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NF-κB has been implicated in mechanisms promoting inflammation in tumor microenvironment leading to breast cancer metastasis. Owing to critical role of CCL2 during metastasis, particularly in its capacity to act as a chemoattractant for macrophages and their precursors i.e monocytes, we decided to explore if pro-metastatic function of NF-κB could be attributable to CCL2 and/or macrophage infiltration. Through our study we provide experimental and clinical evidence in support of co-ordinated expression of chemokines CCL2, NF-κB and intratumoral macrophage content particularly with respect to breast cancer, with an additional evidence of these three variables being key determinant for poor prognosis and diminished survival amongst breast cancer patients both independently as well in a coordinated manner. The mean fold increase in mRNA expression level of NF-κB and CCL2 indicated that it was over expressed 13.57 and 13.18 fold respectively in tumor tissue as compared to adjacent normal tissue. Among these Immunohistochemistry expression of CD68 marker showed that 62 patients (66.7%) had low/moderate CD68 expression while 31 patients (33.3%) had strong expression. All three variables viz.NF-κB, CCL2 and CD68 showed significant (p<0.05 or p<0.01 or p<0.001) respectively associations with both clinicopathological (except CD68 with stage) and hormone receptors (ER, PR and Her2/neu) and their co-expressions indicating these as predictors of breast cancer. In this study we decipher the possible molecular mechanism by way of which NF-κB may promote breast cancer metastasis. Our study has clinical relevance as it establishes significance of these three variables as potential predictive markers to be employed in breast cancer.

Sorafenib is the first and currently the only standard treatment for advanced hepatocellular carcinoma (HCC). We previously developed a sorafenib derivative SC-43, which exhibits much more enhanced anti-HCC activity than sorafenib and also promotes apoptosis in sorafenib-resistant HCC cells. Herein, a novel "sorafenib plus" combination therapy was developed by coupling sorafenib treatment with SC-43. Both sorafenib and SC-43 are proven Src homology region 2 domain containing phosphatase 1 (SHP-1) agonists. The combined actions of sorafenib and SC-43 enhanced SHP-1 activity, which was associated with diminished STAT3-related signals and stronger expression of apoptotic genes above that of either drug alone, culminating in increased cell death. Decreased p-STAT3 signaling and tumor size, as well as increased SHP-1 activity were observed in mice receiving the combination therapy in a subcutaneous HCC model. More reduced orthotopic HCC tumor size and prolonged survival were also observed in mice in the combination treatment arm compared to mice in either of the monotherapy arms. These results in the preclinical setting pave the way for further clinical studies to treat unresectable HCC., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background: Lolium rigidum populations in Australia and globally have demonstrated rapid and widespread evolution of resistance to acetyl coenzyme A carboxylase (ACCase)-inhibiting and acetolactate synthase (ALS)-inhibiting herbicides. Thirty-three resistant L. rigidum populations, randomly collected from crop fields in a most recent resistance survey, were analysed for non-target-site diclofop metabolism and all known target-site ACCase gene resistance-endowing mutations., Results: The HPLC profile of [(14) C]-diclofop-methyl in vivo metabolism revealed that 79% of these resistant L. rigidum populations showed enhanced capacity for diclofop acid metabolism (metabolic resistance). ACCase gene sequencing identified that 91% of the populations contain plants with ACCase resistance mutation(s). Importantly, 70% of the populations exhibit both non-target-site metabolic resistance and target-site ACCase mutations., Conclusions: This work demonstrates that metabolic herbicide resistance is commonly occurring in L. rigidum, and coevolution of both metabolic resistance and target-site resistance is an evolutionary reality. Metabolic herbicide resistance can potentially endow resistance to many herbicides and poses a threat to herbicide sustainability and thus crop production, calling for major research and management efforts., (© 2015 Society of Chemical Industry.)

Five new polyketides including three new butenolides (1-3), one new diphenyl ether (4), and one new benzophenone (5), together with eleven known compounds (6-16) were isolated from a wetland fungus Aspergillus flavipes PJ03-11. Their structures were elucidated on the basis of detailed spectroscopic analysis. All the isolated compounds were tested for their α-glucosidase inhibitory activities. The results showed that compounds 1-3, 6, 7, 11, 15 and 16 exhibited stronger inhibitory activities than acarbose. And the preliminary structure-activity relationships of aspulvinone and diphenyl ether compounds on the α-glucosidase inhibitory activity were reported., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Poor outcome in response to hepatitis C virus, including higher viral load, hepatocellular carcinoma and cirrhosis, is more associated with men and postmenopausal women than with premenopausal women and women receiving hormone replacement therapy, suggesting that β-estradiol plays an innate role in preventing viral infection and liver disease. Consequently, most research in the field has concluded that estrogen affects HCV replication through viral interactions with estrogen receptor-α. Previously, estrogen-like antagonists, including Tamoxifen, were shown to reduce HCV RNA production and prevent viral entry, although the authors did not identify host factors involved. Estrogen can act alternatively through the membrane-bound G-protein-coupled estrogen receptor, GPR30. Here, human hepatoma Huh7.5 cells were infected with HCV J6/JFH-1 and treated with estrogen or Tamoxifen, resulting in a marked decrease in detectable virus. The effect was mimicked by G1, a GPR30-specific agonist, and was reversed by the GPR30-specific antagonist, G15. While previous studies have demonstrated that estrogen down-regulated occludin in cervical cancer cells, its action on liver cells was unknown. Occludin is a tight junction protein and HCV receptor and here we report that activation and cellular export of MMP-9 led to the cleavage of occludin upon estrogen treatment of liver cells. This is the first report of the cleavage of an HCV receptor in response to estrogen. We also identify the occludin cleavage site in extracellular Domain D; the motif required for HCV entry and spread. This pathway gives new insight into a novel innate antiviral pathway and the suboptimal environment that estrogen provides for the proliferation of the virus. It may also explain the disparate host-virus responses to HCV demonstrated by the two sexes. Moreover, these data suggest that hormone replacement therapy may have beneficial antiviral enhancement properties for HCV-infected postmenopausal women and show promise for new antiviral treatments for both men and women.

Context: Bisbibenzyl compounds have gained our interests for their potential antitumor activity in malignant cell-types., Objective: The objective of this study is to investigate the effect of bisbibenzyl compounds riccardin C (RC), marchantin M (MM), and riccardin D (RD) on androgen receptor (AR) in prostate cancer (PCa) cells., Materials and Methods: After exposure to 10 μM of the compounds for 24 h, cell cycle and cell survival analyses were performed using FACS and MTT assay to confirm the effect of these bisbibenzyls on PCa LNCaP cells. Changes in the AR expression and function, as the result of exposure to the compounds, were investigated using real-time PCR, ELISA, transient transfection, western blotting (WB), immunoprecipitation, and immunofluorescence staining (IF). Chemical-induced autophagy was examined by WB, IF, and RNAi., Results: RC, MM, and RD reduced the viability of LNCaP cells accompanied with arrested cell cycle in the G0/G1 phase and induction of apoptosis. Further investigation revealed that these compounds significantly inhibited AR expression at mRNA and protein levels, leading to the suppression of AR transcriptional activity. Moreover, inhibition of proteasome activity by bisbibenzyls, which in turn caused the induction of autophagy, as noted by induction of LC3B expression, conversion, and accumulation of punctate dots in treated cells. Co-localization of AR/LC3B and AR/Ub suggested that autophagy contributed to the degradation of polyubiquitinated-AR when proteasome activity was suppressed by the bisbibenzyls., Discussion and Conclusion: Suppression of proteasome activity and induction of autophagy were involved in bisbibenzyl-mediated modulation of AR activities and apoptosis, suggesting their potential in treating PCa.

