Selective Inhibition of Collagen‐Induced Platelet Aggregation by a Cyclic Peptide from Drymaria diandra

Four cyclic peptides, diandrine A–D ( 1 – 4 ), were isolated from the MeOH extract of Formosan Drymaria diandra. Their structures were elucidated by chemical and spectroscopic analyses as cyclo(‐Gly¹‐Pro²‐Trp³‐Pro⁴‐Tyr⁵‐Phe⁶‐), cyclo(‐Gly¹‐Pro²‐Leu³‐Pro⁴‐Leu⁵‐Trp⁶‐Ser⁷‐Ser⁸‐), cyclo(Gly¹‐Gly²‐Pro³‐Tyr⁴‐Trp⁵‐Pro⁶‐), and cyclo(Gly¹‐Gly²‐Pro³‐Tyr⁴‐Trp⁵‐Pro⁶‐), respectively. Compounds 3 and 4 were stable conformational isomers. Cyclopeptide 1 showed a selective inhibitory effect on collagen‐induced platelet aggregation with an IC₅₀ value of 44.2 μM .     

Sequence and solution structure of cherimolacyclopeptides A and B, novel cyclooctapeptides from the seeds of Annona cherimola

Two new cyclooctapeptides, cherimolacyclopeptide A, cyclo(Pro¹-Gln²-Thr³-Gly⁴-Met⁵-Leu⁶-Pro⁷-Ile⁸-) (1) and the related cherimolacyclopeptide B, cyclo(Pro¹-Gln²-Thr³-Gly⁴-Mso⁵-Leu⁶-Pro⁷-Ile⁸-) (2), have been isolated from the methanol extract of the seeds of Annona cherimola Miller. The sequences were elucidated on the basis of the MS/MS fragmentation, using a Q-TOF mass spectrometer equipped with an ESI source, chemical degradation and extensive 2D-heteronuclear NMR. The three-dimensional solution structure of cherimolacyclopeptide A (1) determined by ¹H NMR data and molecular modelling is characterised by the presence of two β turns and a new type of β-bulge.     

Rapid identification of diarrheagenic Escherichia coli based on barcoded magnetic bead hybridization

Diarrhea, as a global public health problem, causes a large number of infections and deaths every year. Although Escherichia coli (E. coli) is one of the normal flora microorganisms in the human intestinal tract, it has five pathogenic bacteria types that can cause human diarrhea, known as diarrheagenic E. coli. When people are infected, rapid and accurate diagnosis, along with timely treatment, are especially important. Here, we introduce a new method to identify and analyze a large number of pathogenic strains in E. coli by multiplex PCR and barcoded magnetic bead hybridization. Results show that the detection sensitivities of enterohemorrhagic E. coli, enterotoxigenic E. coli, enteropathogenic E. coli, enteroinvasive E. coli and enteroaggregative E. coli were 1.3 × 10³ CFU/mL, 2 × 10⁴ CFU/mL, 4 × 10⁴ CFU/mL, 7.2 × 10⁴ CFU/mL and 1.7 CFU/mL respectively. This method has strong specificity and high sensitivity and detects multiple target sequences in one experiment. Compared with other methods, BMB array has great application potential.     

3D-structure of cycloreticulin C and glabrin A, cyclopeptides from the seeds of Annona reticulata

Two cyclopeptides, the cycloheptapeptide cycloreticulin C, cyclo(Pro¹-Gly²-Gln³-Pro⁴-Pro⁵-Tyr⁶-Val⁷) (1), and the cyclohexapeptide glabrin A, cyclo(Pro¹-Gly²-Leu³-Val⁴-Ile⁵-Tyr⁶) (2), have been isolated from the methanol extract of the seeds of Annona reticulata. Their structures were elucidated on the basis of the MS/MS fragmentation using a Q-TOF mass spectrometer equipped with an ESI source, chemical degradation and extensive 2D-NMR. The solution conformation of cycloreticulin C involves two β-turns, one of type II with trans-Pro¹ and Gly² at the corners, another of type VIa with trans-Pro⁴ and cis-Pro⁵ at the corners, and followed by a β-bulge at the Tyr⁶-Val⁷ level. The solid state and solution conformations of glabrin A have been analyzed by X-ray and 2D-NMR studies, respectively, and are characterized by the presence of two β-turns, the first of type VIa and the second intermediary between types I and III at the solid state and a γ-turn in solution.     

