Complement C1q activates canonical Wnt signaling and promotes aging-related phenotypes.

Wnt signaling plays critical roles in development of various organs and pathogenesis of many diseases, and augmented Wnt signaling has recently been implicated in mammalian aging and aging-related phenotypes. We here report that complement C1q activates canonical Wnt signaling and promotes aging-associated decline in tissue regeneration. Serum C1q concentration is increased with aging, and Wnt signaling activity is augmented during aging in the serum and in multiple tissues of wild-type mice, but not in those of C1qa-deficient mice. C1q activates canonical Wnt signaling by binding to Frizzled receptors and subsequently inducing C1s-dependent cleavage of the ectodomain of Wnt coreceptor low-density lipoprotein receptor-related protein 6. Skeletal muscle regeneration in young mice is inhibited by exogenous C1q treatment, whereas aging-associated impairment of muscle regeneration is restored by C1s inhibition or C1qa gene disruption. Our findings therefore suggest the unexpected role of complement C1q in Wnt signal transduction and modulation of mammalian aging.

Establishment of high-performance liquid chromatography-photodiode array method for 43 weight loss agents and effect of basic environment on bisacodyl degradation.

RATIONALE: The demand for weight loss products is increasing as slimness emerges as the new aesthetic standard and people's desire to achieve it increases. In addition, the distribution and sale of products containing illegal ingredients, pharmaceuticals, and chemicals for which safety is not guaranteed and that cannot be used as foods or dietary supplements are increasing. Thus, the development of an analytical method that could monitor these illegal products is required. METHODS: A high-performance liquid chromatography-photodiode array method capable of rapid and reliable qualitative and quantitative analyses of 43 weight loss agents was established and validated. RESULTS: The process involved dividing analytes into three groups for rapid analysis; when bisacodyl was mixed with chlorocyclopentylsibutramine, it decomposed into its metabolites: monoacetyl bisacodyl and bis-(p-hydroxypheny)-pyridyl-2-methane. This decomposition was due to NaOH that was used to prepare the chlorocyclopentylsibutramine standard solution. Bisacodyl did not degrade when mixed with neutralized chlorocyclopentylsibutramine, whereas when NaOH was added, it rapidly degraded. We identified the bisacodyl degradation products using liquid chromatography-quadrupole-Orbitrap/mass spectrometry. MS2 spectra with proposed structures of fragment peaks were also obtained. CONCLUSIONS: The developed method could be used to regulate slimming products that threaten public health, and knowledge of bisacodyl degradation will be used as the basis for developing an analytic method.

Effects of structural changes on antibacterial activity and cytotoxicity due to proline substitutions in chimeric peptide HnMc.

Owing to the rapidly increasing emergence of multidrug-resistant pathogens, antimicrobial peptides (AMPs) are being explored as next-generation antibiotics. However, AMPs present in nature are highly toxic and exhibit low antibacterial activity. Simple modifications, such as amino acid substitution, can enhance antimicrobial activity and cell selectivity. Herein, we show that HnMc-W, substituted by the Phe1Trp analog of HnMc, a chimeric peptide, resulted in membranolytic antibacterial action and enhanced salt tolerance, whereas HnMc-WP1 with one Ser9Pro substitution resulted in a proline-kink helical structure that increased salt-tolerant antibacterial effects and reduced cytotoxicity. In addition, the HnMc-WP2 peptide, designed with a PXXP motif, had a flexible central hinge in its α-helical structure due to the introduction of two Pro and two Gln (X positions, by deletion of two Gln at positions 16 and 17) residues instead of Ser at position. HnMc-WP2 exhibited excellent antibacterial effects without cytotoxicity in vitro. Moreover, its potent antibacterial activity was demonstrated in a drug-resistant Pseudomonas aeruginosa-infected mouse model in vivo. Our findings provide valuable information for the design of peptides with high antibacterial activity and cell selectivity.