Trypanosomatids possess an unremitting requirement for distinctive endogenous sterols for their life cycle and cannot use the copious availability of cholesterol existing in their mammalian hosts. Exhaustion of endogenous sterols such as ergosterol or of its next biosynthetic product 24-ethylcholesta-5,7,22-trien- 3β-ol brings forth an inhibition of proliferation on Trypanosoma cruzi, the etiologic agent of American trypanosomiasis or Chagas disease. These metabolites are crucial; consequently, the enzymes implicated in catalyzing their formation constitute interesting molecular targets for drug design. Selective inhibition of an enzyme associated to the ergosterol biosynthesis will produce T. cruzi cell arrest. Trypanosomatids, fungi, and yeasts have need of these endogenous sterols for cell viability and growth. In fact, some effective ergosterol biosynthesis inhibitors bearing suitable pharmacokinetic properties in mammals have become putative antiparasitic agents by inducing almost complete parasitological cure in both acute and chronic experimental Chagas disease. WC-9 (compound 7; 4-phenoxyphenoxyethyl thiocyanate) holds our attention bearing in mind that this compound exhibits ED50values at the low nanomolar range against the clinically more relevant replicative form of T. cruzi (amastigotes). The cellular activity of WC-9 is due to an exhaustion of endogenous sterols demonstrating a blockade of the biosynthetic pathway at a pre-squalene level.

LG101506 was originally synthesized to overcome some of the undesirable side effects of rexinoids. We compared the anticarcinogenic action of LG101506 and LG100268 and for the first time showed that both drugs are useful for prevention of lung cancer in A/J mice. These molecules markedly reduced tumor number, tumor size, and total tumor burden, when chronically administered to A/J mice that had been initiated with the mutagenic carcinogen, vinyl carbamate. Moreover, LG100268 synergized with the histone deacetylase inhibitor, vorinostat, for prevention of experimental lung cancer and enhanced the effect of carboplatin/paclitaxel for treatment of experimental lung cancer. Both rexinoids diminished the percentage of high-grade, highly malignant adenocarcinomas found at autopsy. In cell culture studies, the rexinoids exhibited potent anti-inflammatory properties at nanoMolar concentrations. These drugs suppressed the ability of lipopolysaccharide to stimulate the synthesis and secretion of nitric oxide and inflammatory cytokines and chemokines, such as IL6, IL1β, CXCL2, and CSF3, in macrophage-like RAW264.7 cells. The present results suggest that LG100268, LG101506, or a related rexinoid may have useful clinical applications in the field of oncology., (©2015 American Association for Cancer Research.)

Most existing antibiotics were discovered through screens of environmental microbes, particularly the streptomycetes, for the capacity to prevent the growth of pathogenic bacteria. This "activity-guided screening" method has been largely abandoned because it repeatedly rediscovers those compounds that are highly expressed during laboratory culture. Most of these metabolites have already been biochemically characterized. However, the sequencing of streptomycete genomes has revealed a large number of "cryptic" secondary metabolic genes that are either poorly expressed in the laboratory or that have biological activities that cannot be discovered through standard activity-guided screens. Methods that reveal these uncharacterized compounds, particularly methods that are not biased in favor of the highly expressed metabolites, would provide direct access to a large number of potentially useful biologically active small molecules. To address this need, we have devised a discovery method in which a chemical elicitor called Cl-ARC is used to elevate the expression of cryptic biosynthetic genes. We show that the resulting change in product yield permits the direct discovery of secondary metabolites without requiring knowledge of their biological activity. We used this approach to identify three rare secondary metabolites and find that two of them target eukaryotic cells and not bacterial cells. In parallel, we report the first paired use of cheminformatic inference and chemical genetic epistasis in yeast to identify the target. In this way, we demonstrate that oxohygrolidin, one of the eukaryote-active compounds we identified through activity-independent screening, targets the V1 ATPase in yeast and human cells and secondarily HSP90.

BACKGROUND Apoptosis is mediated by the endoplasmic reticulum (ER) stress pathway, mitochondrial pathway, and death receptor. Data herein suggested an inhibitory effect of marchantin M on tumor formation in nude mice as well as the impact on CHOP and GRP78 expression. MATERIAL AND METHODS The role of marchantin M on proliferation and apoptosis of DU145 cells were measured by MTT and flow cytometry, respectively. Western blot was applied to detect the expression of GRP78 and CHOP. The mice received abdominal injection at 1 time/2 d and 2 ml/time. Tumor volume was measured every 6 days. The mice were euthanatized 30 days after marchantin injection and tumor weight was measured. Cell apoptosis was determined by TUNEL. The expressions of CHOP and GRP78 were detected by immunohistochemistry. RESULTS Tumor size and weight in marchantin groups were significantly lower than in the control group (A, B) (P<0.05), and the inhibitory rate presented a dose-dependent increase. Compared with controls, the levels of CHOP and GRP78 expression elevated obviously following the treatment with marchantin (P<0.05). It showed statistically significant difference among groups C, D, E, with different levels of apoptosis indexes incremented in groups of marchantin H, M, L, compared with groups A and B (P<0.05). CONCLUSIONS Overall, this study shows that marchantin M circumvents the growth of prostate cancer PC-3 tumor and up-regulates expressions of CHOP and GRP78. Our data also indicate that marchantin M limits the proliferation and favors apoptosis of DU145 cells in a time- and dose-dependent manner.

Excess nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS) is implicated in neurodegenerative disorders. As a result, inhibition of nNOS and reduction of NO levels is desirable therapeutically, but many nNOS inhibitors are poorly bioavailable. Promising members of our previously reported 2-aminoquinoline class of nNOS inhibitors, although orally bioavailable and brain-penetrant, suffer from unfavorable off-target binding to other CNS receptors, and they resemble known promiscuous binders. Rearranged phenyl ether- and aniline-linked 2-aminoquinoline derivatives were therefore designed to (a) disrupt the promiscuous binding pharmacophore and diminish off-target interactions and (b) preserve potency, isoform selectivity, and cell permeability. A series of these compounds was synthesized and tested against purified nNOS, endothelial NOS (eNOS), and inducible NOS (iNOS) enzymes. One compound, 20, displayed high potency, selectivity, and good human nNOS inhibition, and retained some permeability in a Caco-2 assay. Most promisingly, CNS receptor counterscreening revealed that this rearranged scaffold significantly reduces off-target binding.

PT70 is a diaryl ether inhibitor of InhA, the enoyl-ACP reductase in the Mycobacterium tuberculosis fatty acid biosynthesis pathway. It has a residence time of 24 min on the target, and also shows antibacterial activity in a mouse model of tuberculosis infection. Due to the interest in studying target tissue pharmacokinetics of PT70, we developed a method to radiolabel PT70 with carbon-11 and have studied its pharmacokinetics in mice and baboons using positron emission tomography., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

