Nasrin (Rina) Mawji, Jean Wang, Marianne Sadar. Preclinical evaluation of novel androgen receptor N-terminal domain inhibitor EPI-002 for the treatment of castration-resistant prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 610. doi:10.1158/1538-7445.AM2014-610

Improved procedures for the isolation of apotryptophanase and its use in estimation of the vitamin B-6 coenzymes are presented. An excess of the apoenzyme is allowed to react with limiting amounts of pyridoxal-P. Estimation of the holotryptophanase thus formed by use of the chromogenic substrate. S-o-nitrophenyl- l -cysteine, provides a sensitive (1–400 pmol) and conveniently direct spectrophotometric assay for pyridoxal-P. For the specific estimation of pyridoxamine 5′-phosphate, samples are first reduced with NaBH4 to convert pyridoxal-P to pyridoxine-P (inactive). By nonenzymatic transamination with glyoxylate, pyridoxamine-P is then converted quantitatively to pyridoxal-P and estimated with apotryptophanase. The method gives excellent recoveries of the added coenzymes and indicates that in many tissue extracts pyridoxamine-P surpasses pyridoxal-P in concentration.

Background Resveratrol, a phytoalexin found in grapes and red wine, exhibits diverse pharmacological activities. However, relatively little is known about whether resveratrol modulates the ion channels in cortical neurons. The large-conductance calcium-activated potassium channels (BKCa) and voltage-gated sodium channels were expressed in cortical neurons and play important roles in regulation of neuronal excitability. The present study aimed to determine the effects of resveratrol on BKCa currents and voltage-gated sodium currents in cortical neurons. Results Resveratrol concentration-dependently increased the current amplitude and the opening activity of BKCa channels, but suppressed the amplitude of voltage-gated sodium currents. Similar to the BKCa channel opener NS1619, resveratrol decreased the firing rate of action potentials. In addition, the enhancing effects of BKCa channel blockers tetraethylammonium (TEA) and paxilline on action potential firing were sensitive to resveratrol. Our results indicated that the attenuation of action potential firing rate by resveratrol might be mediated through opening the BKCa channels and closing the voltage-gated sodium channels. Conclusions As BKCa channels and sodium channels are critical molecular determinants for seizure generation, our findings suggest that regulation of these two channels in cortical neurons probably makes a considerable contribution to the antiseizure activity of resveratrol.