Aberrant accumulation of TMEM43 accompanied by perturbed transmural gene expression in arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) caused by TMEM43 p.S358L is a fully penetrant heart disease that results in impaired cardiac function or fatal arrhythmia. However, the molecular mechanism of ACM caused by the TMEM43 variant has not yet been fully elucidated. In this study, we generated knock-in (KI) rats harboring a Tmem43 p.S358L mutation and established induced pluripotent stem cells (iPSCs) from patients based on the identification of TMEM43 p.S358L variant from a family with ACM. The Tmem43-S358L KI rats exhibited ventricular arrhythmia and fibrotic myocardial replacement in the subepicardium, which recapitulated the human ACM phenotype. The four-transmembrane protein TMEM43 with the p.S358L variant (TMEM43S358L ) was found to be modified by N-linked glycosylation in both KI rat cardiomyocytes and patient-specific iPSC-derived cardiomyocytes. TMEM43S358L glycosylation increased under the conditions of enhanced endoplasmic reticulum (ER) stress caused by pharmacological stimulation or age-dependent decline of the ER function. Intriguingly, the specific glycosylation of TMEM43S358L resulted from the altered membrane topology of TMEM43. Moreover, unlike TMEM43WT , which is mainly localized to the ER, TMEM43S358L accumulated at the nuclear envelope of cardiomyocytes with the increase in glycosylation. Finally, our comprehensive transcriptomic analysis demonstrated that the regional differences in gene expression patterns between the inner and outer layers observed in the wild type myocardium were partially diminished in the KI myocardium prior to exhibiting histological changes indicative of ACM. Altogether, these findings suggest that the aberrant accumulation of TMEM43S358L underlies the pathogenesis of ACM caused by TMEM43 p.S358L variant by affecting the transmural gene expression within the myocardium.

Synthesis of 7,2'-Dihydroxy-4',5'-Dimethoxyisoflavanone, a Phytoestrogen with Derma Papilla Cell Proliferative Activity.

This paper reports a concise and scalable method for the synthesis of the phytoestrogen 7,2'-dihydroxy-4',5'-dimethoxyisoflavanone 1 via an optimized synthetic route. Compound 1 was readily obtained in 11 steps and 11% overall yield on a gram scale from commercially available 3,4-dimethoxyphenol. The key features of the synthesis include the construction of the deoxybenzoin unit through a sequence of Claisen rearrangement, oxidative cleavage, and aryllithium addition and the efficient synthesis of the isoflavanone architecture from highly functionalized 2-hydroxyketone.

Synthesis, in vitro evaluation, and computational simulations studies of 1,2,3-triazole analogues as DPP-4 inhibitors.

Novel 1,2,3-triazole analogues (S7 ~ S10) were synthesized and evaluated for their inhibitory activity against hDPP-4. All the 1,2,3-triazole analogues exhibited moderate in vitro hDPP-4 inhibitory activities (265 ~ 780 nM). These results are somewhat less potent compared to those of known 1,2,3-triazole analogues (S1 ~ S6, 14 ~ 254 nM). S2 and S3 manifested excellent potency against hDPP-4 with IC50s of 28 and 14 nM, respectively. The role of the 1,2,3-triazole moiety in binding the molecule to the target was investigated using combined 10 1,2,3-triazole analogues (S1 ~ S10). Molecular dynamics (MD) simulations following the aforementioned docking phase were performed to elucidate potential binding modes of sitagliptin's 1,2,3-triazole analogues in hDPP-4, with the use of a cocrystal structure of hDPP-4 with sitagliptin (PDB ID: 1X70). Docking and MD simulations of the complexes of hDPP-4 with sitagliptin, S2 and S3 suggest that Glu205, Glu206, Tyr662, and Tyr666 would be the key amino acid residues for the binding of the molecules with the receptor. Especially, S2 and S3 showed additional strong π-π interaction between Phe357 and 1,2,3-triazole. Same strong π-π interaction is also observed between Phe357 and the 1,2,4-triazole ring of sitagliptin. Furthermore, additional interactions with Tyr547, Cys551, and especially Arg358 would enhance the binding affinity of the compounds for the pocket of the enzyme. In overall, in vitro hDPP-4 inhibitory activities of synthetic 1,2,3-triazole analogues were well matched with results of computational simulations studies.

KRCA-0008 suppresses ALK-positive anaplastic large-cell lymphoma growth.

Anaplastic lymphoma kinase (ALK), which belongs to the insulin receptor tyrosine kinase superfamily, plays an important role in nervous system development. Due to chromosomal translocations, point mutations, and gene amplification, constitutively activated ALK has been implicated in a variety of human cancers, including anaplastic large-cell lymphoma (ALCL), non-small cell lung cancer, and neuroblastoma. We evaluated the anti-cancer activity of the ALK inhibitor KRCA-0008 using ALCL cell lines that express NPM (nucleophosmin)-ALK. KRCA-0008 strongly suppressed the proliferation and survival of NPM-ALK-positive ALCL cells. Additionally, it induced G0/G1 cell cycle arrest and apoptosis by blocking downstream signals including STAT3, Akt, and ERK1/2. Tumor growth was strongly suppressed in mice inoculated with Karpas-299 tumor xenografts and orally treated with KRCA-0008 (50 mg/kg, BID) for 2 weeks. Our results suggest that KRCA-0008 will be useful in further investigations of ALK signaling, and may provide therapeutic opportunities for NPM-ALK-positive ALCL patients.