It has become clear that histamine H3 receptors (H3R) have been implicated in modulating ethanol intake and preference in laboratory animals. The novel non-imidazole H3R antagonist DL77 with excellent selectivity profile shows high in-vivo potency as well as in-vitro antagonist affinity with ED50 of 2.1 ± 0.2 mg/kg and pKi=8.08, respectively. In the present study, and applying an unlimited access two-bottle choice procedure, the anti-alcohol effects of the H3R antagonist, DL77 (0, 3, 10 and 30 mg/kg; i.p.), were investigated in adult mice. In this C57BL/6 line, effects of DL77 on voluntary alcohol intake and preference, as well as on total fluid intake were evaluated. Results have shown that DL77, dose-dependently, reduced both ethanol intake and preference. These effects were very selective as both saccharin and quinine, used to control for taste sensitivity, and intakes were not affected following DL77 pre-application. More importantly, systemic administration of DL77 (10 mg/kg) during acquisition inhibited ethanol-induced conditioned-place preference (EtOH-CPP) as measured using an unbiased protocol. The anti-alcohol activity observed for DL77 was abrogated when mice were pretreated with the selective H3R agonist R-(α)-methyl-histamine (RAMH) (10 mg/kg), or with the CNS penetrant H1R antagonist pyrilamine (PYR) (10mg/kg). These results suggest that DL77 has a predominant role in two in vivo effects of ethanol. Therefore, signaling via H3R is essential for ethanol-related consumption and conditioned reward and may represent a novel therapeutic pharmacological target to tackle ethanol abuse and alcoholism., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Compounds 10 (ND-322) and 15 (ND-364) are potent selective inhibitors for gelatinases, matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9). However, both of them are racemates. Herein we report facile synthesis of optically active (R)- and (S)-enantiomers of compounds 10 and 15. And the sulfonyl of 15 was transformed to sulfinyl to obtain four epimeric mixtures. All synthesized thiirane-based compounds were evaluated in MMP2 and MMP9 inhibitory assays. Our results indicated that the configuration of thiirane moiety had little effects on gelatinase inhibition, but the substitution of sulfinyl for sulfonyl was detrimental to gelatinase inhibition. Besides, all target compounds exhibited no inhibition against other two Zn(2+) dependant metalloproteases, aminopeptidase N (APN) and histone deacetylases (HDACs), which confirmed the unique Zn(2+) chelation mechanism of thiirane moiety against gelatinases., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Molecular dynamics (MD) simulations of 12 aqueous systems of the NADH-dependent enoyl-ACP reductase from Mycobacterium tuberculosis (InhA) were carried out for up to 20-40 ns using the GROMACS 4.5 package. Simulations of the holoenzyme, holoenzyme-substrate, and 10 holoenzyme-inhibitor complexes were conducted in order to gain more insight about the secondary structure motifs of the InhA substrate-binding pocket. We monitored the lifetime of the main intermolecular interactions: hydrogen bonds and hydrophobic contacts. Our MD simulations demonstrate the importance of evaluating the conformational changes that occur close to the active site of the enzyme-cofactor complex before and after binding of the ligand and the influence of the water molecules. Moreover, the protein-inhibitor total steric (ELJ) and electrostatic (EC) interaction energies, related to Gly96 and Tyr158, are able to explain 80% of the biological response variance according to the best linear equation, pKi=7.772-0.1885×Gly96+0.0517×Tyr158 (R²=0.80; n=10), where interactions with Gly96, mainly electrostatic, increase the biological response, while those with Tyr158 decrease. These results will help to understand the structure-activity relationships and to design new and more potent anti-TB drugs.

During Leishmania infection, host immune response is important to prevent the growth/survival of intracellular amastigotes. In this study, we evaluated in vitro and in vivo whether or not during Leishmania amazonensis infection, pentavalent antimonial treatment/therapy could be more effective under TNF-α inhibition. Both L. amazonensis-infected macrophages (in vitro model) and mice (in vivo model) were treated with a nuclear factor-κB (NF-κB) inhibitor and with Glucantime®, alone and in combined administrations. The in vitro amastigote counts, cytokines and nitrites' production were assessed after 48h incubation with the drugs. Paw lesion sizes and amastigote counts were also evaluated in vivo. Quantification of IL-1β from the infected tissue was performed. In vitro results show that when infected macrophages were incubated with QNZ+Glucantime®, a greater clearance was observed for the amastigotes' growth and this was related to greater nitrite production compared to the group that was only infected. In vivo results show that mice that received the combined treatment had their paw lesion sizes and amastigote nests inside the macrophages greatly diminished, correlating with increased IL-1β levels., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Tesmilifene, a tamoxifen analog with antihistamine action, has chemopotentiating properties in experimental and clinical cancer studies. In our previous works, tesmilifene increased the permeability of the blood-brain barrier (BBB) in animal and culture models. Our aim was to investigate the effects of tesmilifene on brain microvessel permeability in the rat RG2 glioma model and to reveal its mode of action in brain endothelial cells. Tesmilifene significantly increased fluorescein extravasation in the glioma. Short-term treatment with tesmilifene reduced the resistance and increased the permeability for marker molecules in a rat triple co-culture BBB model. Tesmilifene also affected the barrier integrity in brain endothelial cells co-cultured with RG2 glioblastoma cells. Tesmilifene inhibited the activity of P-glycoprotein and multidrug resistance-associated protein-1 efflux pumps and down-regulated the mRNA expression of tight junction proteins, efflux pumps, solute carriers, and metabolic enzymes important for BBB functions. Among the possible signaling pathways that regulate BBB permeability, tesmilifene activated the early nuclear translocation of NFκB. The MAPK/ERK and PI3K/Akt kinase pathways were also involved. We demonstrate for the first time that tesmilifene increases permeability marker molecule extravasation in glioma and inhibits efflux pump activity in brain endothelial cells, which may have therapeutic relevance. Tesmilifene, a chemopotentiator in experimental and clinical cancer studies increases vascular permeability in RG2 glioma in rats and permeability for marker molecules in a culture model of the blood-brain barrier. Tesmilifene inhibits the activity of efflux pumps and down-regulates the mRNA expression of tight junction proteins, transporters, and metabolic enzymes important for the blood-brain barrier functions, which may have therapeutic relevance., (© 2015 International Society for Neurochemistry.)

Some unichiral analogues of 2R,2'S-2-(1'-methyl-2'-pyrrolidinyl)-7-hydroxy-1,4-benzodioxane, a potent and selective α4β2-nAChR partial agonist, were designed by opening dioxane and replacing hydroxyl carbon with nitrogen. The resulting 3-pyridyl and m-hydroxyphenyl ethers have high α4β2 affinity and good subtype selectivity, which get lost if OH is removed from phenyl or the position of pyridine nitrogen is changed. High α4β2 affinity and selectivity are also attained by meta hydroxylating the 3-pyridyl and the phenyl ethers of (S)-N-methylprolinol and the phenyl ether of (S)-2-azetidinemethanol, known α4β2 agonists, although the interaction mode of the aryloxymethylene substructure cannot be assimilated to that of benzodioxane. Indeed, the α4β2 and α3β4 functional tests well differentiate behaviors that the binding tests homologize: both the 3-hydroxyphenyl and the 5-hydroxy-3-pyridyl ether of N-methylprolinol are α4β2 full agonists, but only the latter is highly α4β2/α3β4 selective, while potent and selective partial α4β2 agonism characterizes the hydroxybenzodioxane derivative and its two opened semirigid analogues.

Objectives: To evaluate whether P2X3 receptors (P2X3R) are expressed in the bladder urothelium and to determine their possible function in modulating purinergic detrusor contractions in the rat urinary bladder., Materials and Methods: The expression of urothelial receptors was determined using conventional immunohistochemistry in bladders from normal Sprague-Dawley rats. The urothelial layer was removed by incubation with protamine, and disruption of the urothelium was confirmed using haematoxylin and eosin staining on bladder sections. Open cystometry was used to determine the effects of both urothelial removal as well as intravesical application of a specific P2X3R antagonist on bladder properties from intact and protamine-treated rats. Isometric contractile responses to potassium chloride (KCl) depolarization, electrical field stimulation (EFS) or chemical P2X activation were determined in normal and urothelium-denuded bladder strips, with and without application of the P2X3R antagonist., Results: Immunohistochemical staining showed high expression of P2X3R in the medial and basal layers of the urothelium. Removal of the urothelial layer disturbed normal bladder performance in vivo and eliminated the effects of the P2X3R antagonist on increasing the contractile interval and reducing the amplitude of voiding contractions. Removal of the urothelium did not affect bladder strip contractile responses to KCl depolarization or EFS. Pharmacological inhibition of P2X3R prevented desensitization to P2X-mediated detrusor muscle contractions during EFS only in the strips with an intact urothelium. A concentration-dependent, specific inhibition of P2X3R also prevented desensitization of purinergic contractile responses in intact bladder strips., Conclusions: In the rat bladder, medial and basal urothelial cells express P2X3R, and specific inhibition of the receptor leads to a more hyporeflexive bladder condition. This pathway may involve P2X3R driving a paracrine amplification of ATP released from umbrella cells to increase afferent transmission in the sub-urothelial sensory plexus and desensitization of P2X1-mediated purinergic detrusor contractions., (© 2014 The Authors BJU International © 2014 BJU International Published by John Wiley & Sons Ltd.)

