Investigating a possible role for the bacterial signal molecules N‐acylhomoserine lactones in Balanus improvisus cyprid settlement

Increased settlement on bacterial biofilms has been demonstrated for a number of marine invertebrate larvae, but the nature of the cue(s) responsible is not well understood. We tested the hypothesis that the bay barnacle Balanus improvisus utilizes the bacterial signal molecules N‐acylhomoserine lactones (AHLs) as a cue for the selection of sites for permanent attachment. Single species biofilms of the AHL‐producing bacteria Vibrio anguillarum, Aeromonas hydrophila and Sulfitobacter sp. BR1 were attractive to settling cypris larvae of B. improvisus. However, when AHL production was inactivated, either by mutation of the AHL synthetic genes or by expression of an AHL‐degrading gene (aiiA), the ability of the bacteria to attract cyprids was abolished. In addition, cyprids actively explored biofilms of E. coli expressing the recombinant AHL synthase genes luxI from Vibrio fischeri (3‐oxo‐C6‐HSL), rhlI from Pseudomonas aeruginosa (C4‐HSL/C6‐HSL), vanI from V. anguillarum (3‐oxo‐C10‐HSL) and sulI from Sulfitobacter sp. BR1 (C4‐HSL, 3‐hydroxy‐C6‐HSL, C8‐HSL and 3‐hydroxy‐C10‐HSL), but not E. coli that did not produce AHLs. Finally, synthetic AHLs (C8‐HSL, 3‐oxo‐C10‐HSL and C12‐HSL) at concentrations similar to those found within natural biofilms (5 μm ) resulted in increased cyprid settlement. Thus, B. improvisus cypris exploration of and settlement on biofilms appears to be mediated by AHL‐signalling bacteria in the laboratory. This adds to our understanding of how quorum sensing inhibition may be used as for biofouling control. Nonetheless, the significance of our results for larvae settling naturally in the field, and the mechanisms that underlay the observed responses to AHLs, is as yet unknown.     

Exogenous N‐acyl‐homoserine lactones enhance the expression of flagella of Pseudomonas syringae and activate defence responses in plants

In order to cope with pathogens, plants have evolved sophisticated mechanisms to sense pathogenic attacks and to induce defence responses. The N‐acyl‐homoserine lactone (AHL)‐mediated quorum sensing in bacteria regulates diverse physiological processes, including those involved in pathogenicity. In this work, we study the interactions between AHL‐producing transgenic tobacco plants and Pseudomonas syringae pv. tabaci 11528 (P. syringae 11528). Both a reduced incidence of disease and decrease in the growth of P. syringae 11528 were observed in AHL‐producing plants compared with wild‐type plants. The present data indicate that plant‐produced AHLs enhance disease resistance against this pathogen. Subsequent RNA‐sequencing analysis showed that the exogenous addition of AHLs up‐regulated the expression of P. syringae 11528 genes for flagella production. Expression levels of plant defence genes in AHL‐producing and wild‐type plants were determined by quantitative real‐time polymerase chain reaction. These data showed that plant‐produced AHLs activated a wide spectrum of defence responses in plants following inoculation, including the oxidative burst, hypersensitive response, cell wall strengthening, and the production of certain metabolites. These results demonstrate that exogenous AHLs alter the gene expression patterns of pathogens, and plant‐produced AHLs either directly or indirectly enhance plant local immunity during the early stage of plant infection.     

Chiral Recognition of N‐Phthaloyl,N‐Tetrachlorophthaloyl,and N‐Naphthaloyl α‐Amino Acids and Their Esters on Polysaccharide‐Derived Chiral Stationary Phases