All d-Lysine Analogues of the Antimicrobial Peptide HPA3NT3-A2 Increased Serum Stability and without Drug Resistance.

Novel antibiotic drugs are urgently needed because of the increase in drug-resistant bacteria. The use of antimicrobial peptides has been suggested to replace antibiotics as they have strong antimicrobial activity and can be extracted from living organisms such as insects, marine organisms, and mammals. HPA3NT3-A2 ([Ala1,8] HPA3NT3) is an antimicrobial peptide that is an analogue of the HP (2-20) peptide derived from Helicobacter pylori ribosomal protein L1. Although this peptide was shown to have strong antimicrobial activity against drug-resistant bacteria, it also showed lower toxicity against sheep red blood cells (RBCs) and HaCaT cells compared to HPA3NT3. The l-Lys residues of HPA3NT3-A2 was substituted with d-Lys residues (HPA3NT3-A2D; [d-Lys2,5,6,9,10,15] HPA3NT3-A2) to prevent the cleavage of peptide bonds by proteolytic enzymes under physiological conditions. This peptide showed an increased half-life and maintained its antimicrobial activity in the serum against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) (pathogen). Furthermore, the antimicrobial activity of HPA3NT3-A2D was not significantly affected in the presence of mono- or divalent ions (Na+, Mg2+, Ca2+). Finally, l- or d-HPA3NT3-A2 peptides exhibited the strongest antimicrobial activity against antibiotic-resistant bacteria and failed to induce resistance in Staphylococcus aureus after 12 passages.

Novel metabolites from Trichoderma atroviride against human prostate cancer cells and their inhibitory effect on Helicobacter pylori and Shigella toxin producing Escherichia coli.

The present study aimed to purify and identify the metabolites from T. atroviride using high-performance liquid chromatography (HPLC) and 1H and 13C nuclear magnetic resonance spectrometer (NMR) followed by analyzing their toxicological, antibacterial and anticancer properties. This work identified two metabolites - TM1 and TM2. TM1 was in two forms: (i) 1, 3-dione-5, 5-dimethylcyclohexane; and, (ii) 2-enone-3hydroxy -5,5-dimethylcylohex, while TM2 was 4H-1,3-dioxin-4-one-2,3,6-trimethyl. These metabolites did not exhibit any irritant or allergic reaction as revealed by HET- CAM test. TM2 significantly inhibited the growth of H. pylori and Shigella toxin producing Escherichia coli (STEC) as evident by in vitro and microscopic observations of bacterial cell death. TM2 also induced the cell death and cytotoxicity, as revealed by cell viability test and western blot analysis. According to microscopic, flow cytometer and western blot analysis, TM2 treated cells displayed higher ROS, cell death, and apoptosis-related protein expression than TM1 and control. This study concluded that TM2 derived from T. atroviride was a potential therapeutic agent for anti-prostate cancer and antibiotic agent against MDR- H. pylori and STEC and it is also recommended to carry out further in vivo animal model experiments with improved stability of the metabolites for future pharmaceutical trails.

Activation of DNA Damage Response and Cellular Senescence in Cardiac Fibroblasts Limit Cardiac Fibrosis After Myocardial Infarction.

Cardiac fibrosis plays an important role in cardiac remodeling after myocardial infarction (MI). The molecular mechanisms that promote cardiac fibrosis after MI are well studied; however, the mechanisms by which the progression of cardiac fibrosis becomes attenuated after MI remain poorly understood. Recent reports show the role of cellular senescence in limiting tissue fibrosis. In the present study, we tested whether cellular senescence of cardiac fibroblasts (CFs) plays a role in attenuating the progression of cardiac fibrosis after MI. We found that the number of γH2AX-positive CFs increased up to day 7, whereas the number of proliferating CFs peaked at day 4 after MI. Senescent CFs were also observed at day 7, suggesting that attenuation of CF proliferation occurred simultaneously with the activation of the DNA damage response (DDR) system and the appearance of senescent CFs. We next cultured senescent CFs with non-senescent CFs and showed that senescent CFs suppressed proliferation of the surrounding non-senescent CFs in a juxtacrine manner. We also found that the blockade of DDR by Atm gene deletion sustained the proliferation of CFs and exacerbated the cardiac fibrosis at the early stage after MI. Our results indicate the role of DDR activation and cellular senescence in limiting cardiac fibrosis after MI. Regulation of cellular senescence in CFs may become one of the therapeutic strategies for preventing cardiac remodeling after MI.