We previously reported the anticancer effects of MHY218, which is a hydroxamic acid derivative, in HCT116 human colon cancer cells. In the present study, the involvement of autophagy in the MHY218-induced apoptotic cell death of AGS human gastric cancer cells was investigated. MHY218 treatment induced growth inhibition and apoptotic cell death in a concentration- and time-dependent manner. The induction of apoptosis was confirmed by observations of decreased viability, DNA fragmentation, and an increase in late apoptosis and sub-G1 DNA, which were detected with a flow cytometric analysis. Western blot analyses showed that MHY218 treatment resulted in decreased protein levels of procaspase-8, -9, and -3; cleavage of poly(ADP-ribose) polymerase (PARP); and alterations in the ratio of Bax/Bcl-2 protein expression. Apoptosis induced by MHY218 was involved in the activation of caspase-8, -9, and -3, and it was blocked by the addition of Z-VAD‑FMK, a pan-caspase inhibitor. In addition, autophagy-inducing effects of MHY218 were indicated by cytoplasmic vacuolation, the accumulation of acidic vesicular organelles, the appearance of green fluorescent protein-light-chain 3 (LC3) punctate dots, and increased levels of Beclin-1 and LC3-II protein expression. Pretreatment with the autophagy inhibitors LY294002, 3-methyladenine, chloroquine, and bafilomycin A1 enhanced the induction of apoptosis by MHY218, and this was accompanied by an increase in PARP cleavage. Taken together, these results provide new insights into the role of MHY218 as a potential antitumor agent. The combination of MHY218 with an autophagy inhibitor might be a useful candidate for the chemoprevention and/or treatment of gastric cancer.

Background: Na(+)/Ca(2+) exchanger 1 (NCX1) controls intracellular Ca(2+) concentration in various cell types. In the kidney, NCX1 is expressed mainly in the distal tubular basolateral membrane as well as in vascular smooth muscle. Tubular NCX1 is involved in Ca(2+) reabsorption, and NCX1 in renal arterioles may control intraglomerular pressure. However, the functions of renal NCX1 have not been studied in vivo. Therefore, this study examined the effects of renal NCX1 blockade on water and solute metabolism, renal function and blood pressure in rats., Methods: Wistar-Kyoto rats were uninephrectomized, and an osmotic mini pump was implanted to infuse the remnant kidney cortex with a specific NCX1 inhibitor, SEA0400 (SEA), or vehicle for 7 days., Results: Serum Ca(2+) concentration and urinary Ca(2+) excretion were similar between the vehicle- and SEA-treated groups. However, serum phosphate was significantly decreased by 8 % in the SEA group, with similar urinary phosphate excretion between the two groups. Systolic blood pressure was higher in the SEA group (117 ± 3 vs. 126 ± 1 mmHg, n = 9-11), with a 1.6-fold increase in plasma aldosterone concentration. However, SEA significantly reduced urinary protein excretion and the glomerular sectional area by 16 and 8 %, respectively. Similar experiment in spontaneously hypertensive rats produced different results., Conclusion: Renal SEA treatment reduced serum phosphate concentration, urinary protein and glomerular size with higher systemic blood pressure compared to control Wistar-Kyoto rats. Further study on renal NCX1 may be beneficial in delineating the pathophysiology of glomerular pressure control and calcium/phosphate regulations.

Aminoglycoside-induced hair cell loss is a major cause of hearing impairment in children and deserves more attention in medical research. Epigenetic mechanisms have been shown to protect hair cells from ototoxic drugs. In this study, we focused on the role of dimethylated histone H3K4 (H3K4me2) in hair cell survival. To investigate the effects of lysine-specific demethylase 1 (LSD1)--the histone demethylase primarily responsible for demethylating H3K4me2--on neomycin-induced hair cell loss, isolated cochleae were pretreated with LSD1 inhibitors followed by neomycin exposure. There was a severe loss of hair cells in the organ of Corti after neomycin exposure, and inhibition of LSD1 significantly protected against neomycin-induced hair cell loss. H3K4me2 expression in the nuclei of hair cells decreased after exposure to neomycin, and blocking the decreased expression of H3K4me2 with LSD1 inhibitors prevented hair cell loss. Local delivery of these inhibitors in vivo also protected hair cells from neomycin-induced ototoxicity and maintained the hearing threshold in mice as determined by auditory brain stem response. This inhibition of neomycin-induced apoptosis occurs via reduced caspase-3 activation. Together, our findings demonstrate the protective role for H3K4me2 against neomycin-induced hair cell loss and hearing loss.

Oxidative cell damage contributes to neuronal degeneration in many central nervous system (CNS) diseases such as Parkinson's disease, Alzheimer's disease, and ischemia. Inducible heme oxygenase (HO)-1 acts against oxidants that are thought to play a key role in the pathogenesis of neuronal diseases. The stem bark of Acer nikoense Maxim (Aceraceae) is indigenous to Japan; it has been used in folk medicine as a treatment of hepatic disorders and eye diseases. Acerogenin A, a natural compound isolated from Japanese folk medicine A. nikoense, showed neuroprotective effects and reactive oxygen species (ROS) reduction on glutamate-induced neurotoxicity by inducing the expression of HO-1 in mouse hippocampal HT22 cells. Furthermore, acerogenin A caused the nuclear accumulation of nuclear factor-E2-related factor 2 (Nrf2) and the activation of the PI3K/AKT signaling pathways. In this study, we demonstrated that acerogenin A effectively prevents glutamate-induced oxidative damage, and HO-1 induction via PI3K/Akt and Nrf2 pathways appears to play a key role in the protection of HT22 cells. Therefore, this study implies that the Nrf2/HO-1 pathway represents a biological target and that acerogenin A might be a candidate for the prevention of neurodegeneration.

Emixustat is a visual cycle modulator that has entered clinical trials as a treatment for age-related macular degeneration (AMD). This molecule has been proposed to inhibit the visual cycle isomerase RPE65, thereby slowing regeneration of 11-cis-retinal and reducing production of retinaldehyde condensation byproducts that may be involved in AMD pathology. Previously, we reported that all-trans-retinal (atRAL) is directly cytotoxic and that certain primary amine compounds that transiently sequester atRAL via Schiff base formation ameliorate retinal degeneration. Here, we have shown that emixustat stereoselectively inhibits RPE65 by direct active site binding. However, we detected the presence of emixustat-atRAL Schiff base conjugates, indicating that emixustat also acts as a retinal scavenger, which may contribute to its therapeutic effects. Using agents that lack either RPE65 inhibitory activity or the capacity to sequester atRAL, we assessed the relative importance of these 2 modes of action in protection against retinal phototoxicity in mice. The atRAL sequestrant QEA-B-001-NH2 conferred protection against phototoxicity without inhibiting RPE65, whereas an emixustat derivative incapable of atRAL sequestration was minimally protective, despite direct inhibition of RPE65. These data indicate that atRAL sequestration is an essential mechanism underlying the protective effects of emixustat and related compounds against retinal phototoxicity. Moreover, atRAL sequestration should be considered in the design of next-generation visual cycle modulators.