Enantiomeric separations of N‐phthaloyl (N‐PHT), N‐tetrachlorophthaloyl (N‐TCPHT), and N‐naphthaloyl (N‐NPHT) α‐amino acids and their esters were examined on several kinds of polysaccharide‐derived chiral stationary phases (CSPs). Resolution capability of CSPs was greater Chiralcel OF than the others for N‐PHT and N‐NPHT α‐amino acids and their esters. In N‐TCPHT α‐amino acids and their esters, good enantioselectivities showed Chiralcel OG for N‐TCPHT α‐amino acids, Chiralpak AD for N‐TCPHT α‐amino acid methyl esters, and Chiralcel OD for N‐TCPHT α‐amino acid ethyl esters, respectively. From the results of liquid chromatography and computational chemistry, it is concluded that l ‐form is preferred and more retained with electrostatic interaction in case of interaction between N‐PHT α‐amino acid derivatives and Chiralcel OF, N‐TCPHT α‐amino acid derivatives and Chiralcel OD, and N‐NPHT α‐amino acid derivatives and Chiracel OF. On the other hand, d ‐form is preferred and more retained with van der Waals interaction in case of interaction between N‐TCPHT α‐amino acid ester derivatives and Chiralcel OG and Chiralpak AD. Chirality 24:1037–1046, 2012. © 2012 Wiley Periodicals, Inc.     

N‐ACYL HOMOSERINE LACTONe LACTONASE,AiiA, INACTIVATION OF QUORUM‐SENSING AGONISTS PRODUCED BY CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA) AND CHARACTERIZATION OF aiiA TRANSGENIC ALGAE1

Eukaryotes such as plants and the unicellular green alga Chlamydomonas reinhardtii P. A. Dang. produce and secrete compounds that mimic N‐acyl homoserine lactone (AHL) bacterial quorum‐sensing (QS) signals and alter QS‐regulated gene expression in the associated bacteria. Here, we show that the set of C. reinhardtii signal‐mimic compounds that activate the CepR AHL receptor of Burkholderia cepacia are susceptible to inactivation by AiiA, an AHL lactonase enzyme of Bacillus. Inactivation of these algal mimics by AiiA suggests that the CepR‐stimulatory class of mimics produced by C. reinhardtii may have a conserved lactone ring structure in common with AHL QS signals. To examine the role of AHL mimic compounds in the interactions of C. reinhardtii with bacteria, the aiiA gene codon optimized for Chlamydomonas was generated for the expression of AiiA as a chimeric fusion with cyan fluorescent protein (AimC). Culture filtrates of transgenic strains expressing the fusion protein AimC had significantly reduced levels of CepR signal‐mimic activities. When parental and transgenic algae were cultured with a natural pond water bacterial community, a morphologically distinct, AHL‐producing isolate of Aeromonas veronii was observed to colonize the transgenic algal cultures and form biofilms more readily than the parental algal cultures, indicating that secretion of the CepR signal mimics by the alga can significantly affect its interactions with bacteria it encounters in natural environments. The parental alga was also able to sequester and/or destroy AHLs in its growth media to further disrupt or manipulate bacterial QS.     

Design,Synthesis and Bioevaluation of Two Series of 3‐[(1‐Benzyl‐1H‐1,2,3‐triazol‐4‐yl)methyl]quinazolin‐4(3H)‐ones and N‐(1‐Benzylpiperidin‐4‐yl)quinazolin‐4‐amines

Two series of 3‐[(1‐benzyl‐1H‐1,2,3‐triazol‐4‐yl)methyl]quinazolin‐4(3H)‐ones and N‐(1‐benzylpiperidin‐4‐yl)quinazolin‐4‐amines were designed initially as potential acetylcholine esterase inhibitors. Biological evaluation demonstrated that N‐(1‐benzylpiperidin‐4‐yl)quinazolin‐4‐amines significantly inhibited AChE activity. Especially, two compounds of them were found to be the most potent with relative AChE inhibition percentages of 87 % in comparison to donepezil. The docking studies with AChE showed similar interactions between donepezil and four derivatives. N‐(1‐Benzylpiperidin‐4‐yl)quinazolin‐4‐amines also exhibited significant DPPH scavenging effects. The two series of compound also exerted moderate to good cytotoxicity against three human cancer cell lines, including SW620 (human colon cancer), PC‐3 (prostate cancer), and NCI?H23 (lung cancer), with 3‐[(1‐benzyl‐1H‐1,2,3‐triazol‐4‐yl)methyl]quinazolin‐4(3H)‐one being the most cytotoxic agent. 3‐[(1‐Benzyl‐1H‐1,2,3‐triazol‐4‐yl)methyl]quinazolin‐4(3H)‐one significantly induced early apoptosis and arrested the SW620 cells at G2/M phase. From this study, two compounds of N‐(1‐benzylpiperidin‐4‐yl)quinazolin‐4‐amines could serve as new leads for further design and AChE inhibitors, while 3‐[(1‐benzyl‐1H‐1,2,3‐triazol‐4‐yl)methyl]quinazolin‐4(3H)‐one could serve as a new lead for the design and development of more potent anticancer agents.     