Fluorescence detection of Escherichia coli on mannose modified ZnTe quantum dots

Rapid detection and identification of Escherichia coli(E.coli) is essential to prevent its quickly spread.In this study,a novel fluorescence probe based on ZnTe quantum dots(QDs) modified by mannose(MAN)had been prepared for the determination of E.coli.The results showed that the obtained QDs showed excellent selectivity toward E.coli,and presented a good linearity in range of 1.0×10⁵～1.0×10⁸ CFU/mL.The optimum fluorescence intensity for detecting E. coli was found to be at...     

A novel and simple biosensor based on poly(indoleacetic acid) film and its application for simultaneous electrochemical determination of dopamine and epinephrine in the presence of ascorbic acid

A novel and simple biosensor based on poly(indoleacetic acid) film-modified electrode (PIAA/CPE) was fabricated by electrochemical polymerization of indoleacetic acid on a carbon paste electrode (CPE) through cyclic voltammetry. The resulting electrode was characterized by scanning electron microscopy, and the electrochemical behaviors of dopamine (DA) and epinephrine (EP) at the electrode were studied. It was illustrated that PIAA/CPE had excellent electrochemical catalytic activities toward DA and EP. The anodic peak currents (I _pa) were dramatically enhanced by about seven-fold for DA and ten times for EP at PIAA/CPE. Thus, the determinations of DA and EP were carried out using PIAA/CPE successfully. The linear responses were obtained in the range of 3.0?×?10^?7～7.0?×?10^?4 and 1.0?×?10^?6 ～8.0?×?10^?4 mol L^?1 with the detection limits (3σ) of 1?×?10^?7 and 4?×?10^?7 mol L^?1 corresponding with DA and EP, respectively. Moreover, the cathodic peaks of DA and EP were well-separated with a potential difference about 325 mV in pH 5.3 phosphate-buffered saline, so simultaneous determination of DA and EP was carried out in this paper. Additionally, the interference studies showed that the PIAA/CPE exhibited excellent selectivity in the presence of ascorbic acid (AA). With good selectivity and sensitivity, the present method has been successfully applied to the determination of DA and EP in pharmaceutical samples.     

Thiaminase I (42 kDa) heterogeneity,sequence refinement,and active site location from high-resolution tandem mass spectrometry

Thiaminase I (E.C. 2.5. 1.2) from Bacillus thiaminolyticus catalyzes the degradation of thiamin (vitamin B₁). Unexpected mass heterogeneity (MW 42,127, 42,197, and 42,254; 1:2:1) in recombinant thiaminase I from Escherichia coli was detected by electrospray ionization Fourier-transform mass spectrometry, resolving power 7×10⁴. Nozzle-skimmer fragmentation data reveal an extra Ala (+71.02; 71.04=theory) and GlyAla (+128.04; 128.06=theory) on the N-terminus, in addition to the fully processed enzyme. However, the fragment ion masses were consistent only with this sequence through 330 N-terminal residues; resequencing of the last 150 bps of the thiaminase I gene yields a sequence consistent with the molecular weight values and all 61 fragment ion masses. Covalently labeling the active site with a 108-Da pyrimidine moiety via mechanism-based inhibition produces a corresponding molecular weight increase in all three thiaminase I components, which indicates that they are all enzymatically active. Inspection of the fragment ions that do and do not increase by 108 Da indicates that the active site nucleophile is located between Pro⁷⁹ and Thr¹⁷⁷ in the 379 amino acid enzyme.     