Reprogramming somatic cells into induced-pluripotent cells (iPSCs) provides new access to all somatic cell types for clinical application without any ethical controversy arising from the use of embryonic stem cells (ESCs). Established protocols for iPSCs generation based on viral transduction with defined factors are limited by low efficiency and the risk of genetic abnormality. Several small molecules have been reported as replacements for defined transcriptional factors, but a chemical able to replace Oct3/4 allowing the generation of human iPSCs is still unavailable. Using a cell-based High Throughput Screening (HTS) campaign, we identified that 2-[4-[(4-methoxyphenyl)methoxy]phenyl]acetonitrile (1), termed O4I1, enhanced Oct3/4 expression. Structural verification and modification by chemical synthesis showed that O4I1 and its derivatives not only promoted expression and stabilization of Oct3/4 but also enhanced its transcriptional activity in diverse human somatic cells, implying the possible benefit from using this class of compounds in regenerative medicine.

Cisplatin is a widely used chemotherapeutic drug, but one of its side effects is ototoxicity. Epigenetic-related drugs, such as lysine-specific demethylase 1 (LSD1) inhibitors, have been reported to protect against cisplatin-induced hair cell loss by preventing demethylation of histone H3K4 (H3K4me2). However, the protective effect of LSD1 inhibitors in spiral ganglion neurons (SGNs) remains unclear. To investigate whether LSD1 inhibitors exert similar protective effects on SGNs, we treated mouse cochlear explant cultures with LSD1 inhibitors (2PCPA, S2101, or CBB1007) together with cisplatin. Low concentrations of cisplatin damaged SGNs much more than high concentrations, and blocking the demethylation of H3K4me2 with LSD1 inhibitors prevented the SGNs from injury. Reactive oxygen species are also involved in the injury process, and LSD1 inhibitors protected SGNs by increasing the expression level of the antioxidant gene Slc7a11 and decreasing the level of the pro-oxidant gene lactoperoxidase (Lpo). Our findings show that LSD1 inhibitors prevent cisplatin-induced SGN loss by regulating the demethylation of H3K4 and preventing increases of reactive oxygen species levels, which might provide a potential therapeutic strategy for cisplatin-induced hearing loss.

Candida albicans, which is the most common human fungal pathogen, causes high mortality among immunocompromised patients. Antifungal drug resistance becomes a major challenge for the management of Candida infection. Diorcinol D (DD), a diphenyl ether derivative isolated from an endolichenic fungus, exerted fungicidal action against Candida species. In this study, we investigated the possible mechanism of its antifungal activity. The change of membrane dynamics and permeability suggested that the cell membrane was disrupted by the treatment of DD. This was further supported by the evidences of intracellular glycerol accumulation, alteration of cell ultrastructure, and down-regulation of genes involved in cell membrane synthesis. In addition, the treatment of C. albicans with DD resulted in the elevation of reactive oxygen species (ROS), which caused the dysfunction of mitochondria. These altogether suggested that DD exerted its antifungal activity through cytoplasmic membrane destruction and ROS accumulation. This finding is helpful to uncover the underlying mechanisms for the diphenyl ether derivatives and provides a potential application in fighting clinical fungal infections.

Background and Purpose: Inhaled amiloride, a blocker of the epithelial sodium channel (ENaC), enhances mucociliary clearance (MCC) in cystic fibrosis (CF) patients. However, the dose of amiloride is limited by the mechanism-based side effect of hyperkalaemia resulting from renal ENaC blockade. Inhaled ENaC blockers with a reduced potential to induce hyperkalaemia provide a therapeutic strategy to improve mucosal hydration and MCC in the lungs of CF patients. The present study describes the preclinical profile of a novel ENaC blocker, NVP-QBE170, designed for inhaled delivery, with a reduced potential to induce hyperkalaemia., Experimental Approach: The in vitro potency and duration of action of NVP-QBE170 were compared with amiloride and a newer ENaC blocker, P552-02, in primary human bronchial epithelial cells (HBECs) by short-circuit current. In vivo efficacy and safety were assessed in guinea pig (tracheal potential difference/hyperkalaemia), rat (hyperkalaemia) and sheep (MCC)., Key Results: In vitro, NVP-QBE170 potently inhibited ENaC function in HBEC and showed a longer duration of action to comparator molecules. In vivo, intratracheal (i.t.) instillation of NVP-QBE170 attenuated ENaC activity in the guinea pig airways with greater potency and duration of action than that of amiloride without inducing hyperkalaemia in either guinea pig or rat. Dry powder inhalation of NVP-QBE170 by conscious sheep increased MCC and was better than inhaled hypertonic saline in terms of efficacy and duration of action., Conclusions and Implications: NVP-QBE170 highlights the potential for inhaled ENaC blockers to exhibit efficacy in the airways with a reduced risk of hyperkalaemia, relative to existing compounds., (© 2015 The British Pharmacological Society.)

We investigated the protective ability of 3-(4'-hydroxyl-3',5'-dimethoxyphenyl)propionic acid (HDMPPA), an active principle in Korean cabbage kimchi, against the production of proinflammatory mediators and cytokines, and the mechanisms involved in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. HDMPPA significantly suppressed the production of nitric oxide (NO) and prostaglandin E2, along with the expression of inducible NO synthase and cyclooxygenase-2 in LPS-stimulated BV2 cells, at concentrations with no cytotoxicity. HDMPPA also attenuated the LPS-induced expression and secretion of proinflammatory cytokines, such as tumor necrosis factor-α and interleukin-1β. Furthermore, HDMPPA inhibited LPS-induced nuclear factor-κB (NF-κB) activation, which was associated with the abrogation of IκB-α degradation and phosphorylation, and subsequent decreases in NF-κB p65 levels. Moreover, the phosphorylation of mitogen-activated protein kinases (MAPKs) and Akt, a downstream molecule of phosphatidylinositol-3-kinase (PI3K), in LPS-stimulated BV2 cells was suppressed markedly by HDMPPA. This effect was associated with a significant reduction in the formation of intracellular reactive oxygen species. The findings in this study suggest that HDMPPA may exert anti-inflammatory responses by suppressing LPS-induced expression of proinflammatory mediators and cytokines through blockage of NF-κB, MAPKs, and PI3K/Akt signaling pathways and oxidative stress in microglia.

Purpose: This study assessed the safety, tolerability, and pharmacodynamics of emixustat hydrochloride (ACU-4429), a novel visual cycle modulator, in subjects with geographic atrophy associated with dry age-related macular degeneration., Methods: Subjects were randomly assigned to oral emixustat (2, 5, 7, or 10 mg once daily) or placebo (3:1 ratio) for 90 days. Recovery of rod photoreceptor sensitivity after a photobleach was measured by electroretinography. Safety evaluations included analysis of adverse events and ophthalmic examinations., Results: Seventy-two subjects (54 emixustat and 18 placebo) were evaluated. Emixustat suppressed rod photoreceptor sensitivity in a dose-dependent manner. Suppression plateaued by Day 14 and was reversible within 7 days to 14 days after drug cessation. Most systemic adverse events were not considered treatment related. Dose-related ocular adverse events (chromatopsia, 57% emixustat vs. 17% placebo and delayed dark adaptation, 48% emixustat vs. 6% placebo) were mild to moderate in severity, and the majority resolved on study or within 7 days to 14 days after study drug cessation. Reversibility of these adverse events with long-term administration, however, is undetermined., Conclusion: In this Phase II study, emixustat produced a dose-dependent reversible effect on rod function that is consistent with the proposed mechanism of action. These results support further testing of emixustat for the treatment of geographic atrophy associated with dry age-related macular degeneration.

Visual function in vertebrates is dependent on the membrane-bound retinoid isomerase RPE65, an essential component of the retinoid cycle pathway that regenerates 11-cis-retinal for rod and cone opsins. The mechanism by which RPE65 catalyzes stereoselective retinoid isomerization has remained elusive because of uncertainty about how retinoids bind to its active site. Here we present crystal structures of RPE65 in complex with retinoid-mimetic compounds, one of which is in clinical trials for the treatment of age-related macular degeneration. The structures reveal the active site retinoid-binding cavity located near the membrane-interacting surface of the enzyme as well as an Fe-bound palmitate ligand positioned in an adjacent pocket. With the geometry of the RPE65-substrate complex clarified, we delineate a mechanism of catalysis that reconciles the extensive biochemical and structural research on this enzyme. These data provide molecular foundations for understanding a key process in vision and pharmacological inhibition of RPE65 with small molecules.