Kinetic resolution of N‐acetyl‐DL‐alanine methyl ester using immobilized Escherichia coli cells bearing recombinant esterase from Bacillus cereus

D‐alanine is widely used in medicine, food, additives, cosmetics, and other consumer items. Esterase derived from Bacillus cereus WZZ001 exhibits high hydrolytic activity and stereoselectivity. In this study, we expressed the esterase gene in Escherichia coli BL21 (DE3). We analyzed the biocatalytic resolution of N‐acetyl‐DL‐alanine methyl ester by immobilized whole E. coli BL21 (DE3) cells, which were prepared through embedding and cross‐linking. We analyzed biocatalytic resolution under the optimal conditions of pH of 7.0, temperature of 40°C and substrate concentration of at 700 mM with an enantiomeric excess of 99.99% and e.e._p of 99.50%. The immobilized recombinant B. cereus esterase E. coli BL21 (DE3) cells exhibited excellent reusability and retained 86.04% of their initial activity after 15 cycles of repeated reactions. The immobilized cells are efficient and stable biocatalysts for the preparation of N‐acetyl‐D‐alanine methyl esters.     

N‐[2‐[(3‐Chlorophenyl)amino]‐phenyl]‐3‐(difluoromethyl)‐1‐methyl‐1H‐pyrazole‐4‐carboxamide: Synthesis,Crystal Structure,Molecular Docking and Biological Activities

In continuation of our previous research on the development of novel pyrazole‐4‐carboxamide with potential antifungal activity, compound SCU2028 , namely N‐[2‐[(3‐chlorophenyl)amino]phenyl]‐3‐(difluoromethyl)‐1‐methyl‐1H‐pyrazole‐4‐carboxamide, was synthesized by new method, structurally characterized by IR, HR‐ESI‐MS, ¹H‐ and ¹³C‐NMR spectra and further identified by single‐crystal X‐ray diffraction. In pot tests, compound SCU2028 showed good in vivo antifungal activity against Rhizoctonia solani (R. solani) and IC₅₀ value of it was 7.48 mg L^?1. In field trials, control efficacy of compound SCU2028 at 200 g.a.i. ha^?1 was 42.30 % on the 7^th day after the first spraying and 68.10 % on the 14^th day after the second spraying, only slightly lower than that of thifluzamide (57.20 % and 71.40 %, respectively). Further in vitro inhibitory activity showed inhibitory ability of compound SCU2028 was 45‐fold higher than that of bixafen and molecular docking of compound SCU2028 to SDH predicted its binding orientation in the active site of the target protein SDH. These results suggested that compound SCU2028 was a potential fungicide for control of rice sheath blight.     

Dopaminergic neurotoxicity of S‐ethyl N,N‐dipropylthiocarbamate (EPTC), molinate,and S‐methyl‐N,N‐diethylthiocarbamate (MeDETC) in Caenorhabditis elegans