Conformational studies of 3,4-dideoxy furanoid sugar amino acid containing analogs of the receptor binding inhibitor of vasoactive intestinal peptide

Conformational analysis of vasoactive intestinal peptide (VIP) receptor binding inhibitor Leu¹-Met²-Tyr³-Pro⁴-Thr⁵-Tyr⁶-Leu⁷-Lys⁸1 by various NMR techniques and constrained molecular dynamics (MD) simulation studies revealed that the molecule had a turn structure involving its Tyr³-Pro⁴-Thr⁵-Tyr⁶ moiety with intramolecular hydrogen bond between Tyr⁶NH→Tyr³CO. In order to mimic the structure of 1, peptidomimetic analogs 2-4 were synthesized using conformationally constrained scaffolds of 3,4-dideoxy furanoid sugar amino acids (2S,5R)-ddSaa1 5 and its enantiomer (2R,5S)-ddSaa2 6. All these analogs displayed well defined three-dimensional structures akin to that found in 1. Peptides 2 and 3, which differed only in the sugar amino acid stereochemistry, show propensity of structures with identical intramolecular hydrogen bonds between ThrNH→MetCO. A similar structure with a hydrogen bond between TyrNH→MetCO was observed in 4.     

Cis→Trans Isomerization of Pro<Superscript>7</Superscript> in Oxytocin Regulates Zn<Superscript>2+</Superscript> Binding

Ion mobility/mass spectrometry techniques are employed to investigate the binding of Zn²⁺ to the nine-residue peptide hormone oxytocin (OT, Cys¹-Tyr²-Ile³-Gln⁴-Asn⁵-Cys⁶-Pro⁷-Leu⁸-Gly⁹-NH₂, having a disulfide bond between Cys¹ and Cys⁶ residues). Zn²⁺ binding to OT is known to increase the affinity of OT for its receptor [Pearlmutter, A. F., Soloff, M. S.: Characterization of the metal ion requirement for oxytocin-receptor interaction in rat mammary gland membranes. J. Biol. Chem. 254, 3899–3906 (1979)]. In the absence of Zn²⁺, we find evidence for two primary OT conformations, which arise because the Cys⁶–Pro⁷ peptide bond exists in both the trans- and cis-configurations. Upon addition of Zn²⁺, we determine binding constants in water of K_A = 1.43 ± 0.24 and 0.42 ± 0.12 μM^?1, for the trans- and cis-configured populations, respectively. The Zn²⁺ bound form of OT, having a cross section of Ω = 235 Ų, has Pro⁷ in the trans-configuration, which agrees with a prior report [Wyttenbach, T., Liu, D., Bowers, M. T.: Interactions of the hormone oxytocin with divalent metal ions. J. Am. Chem. Soc. 130, 5993–6000 (2008)], in which it was proposed that Zn²⁺ binds to the peptide ring and is further coordinated by interaction of the C-terminal, Pro⁷-Leu⁸-Gly⁹-NH₂, tail. The present work shows that the cis-configuration of OT isomerizes to the trans-configuration upon binding Zn²⁺. In this way, the proline residue regulates Zn²⁺ binding to OT and, hence, is important in receptor binding.

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Nanofibrils in 3D aligned channel arrays with synergistic effect of Ag/NPs for rapid and highly efficient electric field disinfection

The disinfection of waterborne pathogens from drinking water is extremely important for human health. Although countless efforts have been devoted for drinking water inactivation, challenges still exist in terms of relative high energy consumption and complicated to implement and maintain. Here, silver nanoparticles anchoring wood carbon (Ag NPs/WC) membrane is developed as cost-effective, high flux, scalable filter for highly efficient electric field disinfection of water. Under electric field of 4 V voltage, the designed membrane achieved more than 5 log (99.999%) disinfection performance for different model bacteria, including Escherichia coli (E. coli), Enterococcus faecalis (E. faecalis), Salmonella enterica serovar Typhimirium (S. Typhimurium) and Bacillus subtilis (B. subtilis) with a high flux of 3.8 × 10³ L m⁻² h⁻¹, extremely low energy consumption of 2 J L⁻¹ m⁻² and fantastic durability (7 days). The high disinfection performance of Ag NPs/WC membrane is attributed to the synergistic disinfection of carbon nanofibrils, Ag nanoparticles as well as the low tortuous structure of the channels in wood carbon. The Ag NPs/WC membrane presents a promising strategy for point-of-use drinking water electric field disinfection treatment.     