Increased exposure to blue or visible light, fluctuations in oxygen tension, and the excessive accumulation of toxic retinoid byproducts places a tremendous amount of stress on the retina. Reduction of visual chromophore biosynthesis may be an effective method to reduce the impact of these stressors and preserve retinal integrity. A class of non-retinoid, small molecule compounds that target key proteins of the visual cycle have been developed. The first candidate in this class of compounds, referred to as visual cycle modulators, is emixustat hydrochloride (emixustat). Here, we describe the effects of emixustat, an inhibitor of the visual cycle isomerase (RPE65), on visual cycle function and preservation of retinal integrity in animal models. Emixustat potently inhibited isomerase activity in vitro (IC50 = 4.4 nM) and was found to reduce the production of visual chromophore (11-cis retinal) in wild-type mice following a single oral dose (ED50 = 0.18 mg/kg). Measure of drug effect on the retina by electroretinography revealed a dose-dependent slowing of rod photoreceptor recovery (ED50 = 0.21 mg/kg) that was consistent with the pattern of visual chromophore reduction. In albino mice, emixustat was shown to be effective in preventing photoreceptor cell death caused by intense light exposure. Pre-treatment with a single dose of emixustat (0.3 mg/kg) provided a ~50% protective effect against light-induced photoreceptor cell loss, while higher doses (1-3 mg/kg) were nearly 100% effective. In Abca4-/- mice, an animal model of excessive lipofuscin and retinoid toxin (A2E) accumulation, chronic (3 month) emixustat treatment markedly reduced lipofuscin autofluorescence and reduced A2E levels by ~60% (ED50 = 0.47 mg/kg). Finally, in the retinopathy of prematurity rodent model, treatment with emixustat during the period of ischemia and reperfusion injury produced a ~30% reduction in retinal neovascularization (ED50 = 0.46mg/kg). These data demonstrate the ability of emixustat to modulate visual cycle activity and reduce pathology associated with various biochemical and environmental stressors in animal models. Other attributes of emixustat, such as oral bioavailability and target specificity make it an attractive candidate for clinical development in the treatment of retinal disease.

Four new nitroasterric acid derivatives, pseudogymnoascins A-C (1-3) and 3-nitroasterric acid (4), along with the two known compounds questin and pyriculamide, were obtained from the cultures of a Pseudogymnoascus sp. fungus isolated from an Antarctic marine sponge belonging to the genus Hymeniacidon. The structures of the new compounds were determined by extensive NMR and MS analyses. These compounds are the first nitro derivatives of the known fungal metabolite asterric acid. Several asterric acid derivatives isolated from other fungal strains have shown antibacterial and antifungal activities. However, the new compounds described in this work were inactive against a panel of bacteria and fungi (MIC > 64 μg/mL).

Eleven new polyphenols namely spiromastols A-K (1-11) were isolated from the fermentation broth of a deep sea-derived fungus Spiromastix sp. MCCC 3A00308. Their structures were determined by extensive NMR data and mass spectroscopic analysis in association with chemical conversion. The structures are classified as diphenyl ethers, diphenyl esters and isocoumarin derivatives, while the n-propyl group in the analogues is rarely found in natural products. Compounds 1-3 exhibited potent inhibitory effects against a panel of bacterial strains, including Xanthomanes vesicatoria, Pseudomonas lachrymans, Agrobacterium tumefaciens, Ralstonia solanacearum, Bacillus thuringensis, Staphylococcus aureus and Bacillus subtilis, with minimal inhibitory concentration (MIC) values ranging from 0.25 to 4 µg/mL. The structure-activity relationships are discussed, while the polychlorinated analogues 1-3 are assumed to be a promising structural model for further development as antibacterial agents.

A chemical investigation of the endolichenic fungus Aspergillus versicolor (125a), which was found in the lichen Lobaria quercizans, resulted in the isolation of four novel diphenyl ethers, named diorcinols F-H (1-3, resp.) and 3-methoxyviolaceol-II (4), eight new bisabolane sesquiterpenoids, named (-)-(R)-cyclo-hydroxysydonic acid (5), (-)-(7S,8R)-8-hydroxysydowic acid (6), (-)-(7R,10S)-10-hydroxysydowic acid (7), (-)-(7R,10R)-iso-10-hydroxysydowic acid (8), (-)-12-acetoxy-1-deoxysydonic acid (9), (-)-12-acetoxysydonic acid (10), (-)-12-hydroxysydonic acid (11), and (-)-(R)-11-dehydrosydonic acid (12), two new tris(pyrogallol ethers), named sydowiols D (13) and E (14), and fifteen known compounds, 15-29. All of the structures were determined by spectroscopic analyses, and a number of them were further identified through chemical transformations and electronic circular dichroism (ECD) calculations. Preliminary bioassays of these isolates for the determination of their inhibitory activities against the fungus Candida albicans, and their cytotoxicities against the human cancer cell lines PC3, A549, A2780, MDA-MB-231, and HEPG2 were also evaluated., (Copyright © 2015 Verlag Helvetica Chimica Acta AG, Zürich.)

Background and Objectives: The creation of cardiomyocytes derived from human induced pluripotent stem cells (hiPS-CMs) has spawned broad excitement borne out of the prospects to diagnose and treat cardiovascular diseases based on personalized medicine. A common feature of hiPS-CMs is their spontaneous contractions but the mechanism(s) remain uncertain., Methods: Intrinsic activity was investigated by the voltage-clamp technique, optical mapping of action potentials (APs) and intracellular Ca(2+) (Cai) transients (CaiT) at subcellular-resolution and pharmacological interventions., Results: The frequency of spontaneous CaiT (sCaiT) in monolayers of hiPS-CMs was not altered by ivabradine, an inhibitor of the pacemaker current, If despite high levels of HCN transcripts (1-4). HiPS-CMs had negligible If and IK1 (inwardly-rectifying K(+)-current) and a minimum diastolic potential of -59.1±3.3mV (n=18). APs upstrokes were preceded by a depolarizing-foot coincident with a rise of Cai. Subcellular Cai wavelets varied in amplitude, propagated and died-off; larger Cai-waves triggered cellular sCaTs and APs. SCaiTs increased in frequency with [Ca(2+)]out (0.05-to-1.8mM), isoproterenol (1μM) or caffeine (100μM) (n≥5, p<0.05). HiPS-CMs became quiescent with ryanodine receptor stabilizers (K201=2μM); tetracaine; Na-Ca exchange (NCX) inhibition (SEA0400=2μM); higher [K(+)]out (5→8mM), and thiol-reducing agents but could still be electrically stimulated to elicit CaiTs. Cell-cell coupling of hiPS-CM in monolayers was evident from connexin-43 expression and CaiT propagation. SCaiTs from an ensemble of dispersed hiPS-CMs were out-of-phase but became synchronous through the outgrowth of inter-connecting microtubules., Conclusions: Automaticity in hiPS-CMs originates from a Ca(2+)-clock mechanism involving Ca(2+) cycling across the sarcoplasmic reticulum linked to NCX to trigger APs., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