Epidemiological studies corroborate a correlation between pesticide use and Parkinson's disease (PD). Thiocarbamate and dithiocarbamate pesticides are widely used and produce neurotoxicity in the peripheral nervous system. Recent evidence from rodent studies suggests that these compounds also cause dopaminergic (DAergic) dysfunction and altered protein processing, two hallmarks of PD. However, DAergic neurotoxicity has yet to be documented. We assessed DAergic dysfunction in Caenorhabditis elegans (C. elegans) to investigate the ability of thiocarbamate pesticides to induce DAergic neurodegeneration. Acute treatment with either S‐ethyl N,N‐dipropylthiocarbamate (EPTC), molinate, or a common reactive intermediate of dithiocarbamate and thiocarbamate metabolism, S‐methyl‐N,N‐diethylthiocarbamate (MeDETC), to gradual loss of DAergic cell morphology and structure over the course of 6 days in worms expressing green fluorescent protein (GFP) under a DAergic cell specific promoter. HPLC analysis revealed decreased DA content in the worms immediately following exposure to MeDETC, EPTC, and molinate. In addition, worms treated with the three test compounds showed a drastic loss of DAergic‐dependent behavior over a time course similar to changes in DAergic cell morphology. Alterations in the DAergic system were specific, as loss of cell structure and neurotransmitter content was not observed in cholinergic, glutamatergic, or GABAergic systems. Overall, our data suggest that thiocarbamate pesticides promote neurodegeneration and DAergic cell dysfunction in C. elegans, and may be an environmental risk factor for PD.     

A convenient microwave‐enhanced solid‐phase synthesis of short chain N‐methyl‐rich peptides

Structural modification of the peptide backbone via N‐methylation is a powerful tool to modulate the pharmacokinetic profile and biological activity of peptides. Here we describe a rapid and highly efficient microwave(MW)‐assisted Fmoc/tBu solid‐phase method to prepare short chain N‐methyl‐rich peptides, using Rink amide p‐methylbenzhydrylamine (MBHA) resin as solid‐phase support. This method produces peptides in high yield and purity, and reduces the time required for Fmoc‐N‐methyl amino acid coupling. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Quorum Sensing in <Emphasis Type="Italic">Aeromonas</Emphasis> Species Isolated from Patients in Malaysia

Bacterial quorum sensing signal molecules called N-acylhomoserine lactone (AHL) controls the expression of virulence determinants in many Gram-negative bacteria. We determined AHL production in 22 Aeromonas strains isolated from various infected sites from patients (bile, blood, peritoneal fluid, pus, stool and urine). All isolates produced the two principal AHLs, N-butanoylhomoserine lactone (C4-HSL) and N-hexanoyl homoserine lactone (C6-HSL). Ten isolates also produced additional AHLs. This report is the first documentation of Aeromonas sobria producing C6-HSL and two additional AHLs with N-acyl side chain longer than C₆. Our data provides a better understanding of the mechanism(s) of this environmental bacterium emerging as a human pathogen.     

Design,Synthesis, and Cholinesterase Inhibition Assay of Coumarin‐3‐carboxamide‐N‐morpholine Hybrids as New Anti‐Alzheimer Agents

A new series of coumarin‐3‐carboxamide‐N‐morpholine hybrids 5a – 5l was designed and synthesized as cholinesterases inhibitors. The synthetic approach for title compounds was started from the reaction between 2‐hydroxybenzaldehyde derivatives and Meldrum's acid to afford corresponding coumarin‐3‐carboxylic acids. Then, amidation of the latter compounds with 2‐morpholinoethylamine or N‐(3‐aminopropyl)morpholine led to the formation of the compounds 5a – 5l . The in vitro inhibition screen against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) revealed that most of the synthesized compounds had potent AChE inhibitory while their BuChE inhibitions are moderate to weak. Among them, propylmorpholine derivative 5g (N‐[3‐(morpholin‐4‐yl)propyl]‐2‐oxo‐2H‐chromene‐3‐carboxamide) bearing an unsubstituted coumarin moiety and ethylmorpholine derivative 5d (6‐bromo‐N‐[2‐(morpholin‐4‐yl)ethyl]‐2‐oxo‐2H‐chromene‐3‐carboxamide) bearing a 6‐bromocoumarin moiety showed the most activity against AChE and BuChE, respectively. The inhibitory activity of compound 5g against AChE was 1.78 times more than that of rivastigmine and anti‐BuChE activity of compound 5d is approximately same as rivastigmine. Kinetic and docking studies confirmed the dual binding site ability of compound 5g to inhibit AChE.     