Generation of Fabry cardiomyopathy model for drug screening using induced pluripotent stem cell-derived cardiomyocytes from a female Fabry patient.

BACKGROUND: Fabry disease is an X-linked disease caused by mutations in α-galactosidase A (GLA); these mutations result in the accumulation of its substrates, mainly globotriaosylceramide (Gb3). The accumulation of glycosphingolipids induces pathogenic changes in various organs, including the heart, and Fabry cardiomyopathy is the most frequent cause of death in patients with Fabry disease. Existing therapies to treat Fabry disease have limited efficacy, and new approaches to improve the prognosis of patients with Fabry cardiomyopathy are required. METHODS AND RESULTS: We generated induced pluripotent stem cell (iPSC) lines from a female patient and her son. Each iPSC clone from the female patient showed either deficient or normal GLA activity, which could be used as a Fabry disease model or its isogenic control, respectively. Erosion of the inactivated X chromosome developed heterogeneously among clones, and mono-allelic expression of the GLA gene was maintained for a substantial period in a subset of iPSC clones. Gb3 accumulation was observed in iPSC-derived cardiomyocytes (iPS-CMs) from GLA activity-deficient iPSCs by mass-spectrometry and immunofluorescent staining. The expression of ANP was increased, but the cell surface area was decreased in iPS-CMs from the Fabry model, suggesting that cardiomyopathic change is ongoing at the molecular level in Fabry iPS-CMs. We also established an algorithm for selecting proper Gb3 staining that could be used for high-content analysis-based drug screening. CONCLUSIONS: We generated a Fabry cardiomyopathy model and a drug screening system by using iPS-CMs from a female Fabry patient. Drug screening using our system may help discover new drugs that would improve the prognosis of patients with Fabry cardiomyopathy.

Anti-endotoxin mechanism of the KW4 peptide in inflammation in RAW 264.7 cells induced by LTA and drug-resistant Staphylococcus aureus 1630.

Drug-resistant microorganism infections cause serious disease and can lead to mortality and morbidity. In particular, Staphylococcus aureus induces pyrogenic and toxigenic infections, and drug-resistance occurs rapidly. Multidrug-resistant S. aureus, such as methicillin-resistant S. aureus and methicillin-sensitive S. aureus, can also cause immunodeficiency and immune deficiency syndrome from lipoteichoic acid. However, antimicrobial peptides, such as KW4, have strong antimicrobial activity, low cytotoxicity, and high neutralization activity against endotoxin substances from Gram-negative bacteria. The objective of this study was to use a synthetic KW4 antimicrobial peptide to evaluate the inhibition of drug-resistance development, antimicrobial activity, and neutralizing activity in S. aureus Gram-positive bacteria. The KW4 peptide showed strong antimicrobial activity against drug-resistant S. aureus strains and significantly increased the anti-neutralizing activity of lipoteichoic acid in S. aureus 1630 drug-resistant bacteria. In addition, S. aureus ATCC 29213 did not develop resistance to KW4 as with other antibiotic drugs. These results suggest that the KW4 peptide is an effective antibiotic and anti-neutralizing agent against multidrug-resistant S. aureus strains.

In vitro platform of allogeneic stem cell-derived cardiomyocyte transplantation for cardiac conduction defects.