During ischaemia/reperfusion, the rise in [Na(+)](i), induced by simultaneous depression of the Na(+)/K(+)-ATPase and activation of the Na(+)/H(+) exchanger (NHE), shifts the Na(+)/Ca(2+) exchanger (NCX) into reverse transport mode, resulting in Ca(2+)(i)overload, which is a critical factor in enhancing the liability to cardiac arrhythmias. The inhibition of NHE, and recently NCX has been suggested to effectively protect the heart from reperfusion-induced arrhythmias. In this study, we investigated and compared the efficacy of individual or the simultaneous inhibition of the NHE and NCX against reperfusion-induced arrhythmias in Langendorff-perfused rat hearts by applying a commonly used regional ischaemia-reperfusion protocol. The NHE and NCX were inhibited by cariporide and SEA0400 or the novel, more selective ORM-10103, respectively. Arrhythmia diagrams calculated for the reperfusion period were analysed for the incidence and duration of extrasystoles (ESs), ventricular tachycardia (VT) and ventricular fibrillation (VF). NHE inhibition by cariporide was highly efficient in reducing the recorded reperfusion-induced arrhythmias. Following the application of SEA0400 or ORM-10103, the number and duration of arrhythmic periods were efficiently or moderately decreased. While both NCX inhibitors effectively reduced ESs, the most frequently triggered arrhythmias, they exerted limited or no effect on VTs and VFs. Of the NCX inhibitors, ORM-10103 was more effective. Surprisingly, the simultaneous inhibition of the NCX and NHE failed to significantly improve the antiarrhythmic efficacy reached by NCX blockade alone. In conclusion, although principal simultaneous NHE+NCX inhibition should be highly effective against all types of the recorded reperfusion-induced arrhythmias, NCX inhibitors, alone or in combination with cariporide, seem to be moderately suitable to provide satisfactory cardioprotection - at least in the present arrhythmia model. Since ORM-10103 and SEA0400 are known to effectively inhibit after-depolarisations, it is suggested that their efficacy and that of other NCX inhibitors may be higher and more pronounced in the predominantly Ca(2+)(i)-dependent triggered arrhythmias.

Three new diphenyl ether derivatives, talaromycins A-C (1-3, resp.), together with six known analogs, 4-9, were isolated from a gorgonian-derived fungus, Talaromyces sp. The structures of the new compounds were determined by analysis of extensive NMR spectroscopic data. All of the isolated metabolites, 1-9, were evaluated for their cytotoxic and antifouling activities. Compound 4 exhibited pronounced cytotoxicity against the tested human cell lines with the IC50 values ranging from 4.3 to 9.8 μM. Compounds 3, 5, 8, and 9 showed potent antifouling activities against the larval settlement of the barnacle Balanus amphitrite with the EC50 values ranging from 2.2 to 4.8 μg/ml., (Copyright © 2015 Verlag Helvetica Chimica Acta AG, Zürich.)

The Na(+)/Ca(2+) exchanger (NCX) is a bidirectional transporter that is controlled by membrane potential and transmembrane gradients of Na(+) and Ca(2+). Although two isoforms of NCX1 and NCX2 are coexpressed on the basolateral membrane of the distal nephron, the functional significance of these isoforms is not entirely clear. Therefore, we used NCX1- and NCX2-heterozygote knockout mice (KO) and their double KO, as well as isoform-selective NCX inhibitors, to determine the roles of NCX isoforms in urine formation and electrolyte excretion in mice. NCX inhibitors, particularly NCX2-sensitive inhibitors, caused a dose-dependent natriuresis and in a higher dose, moreover, hypercalciuria. Consistently, NCX1-KO possessed normal renal function similar to wild-type mice (WT), whereas NCX2-KO and double KO exhibited moderate natriuresis and hypercalciuria. Notably, renal responses to YM-244769 were equivalently observed in NCX1-KO and WT, but disappeared in NCX2-KO and double KO. Thus, functional inhibition of NCX2 initially causes natriuresis, and further inhibition of NCX2 produces hypercalciuria, suggesting that the functional significance of NCX2 lies in Na(+) and Ca(2+) reabsorption of the kidney., (Copyright © 2014 Elsevier Inc. All rights reserved.)

We examined the effects of SEA0400 and CGP-37157 on the plasmalemmal Na(+)-Ca(2+) exchanger (NCX) and mitochondrial NCX using H9c2 cardiomyocytes loaded with Ca(2+)-sensitive fluorescent probes. The plasmalemmal NCX activity, which was measured as the increase in cytoplasmic Ca(2+) concentration after application of low Na(+) extracellular solution, was inhibited by SEA0400 but not by CGP-37157. The mitochondrial NCX activity, which was measured in permeabilized H9c2 cells as the decrease in mitochondrial Ca(2+) concentration after application of Ca(2+)-free extramitochondrial solution, was inhibited by CGP-37157 but not by SEA0400. These results indicate that SEA0400 and CGP-37157 act as selective inhibitors towards plasmalemmal and mitochondrial NCX, respectively, and provide pharmacological evidence that the plasmalemmal and mitochondrial NCX are distinct molecular entities.

A highly convenient method has been developed for the synthesis of (prop-2-ynyloxy) benzene and its derivatives. Differently substituted phenol and aniline derivatives were allowed to react with propargyl bromide in the presence of K2CO3 base and acetone as solvent. The compounds were synthesized in good yields (53-85%). Low cost, high yields and easy availability of compounds helped in the synthesis. Electron withdrawing groups favor the formation of stable phenoxide ion thus in turn favors the formation of product while electron donating groups do not favor the reaction. Phenol derivatives gave good yields as compared to that of aniline. As aprotic polar solvents favor SN2 type reactions so acetone provided best solvation for the reactions. K2CO3 was proved to be good for the synthesis. Antibacterial, Antiurease and NO scavenging activity of synthesized compounds were also examined. 4-bromo-2-chloro-1-(prop-2-ynyloxy)benzene 2a was found most active compound against urease enzyme with a percentage inhibition of 82.00±0.09 at 100 µg/mL with IC50 value of 60.2. 2-bromo-4-methyl-1-(prop-2-ynyloxy)benzene 2d was found potent antibacterial against Bacillus subtillus showing excellent inhibitory action with percentage inhibition of 55.67±0.26 at 100 µg/ml wih IC50 value of 79.9. Based on results, it can be concluded that some of the synthesized compounds may have potential antiurease and antibacterial effects against several harmful substances.

Background: Monoamine reuptake inhibitors exhibit unique clinical profiles that reflect distinct engagement of the central nervous system (CNS) transporters., Methods: We used a translational strategy, including rodent pharmacokinetic/pharmacodynamic modeling and positron emission tomography (PET) imaging in humans, to establish the transporter profile of TD-9855, a novel norepinephrine and serotonin reuptake inhibitor., Results: TD-9855 was a potent inhibitor of norepinephrine (NE) and serotonin 5-HT uptake in vitro with an inhibitory selectivity of 4- to 10-fold for NE at human and rat transporters. TD-9855 engaged norepinephrine transporters (NET) and serotonin transporters (SERT) in rat spinal cord, with a plasma EC50 of 11.7 ng/mL and 50.8 ng/mL, respectively, consistent with modest selectivity for NET in vivo. Accounting for species differences in protein binding, the projected human NET and SERT plasma EC50 values were 5.5 ng/mL and 23.9 ng/mL, respectively. A single-dose, open-label PET study (4-20mg TD-9855, oral) was conducted in eight healthy males using the radiotracers [(11)C]-3-amino-4- [2-[(di(methyl)amino)methyl]phenyl]sulfanylbenzonitrile for SERT and [(11)C]-(S,S)-methylreboxetine for NET. The long pharmacokinetic half-life (30-40 h) of TD-9855 allowed for sequential assessment of SERT and NET occupancy in the same subject. The plasma EC50 for NET was estimated to be 1.21 ng/mL, and at doses of greater than 4 mg the projected steady-state NET occupancy is high (>75%). After a single oral dose of 20mg, SERT occupancy was 25 (±8)% at a plasma level of 6.35 ng/mL., Conclusions: These data establish the CNS penetration and transporter profile of TD-9855 and inform the selection of potential doses for future clinical evaluation., (© The Author 2015. Published by Oxford University Press on behalf of CINP.)