Rapid voltammetric determination of maltol in some foods and beverages using a poly(methylene blue)/graphene-modified glassy carbon electrode

A novel poly(methylene blue)/graphene composite glassy carbon electrode was fabricated and the electrochemical behavior of maltol at the modified electrode was studied by cyclic voltammetry. In phosphate-buffered solution, the modified electrode exhibited excellent electrocatalytic activity towards the electrochemical oxidation of maltol. Under optimized conditions, the oxidation peak current showed a linear relationship with the concentrations of maltol in the ranges of 8.00?×?10^?7 to 4.00?×?10^?5 and 4.00?×?10^?5 to 5.40?×?10^?4 mol L^?1, with a detection limit of 6.50?×?10^?8 mol L^?1. The performance of the developed method was validated in terms of linearity (r?=?0.9981 and 0.9955), recovery (97.0?99.3 %), reproducibility (relative standard deviations?≤?3.1 %, n?=?6), and robustness. The method shows excellent sensitivity, selectivity, and reproducibility and has been successfully applied to analyzing maltol in a wide variety of food products.     

Rapid determination of microorganisms using a flow-injection system

An automated method for the rapid determination of microorganisms using a flow-injection system is presented. Electrochemical measurement of a mediator reduced by microbial metabolism allowed the determination of fungi and bacteria in a few minutes. The lowest detection limit was 5 × 10⁶ colony-forming units (cfu) ml^?1 for Escherichia coli. Correlation between the flow-injection method and standard microbiological methods was excellent (r = 0.997, n = 4 for Beauveria bassiana; r = 0.997, n = 7 for E. coli). The flow-injection system was applied to the on-line control of an E. coli cultivation.     

The Synthesis of [4-Carboranylalanine, 5-Leucine]-Enkephalin (Including an Improved Preparation of t-Butoxycarbonyl-L-o-carboranylalnine,New Derivatives of L-Propargylglycine,and a Note on Melanotropic and Opiate Receptor Binding Characteristics)

The title compound, an analogue of [Leu⁵]-enkephalin with L -o-carboranylalanine replacing L -phenylalanine in position 4, was prepared by fragment condensation. The analogue has a 3-fold higher affinity for rat brain opiate receptors in the [³H]naloxone competition assay than natural [Leu⁵]-enkephalin. Like [Leu⁵]-enkephalin and N^a-acetyl-[Leu⁵]-enkephalin, the N-terminal tripeptide fragment, H · Tyr-Gly-Gly · OH, had no melanotropic activity in the Rana pipiens frog skin assay. A convenient, direct synthesis of methyl t-butoxycarbonyl-L -propargylglycinate is described, and the ¹³C-NMR. spectra of L -o-carboranylalanine recorded. The procedure was extended to the preparation of BOC · Car-Leu · OMe from BOC · Pra-Leu · OMe. A number of new propargylglycine derivatives are reported.     

Amperometric method for rapid detection of Escherichia coli by flow injection analysis using a bismuth nano-film modified glassy carbon electrode

A new method for rapid detection of Escherichia coli (E. coli) was developed by flow injection analysis (FIA) using bismuth nano-film modified glassy carbon electrode (BiNFE) in this paper. The method depended on a good marker β-d-glucuronidase which is found in E. coli strains. β-d-Glucuronidase was produced by the induction of methyl-β-d-glucuronide sodium (MetGlu), then released from E. coli cells through the permeabilization of cell membrane caused by polymyxin B nonapeptide and lysozyme. The released β-d-glucuronidase could catalyze the hydrolysis of the substrate 4-nitrophenyl β-d-glucuronide (PNPG) in the culture medium to produce 4-nitrophenol. Since 4-nitrophenol is electroactive and its quantity is proportional to the concentration of E. coli, E. coli could be determined by electroanalysis of 4-nitrophenol. The BiNFE was fabricated by an electrodeposition of metallic bismuth onto a glassy carbon electrode, which showed a high sensitivity in determination of 4-nitrophenol when used in conjunction with FIA system. Experimental results showed that the amplified response current of 4-nitrophenol obtained at the BiNFE was linear with the concentration of E. coli ranging from 1.5 × 10² to 1.0 × 10⁶ cfu/ml, the detection limit of this method for E. coli is 100 cfu/ml, and the complete assay was performed in 3 h.     