Aims: The aim of the present study is to develop in vitro experimental analytical method for the electrophysiological properties of allogeneic induced pluripotent stem cell-derived cardiomyocytes (CMs) in cardiac conduction defect model. Methods and results: Cardiomyocytes were derived from rat induced pluripotent stem cells CMs (riPSC-CMs) using an embryoid body-based differentiation method with the serial application of growth factors including activin-A, bone morphogenetic protein 4 (BMP-4), and inhibitor of wnt production 2 (IWP-2). Flow cytometry analysis showed that 74.0 ± 2.7% of riPSC-CMs expressed cardiac troponin-T (n = 3). Immunostaining analysis revealed organized sarcomeric structure in riPSC-CMs and the expression of connexin 43 between riPSC-CMs and neonatal rat ventricular CMs (NRVMs). Ca2+ transient recordings revealed the simultaneous excitement of riPSC-CMs and NRVMs, and prolonged Ca2+ transient duration of riPSC-CMs as compared with NRVMs (731 ± 15.9 vs. 610 ± 7.72 ms, P < 0.01, n = 3). Isolated NRVMs were cultured in two discrete regions to mimic cardiac conduction defects on multi-electrode array dish, and riPSC-CMs were seeded in the channel between the two discrete regions. Membrane potential imaging with di-8-ANEPPS discerned the propagation of the electrical impulse from one NRVM region to the other through a riPSC-CM pathway. This pathway had significantly longer action potential duration as compared with NRVMs. Electrophysiological studies using a multi-electrode array platform demonstrated the longer conduction time and functional refractory period of the riPSC-CM pathway compared with the NRVM pathway. Conclusion: Using an in vitro experimental system to mimic cardiac conduction defect, transplanted allogeneic riPSC-CMs showed electrical coupling between two discrete regions of NRVMs. Electrophysiological testing using our platform will enable electrophysiological screening prior to transplantation of stem cell-derived CMs.

2-Formyl-komarovicine promotes adiponectin production in human mesenchymal stem cells through PPARγ partial agonism.

Adiponectin is a major adipocytokine secreted from mammalian adipocytes. Relatively low expression of adiponectin is associated with various human metabolic diseases and some cancers. Adiponectin-secreting compounds have therapeutic potential for these diseases. Adipogenesis of human bone marrow-mesenchymal stem cells (hBM-MSCs) has been used as a phenotypic assay to find adiponectin secreting compounds. In a phytochemical library screen, 2-formyl-komarovicine, 1-(quinolin-8-yl)-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole-2-carbaldehyde, isolated from Nitraria komarovii was identified as a potential adiponectin-secreting compound. To validate the results of the impure phytochemical, we synthesized 2-formyl-komarovicine. The synthetic 2-formyl-komarovicine significantly promoted adiponectin production during adipogenesis in hBM-MSCs. In a target identification experiment, 2-formyl-komarovicine bound to peroxisome proliferator-activated receptor γ (PPARγ) in a concentration-dependent manner. Notably, 2-formyl-komarovicine competitively inhibited the adiponectin-promoting activity of a full PPARγ agonist, troglitazone, in hBM-MSCs, which is a pharmacological feature of a partial agonist. The ligand-docking model showed that 2-formyl-komarovicine interacted with the hydrophobic pocket of the PPARγ ligand-binding domain, but lacked an interaction to stabilize helix H12, which is one of the major binding themes of PPARγ partial agonists. We concluded that 2-formyl-komarovicine provides a novel pharmacophore for PPARγ partial agonists to increase adiponectin production.

Phenotypic Screening Using Patient-Derived Induced Pluripotent Stem Cells Identified Pyr3 as a Candidate Compound for the Treatment of Infantile Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a genetic disorder that is characterized by hypertrophy of the myocardium. Some of the patients are diagnosed for HCM during infancy, and the prognosis of infantile HCM is worse than general HCM. Nevertheless, pathophysiology of infantile HCM is less investigated and remains largely unknown. In the present study, we generated induced pluripotent stem cells (iPSCs) from two patients with infantile HCM: one with Noonan syndrome and the other with idiopathic HCM. We found that iPSC-derived cardiomyocytes (iPSC-CMs) from idiopathic HCM patient were significantly larger and showed higher diastolic intracellular calcium concentration compared with the iPSC-CMs from healthy subject. Unlike iPSC-CMs from the adult/adolescent HCM patient, arrhythmia was not observed as a disease-related phenotype in iPSC-CMs from idiopathic infantile HCM patient. Phenotypic screening revealed that Pyr3, a transient receptor potential channel 3 channel inhibitor, decreased both the cell size and diastolic intracellular calcium concentration in iPSC-CMs from both Noonan syndrome and idiopathic infantile HCM patients, suggesting that the target of Pyr3 may play a role in the pathogenesis of infantile HCM, regardless of the etiology. Further research may unveil the possibility of Pyr3 or its derivatives in the treatment of infantile HCM.

Isolation and characterisation of a novel sildenafil analogue adulterant, desmethylpiperazinyl propoxysildenafil, in a dietary supplement.

A new sildenafil analogue was detected during routine screening of dietary supplements suspected to be adulterated with an erectile dysfunction drug(s) using HPLC-DAD. The UV spectrum of this compound was highly similar to that of sildenafil and almost identical to that of desmethylpiperazinyl sildenafil. The analogue was purified by using semi-preparative HPLC and structurally elucidated by performing mass spectrometric and NMR spectroscopic experiments. The spectral data revealed that this sildenafil analogue bears an n-propoxy group instead of an ethoxy group and possesses no methylpiperazinyl moiety. The isolated compound, structure of which was further confirmed by spectral comparison with synthetic one, was thus named as desmethylpiperazinyl propoxysildenafil.