Rapid high‐yield N‐acylation of aminothiols: N‐acetylglutathione and N‐acetylhomocysteine and their thiol pKa values

Methodology for the rapid N‐acylation of aminothiols in aqueous solution using procedures commonly employed in biochemical studies is described here. Glutathione disulfide (GSSG) and homocystine were diN‐acetylated in ~100% yield in 0.1 M aqueous NaHCO₃ (pH 8.5) at room temperature by 2.5 equiv of the activated ester, N‐hydroxysulfosuccinimidyl acetate, an efficient water‐soluble acetylating reagent. Following acetone precipitation, diN‐acetylGSSG was further purified and desalted on a strong anion‐exchange (SAX) cartridge. DiN‐acetylhomocystine was simultaneously purified and desalted on a C₁₈ cartridge. The N‐acetylated aminothiols were generated using gel‐immobilized tris(2‐carboxyethyl)phosphine as a reductant, which obviated the need for further purification. Alternatively, disulfide exchange with dissolved dithiothreitol yielded N‐acetylglutathione, which was purified on the SAX cartridge. pH titrations of N‐acetylglutathione (8.99) and N‐acetylhomocysteine (9.66) as well as those of commercially available N‐acetylcysteine (9.53) and N‐acetylpenicillamine (10.21) yielded pK_a(SH) values of importance for biological studies. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Reducing infectivity of HIV upon exposure to surfaces coated with N,N‐dodecyl,methyl‐polyethylenimine

The infectivity of high‐titer, cell‐free HIV in culture media and human milk is rapidly reduced upon exposure to polyethylene slides painted with the linear hydrophobic polycation N,N‐dodecyl,methyl‐polyethylenimine (DMPEI). Accompanying viral p24 protein and free viral RNA analysis of solutions exposed to DMPEI‐coated surfaces suggests that virion attachment to the polycationic surface and its subsequent inactivation are the likely mechanism of this phenomenon. Biotechnol. Bioeng. 2013; 110: 2058–2062. © 2013 Wiley Periodicals, Inc.     

Solvent effects of N‐nitroso,N‐(2‐chloroethyl), N′,N′‐dibenzylsulfamid and its copper(II) and cobalt(II) complexes: fluorescence studies

The structure of N‐nitroso, N‐(2‐chloroethyl), N′,N′‐dibenzylsulfamid (CENS) was established by X‐ray crystallography. The atomic coordinates, factors of isotropic thermal agitation, bond lengths and valence angles were determined. The solvent effects on the electronic absorption and fluorescence spectra of CENS were investigated at room temperature. The effects of solvent polarity and of hydrogen bonding were interpreted by means of linear solvation energy relationships (LSERs). Multiple linear regression analysis indicated that the hydrogen donation properties of the solvent play an important role in determining the position of the absorption maximum, while the classical polarity of the medium is the only dominating parameter in determining the emission maximum and the Stokes' shift. Complexation of the investigated compound by two different transition metal ions was studied. Fluorescence measurements show that fluorescence quenching by cobalt(II) is more important than that by copper(II). This phenomenon can be attributed to good stereo‐structural matching between the electronic configuration of the Co²⁺ ion and the active site distribution of CENS in aqueous solution. Copyright © 2012 John Wiley & Sons, Ltd.     

Influence of N‐hexane inhalation on the enantioselective pharmacokinetics and metabolism of verapamil in rats

Verapamil (VER) is commercialized as a racemic mixture of the (+)‐(R)‐VER and (?)‐(S)‐VER enantiomers. VER is biotransformed into norverapamil (NOR) and other metabolites through CYP‐dependent pathways. N‐hexane is a solvent that can alter the metabolism of CYP‐dependent drugs. The present study investigated the influence of n‐hexane (nose‐only inhalation exposure chamber at concentrations of 88, 176, and 352 mg/m³) on the kinetic disposition of the (+)‐(R)‐VER, (?)‐(S)‐VER, (R)‐NOR and (S)‐NOR in rats treated with a single dose of racemic VER (10 mg/kg). VER and NOR enantiomers in rat plasma was analyzed by LC‐MS/MS (m/z = 441.3 > 165.5 for the NOR and m/z 455.3 > 165.5 for the VER enantiomers) using a Chiralpak® AD column. Pharmacokinetic analysis was performed using a monocompartmental model. The pharmacokinetics of VER was enantioselective in control rats, with higher plasma proportions of the (?)‐(S)‐VER eutomer (AUC^0?∞ = 250.8 vs. 120.4 ng/ml/h; P ≤ 0.05, Wilcoxon test). The (S)‐NOR metabolite was also found to accumulate in plasma of control animals, with an S/R AUC^0?∞ ratio of 1.5. The pharmacokinetic parameters AUC^0?∞, Cl/F, Vd/F, and t_1/2 obtained for VER and NOR enantiomers were not altered by nose‐only exposure to n‐hexane at concentrations of 88, 176, or 352 mg/m³ (P > 0.05, Kruskal‐Wallis test). However, the verapamil kinetic disposition was not enantioselective for the animals exposed to n‐hexane at concentrations equal to or higher than the TLV‐TWA. This finding is relevant considering that the (?)‐(S)‐VER eutomer is 10–20 times more potent than R‐(+)‐VER in terms of its chronotropic effect on atrioventricular conduction in rats and humans. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Three‐dimensional structure of a sugar N‐formyltransferase from Francisella tularensis