Obovatol, a compound isolated from the bark cortex of Magnolia officinalis (cortex Magnoliae officinalis; M. officinalis), has been studied for use in the treatment of solid cancers. However, the mechanisms of action and the effects of obovatol against acute myeloid leukemia (AML) remain unclear and require further investigation. Therefore, this study was conducted using a human AML cell line (MM6). Obovatol increased pro-apoptotic (Bax) and decreased anti-apoptotic (Bcl-2) protein expression, resulting in caspase-3 and caspase-9 activation measured by caspase-Glo 3/7 assay. Furthermore, obovatol activated the mitogen-activated protein kinase (MAPK) signaling pathway [c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38] and inhibited the activation of the nuclear factor-κB (NF-κB) signaling pathway analyzed by western blot analysis. Taken together, these findings provide evidence that obovatol inhibits cell proliferation in AML and induces apoptosis through the activation of the MAPK pathway in addition to the intrinsic apoptotic pathway. In addition, obovatol suppressed the expression of mixed-lineage leukemia (MLL) target genes by inhibiting the activation of the NF-κB pathway. Therefore, these results suggest that obovatol may have potential for use in the treatment of leukemia.

Background and Purpose: Augmented Na(+) /Ca(2+) exchanger (NCX) activity may play a crucial role in cardiac arrhythmogenesis; however, data regarding the anti-arrhythmic efficacy of NCX inhibition are debatable. Feasible explanations could be the unsatisfactory selectivity of NCX inhibitors and/or the dependence of the experimental model on the degree of Ca(2+) i overload. Hence, we used NCX inhibitors SEA0400 and the more selective ORM10103 to evaluate the efficacy of NCX inhibition against arrhythmogenic Ca(2+) i rise in conditions when [Ca(2+) ]i was augmented via activation of the late sodium current (INaL ) or inhibition of the Na(+) /K(+) pump., Experimental Approach: Action potentials (APs) were recorded from canine papillary muscles and Purkinje fibres by microelectrodes. NCX current (INCX ) was determined in ventricular cardiomyocytes utilizing the whole-cell patch clamp technique. Ca(2+) i transients (CaTs) were monitored with a Ca(2+) -sensitive fluorescent dye, Fluo-4., Key Results: Enhanced INaL increased the Ca(2+) load and AP duration (APD). SEA0400 and ORM10103 suppressed INCX and prevented/reversed the anemone toxin II (ATX-II)-induced [Ca(2+) ]i rise without influencing APD, CaT or cell shortening, or affecting the ATX-II-induced increased APD. ORM10103 significantly decreased the number of strophanthidin-induced spontaneous diastolic Ca(2+) release events; however, SEA0400 failed to restrict the veratridine-induced augmentation in Purkinje-ventricle APD dispersion., Conclusions and Implications: Selective NCX inhibition - presumably by blocking rev INCX (reverse mode NCX current) - is effective against arrhythmogenesis caused by [Na(+) ]i -induced [Ca(2+) ]i elevation, without influencing the AP waveform. Therefore, selective INCX inhibition, by significantly reducing the arrhythmogenic trigger activity caused by the perturbed Ca(2+) i handling, should be considered as a promising anti-arrhythmic therapeutic strategy., (© 2014 The British Pharmacological Society.)

The cabbage looper, Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae), is one of the most damaging insect pests of cabbage (Brassica oleracea variety capitata) and broccoli (B. oleracea variety italica) in North America. Leaf-feeding larvae attack crucifer and vegetable crops in greenhouses and fields. Here, we have studied a synthetic feeding deterrent, 1-allyloxy-4-propoxybenzene, and a botanical deterrent, neem (an extract from seeds of Azadirachta indica A. de Jussieu (Meliaceae)), in leaf disc choice bioassays with T. ni. We tested the two deterrents and the combination, and we found that the blend exhibits synergy between the two deterrents. We also tested the deterrents in assays with whole cabbage plants in ventilated enclosures and found that 1-allyloxy-4-propoxybenzene evaporated and, therefore, in that context addition of 1-allyloxy-4-propoxybenzene to neem did not enhance deterrence against T. ni., (© 2014 Entomological Society of America.)

A new family of poly-guanidinium species with a benzene core as the organising agent has been developed. Their antibacterial properties have been evaluated against reference Gram positive and Gram negative bacteria, and their cytotoxicity against eukaryotic cells. Most of these compounds exhibited Minimum Inhibitory Concentrations in the micromolar range., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: It has been shown that pulmonary retinol level is decreased during lung morphogenesis in the nitrofen-induced PH in congenital diaphragmatic hernia (CDH). Placenta has a major role in the retinol homeostasis in fetal life. Since there is no fetal retinol synthesis, maternal retinol has to cross the placenta. Placenta is the main fetal retinol store where retinol is stored in retinyl-ester formation. Trophoblasts have to produce its own retinol-binding protein (RBP) for retinol transport from placenta to fetus. Recently, we demonstrated that trophoblastic RBP expression is decreased in the nitrofen model of CDH. The aim of this study was to investigate the retinol transfer from mother to the placenta in nitrofen model of CDH., Methods: Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetal placenta harvested on D21 and divided into two groups: control (n = 11) and nitrofen with CDH (n = 11). Retinoid levels in placenta were measured using HPLC. Immunohistochemistry was performed to evaluate trophoblastic expression of main RSP genes., Results: Total retinol levels in the placenta were significantly increased in CDH placenta compared to control placenta. The retinyl-ester levels were significantly increased in CDH placenta compared to control placenta. Markedly, decreased immunoreactivity of retinoid signaling pathway was observed in trophoblast cells in CDH compared to control placenta., Conclusions: Increased placental retinol levels show that retinol is transferred from mother to placenta and stored in the placenta in nitrofen model of CDH during lung morphogenesis. Nitrofen may disturb the mobilization of retinol from placenta to fetal circulation causing PH in CDH.

We report the synthesis of 3-phenethylazetidine derivatives 2 and their biological activities against 5-HT, NE and DA transporters as well as microsomal stability, CYP inhibition, and hERG inhibition profiles. Compound 2at showed most potent triple reuptake inhibitor with good selectivity as a candidate for depression., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Macrophage foam cell formation by oxidized low-density lipoprotein (oxLDL) is a key step in the progression of atherosclerosis, which is involved in cholesterol influx and efflux in macrophages mediated by related proteins such as peroxisome proliferator-activated receptor γ (PPARγ), CD36, PPARα, liver-X receptor α (LXRα), and ATP-binding cassette transporter A1 (ABCA1). The aim of this study was to investigate the beneficial effects of kimchi methanol extract (KME) and a kimchi active compound, 3-(4'-hydroxyl-3',5'-dimethoxyphenyl)propionic acid (HDMPPA) on cholesterol flux in THP-1-derived macrophages treated with oxLDL. The effects of KME and HDMPPA on cell viability and lipid peroxidation were determined. Furthermore, the protein expression of PPARγ, CD36, PPARα, LXRα, and ABCA1 was examined. OxLDL strongly induced cell death and lipid peroxidation in THP-1-derived macrophages. However, KME and HDMPPA significantly improved cell viability and inhibited lipid peroxidation induced by oxLDL in THP-1-derived macrophages (P<.05). Moreover, KME and HDMPPA suppressed CD36 and PPARγ expressions, both of which participate in cholesterol influx. In contrast, KME and HDMPPA augmented LXRα, PPARα, and ABCA1 expression, which are associated with cholesterol efflux. Consequently, KME and HDMPPA suppressed lipid accumulation. These results indicate that KME and HDMPPA may inhibit lipid accumulation, in part, by regulating cholesterol influx- and efflux-related proteins. These findings will thus be useful for future prevention strategies against atherosclerosis.