Antitumor effect of the integrin α4 signaling inhibitor JK273 in non-small cell lung cancer NCI-H460 cells.

Lung cancer accounts for the highest death rate among cancers worldwide, with most patients being diagnosed with non-small cell lung cancer (NSCLC), urging more effective therapies. We report that JK273, a pyrrolo[2,3-d]pyrimidine analog, which inhibits α4 integrin signaling, showed a selective cytotoxic effect against HCI-H460 NSCLC cells, with an IC50 of 0.98 ± 0.15 µM, but showed less sensitivity to fibroblasts with a selectivity index (SI) greater than 30. This effect was attributed to cell cycle arrest at S phase by JK273 treatment, resulting in the apoptosis of NCI-H460 cells, further confirmed by exposing phosphatidylserine and morphological changes. Taken together with the previous study of JK273 inhibiting cell migration, we propose that JK273 could serve as an antitumor compound to specifically target cancer cells but not non-cancerous cells by triggering programmed cell death, in addition to anti-metastatic effects in cancer therapy.

Novel 5-nitropyrimidine derivatives bearing endo-azabicyclic alcohols/amines as potent GPR119 agonists.

A series of GPR119 agonists based on a 5-nitropyrimidine scaffold bearing endo-azabicyclic substituents were synthesized and evaluated for their GPR119 agonistic activities. Most compounds exhibited much stronger EC50 values than that of oleoylethanolamide (OEA). Among them, derivatives from endo-azabicyclic alcohols displayed more potent GPR119 agonistic activities than compounds with endo-azabicyclic amines. Especially the optimized compounds (6, 7, 8, 12, 17) were shown to have potent biological activities and were identified as full agonists. Isopropyl carbamate compound 8 synthesized from endo-azabicyclic alcohol was observed to have the best EC50 value (0.6nM). Generally 2-fluoro substitution of the aryl group at the C4 position of 5-nitropyrimidine scaffold resulted in the increase of biological activity.

The topogenic function of S4 promotes membrane insertion of the voltage-sensor domain in the KvAP channel.

Voltage-dependent K+ (KV) channels control K+ permeability in response to shifts in the membrane potential. Voltage sensing in KV channels is mediated by the positively charged transmembrane domain S4. The best-characterized KV channel, KvAP, lacks the distinct hydrophilic region corresponding to the S3-S4 extracellular loop that is found in other K+ channels. In the present study, we evaluated the topogenic properties of the transmembrane regions within the voltage-sensing domain in KvAP. S3 had low membrane insertion activity, whereas S4 possessed a unique type-I signal anchor (SA-I) function, which enabled it to insert into the membrane by itself. S4 was also found to function as a stop-transfer signal for retention in the membrane. The length and structural nature of the extracellular S3-S4 loop affected the membrane insertion of S3 and S4, suggesting that S3 membrane insertion was dependent on S4. Replacement of charged residues within the transmembrane regions with residues of opposite charge revealed that Asp72 in S2 and Glu93 in S3 contributed to membrane insertion of S3 and S4, and increased the stability of S4 in the membrane. These results indicate that the SA-I function of S4, unique among K+ channels studied to date, promotes the insertion of S3 into the membrane, and that the charged residues essential for voltage sensing contribute to the membrane-insertion of the voltage sensor domain in KvAP.

Synthesis and Structure Revision of Dimeric Tadalafil Analogue Adulterants in Dietary Supplements.

A number of phosphodiesterase 5 (PDE5) inhibitors approved by authorities have been used successfully in the treatment of erectile dysfunction. These medicines must be prescribed carefully due to their adverse effects, but they and their analogues are being illegally added to dietary supplements. These illegal dietary supplements pose a significant risk to public health. Several dimeric tadalafil analogues have been synthesized for use as reference standards in the inspection of functional foods that are mainly advertised as sexual enhancement products. During the course of this synthesis, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) was proven to be the reagent of choice for amide coupling to produce these dimeric tadalafil analogues. Moreover, the trans-isomer structures tentatively assigned for the isolated dimeric tadalafil analogues (bisprehomotadalafil and bisprecyclopentyltadalafil) found in dietary supplements are now revised to cis-isomer structures.