N‐formylated sugars have been observed on the O‐antigens of such pathogenic Gram‐negative bacteria as Campylobacter jejuni and Francisella tularensis. Until recently, however, little was known regarding the overall molecular architectures of the N‐formyltransferases that are required for the biosynthesis of these unusual sugars. Here we demonstrate that the protein encoded by the wbtj gene from F. tularensis is an N‐formyltransferase that functions on dTDP‐4‐amino‐4,6‐dideoxy‐d ‐glucose as its substrate. The enzyme, hereafter referred to as WbtJ, demonstrates a strict requirement for N¹⁰‐formyltetrahydrofolate as its carbon source. In addition to the kinetic analysis, the three‐dimensional structure of the enzyme was solved in the presence of dTDP‐sugar ligands to a nominal resolution of 2.1 Å. Each subunit of the dimeric enzyme is dominated by a “core” domain defined by Met 1 to Ser 185. This core motif harbors the active site residues. Following the core domain, the last 56 residues fold into two α‐helices and a β‐hairpin motif. The hairpin motif is responsible primarily for the subunit:subunit interface, which is characterized by a rather hydrophobic pocket. From the study presented here, it is now known that WbtJ functions on C‐4′ amino sugars. Another enzyme recently investigated in the laboratory, WlaRD, formylates only C‐3′ amino sugars. Strikingly, the quaternary structures of WbtJ and WlaRD are remarkably different. In addition, there are several significant variations in the side chains that line their active site pockets, which may be important for substrate specificity. Details concerning the kinetic and structural properties of WbtJ are presented.     

Brain region‐specific changes in N‐acylethanolamine contents with time of day

The N‐acylethanolamines (NAEs) exert important behavioral, physiological, and immunological effects through actions at cannabinoid and other receptors. We measured concentrations of three NAEs, the K_m and V_max for fatty acid amide hydrolysis (FAAH), FAAH protein and FAAH mRNA in prefrontal cortex, hippocampus, hypothalamus, amygdala, striatum, and cerebellum at 4 h intervals, starting at 03:00. Significant differences in N‐arachidonylethanolamine contents among the times examined occur in the prefrontal cortex (PFC), hippocampus, hypothalamus, and striatum. N‐Oleoylethanolamine concentrations exhibit large fluctuations over the day in the cerebellum, including a threefold decrease between 19:00 and 23:00. N‐Palmitoylethanolamine and N‐oleoylethanolamine were significantly, positively correlated in all regions examined except the hypothalamus. FAAH K_m values are significantly affected by time of day in PFC, hippocampus and amygdala and FAAH V_max values are significantly affected in PFC, hippocampus and cerebellum. However, correlational data indicate that FAAH does not play a primary role in the circadian regulation of the NAE concentrations. FAAH protein expression is not significantly different among the harvest times in any brain region examined. Concentrations of 2‐arachidonoylglycerol are significantly affected by time of harvest in the striatum and cerebellum, but not in other brain regions. Together, these data indicate that the NAEs exhibit diverse patterns of change with time of day that are likely the result of alterations in biosynthesis, and support the hypothesis that N‐arachidonylethanolamine is a tonic activator of cannabinoid receptor signaling.