Peptide stapling with the retention of double native side-chains

All-hydrocarbon stapling strategy has been widely applied for enhancing the proteolytic stability of peptides. However, two major technical hurdles to some extent limit the development of stapled peptides for therapeutic usage: rational selection of the stapling sites and the corresponding deletion of the native side chains. Previously we described the development of the olefin-terminated amino acids with the retention of native side chains and successfully applied them in the synthesis of hydrocarbon stapled peptides with single side-chain retention. Here, we explored the feasibility and effectiveness of hydrocarbon stapling strategy characterized as double side-chains retention. Modeled after a lengthy human immunodeficiency virus-1 (HIV-1) fusion inhibitor SC34EK, Leuⁱ, Serⁱ⁺⁴ and Lysⁱ, Leuⁱ⁺⁴ stapled peptides with the retention of double side-chains were effectively obtained. Our complementary study provided a convenient alternative to address where to install the staple in sequence for conventional all-hydrocarbon peptide stapling. Furthermore, this method not only conferred conformational reinforcement for SC34EK with high α-helicity and protease resistance, but also preserved the structural characteristic (key peripheral residues, charge and solubility) of the linear peptide to the maximum, which are crucial for anti-HIV-1 activity.     

Rapid detection of Escherichia coli by flow injection analysis coupled with amperometric method using an IrO2–Pd chemically modified electrode

An amperometric method for the rapid detection of Escherichia coli (E. coli) by flow injection analysis (FIA) using an IrO₂–Pd chemically modified electrode (CME) was developed in this paper. The method is based on a good marker β-d-galactosidase which was found in E. coli strains. β-d-galactosidase was produced by the induction of isopropyl β-d-thiogalactopyranoside (IPTG) and released from E. coli cells through the permeabilization of both polymyxin B nonapeptide and lysozyme to E. coli cells wall. The released β-d-galactosidase could catalyze the hydrolysis of the substrate p-aminophenyl β-d-galactopyranoside (PAPG) in the culture medium to produce 4-aminophenol which was proportional to the concentration of E. coli. Hence, E. coli could be detected by the determination of 4-aminophenol. An IrO₂–Pd CME, which showed high sensitivity in determination of 4-aminophenol, was prepared as the electro-detector in FIA. The amplified response current of 4-aminophenol obtained at the IrO₂–Pd CME was linear with the concentration of E. coli ranging from 2.0 × 10² to 1.0 × 10⁶ cfu/mL, the detection limit of this method to E. coli was 150 cfu/mL and the complete assay could be performed in 3 h.     

Enhanced production of poly-3-hydroxybutyrate by recombinant Escherichia coli containing NAD kinase and phbCAB operon

With the help of Tn5 transposon technique, gene yfjB encoding NAD kinase in Escherichia coli (E. coli) was inserted into chromosome of recombinant E. coli polyhydroxybutyrate (PHB) containing PHB synthesis operon integrated in the host genome. After successful transposition of an extra yfjB gene copy into genome, the selected recombinant named E. coli PHBTY4 showed stronger NAD kinase activity than that of E. coli PHB. Shake flask studies suggested that both cell dry weight and PHB accumulation were significantly increased in E. coli PHBTY4 compared with that of the control. E. coli PHBTY4 produced approximately 23 g/L PHB compared with its control which synthesized only 10 g/L PHB when grown under the same conditions in a 6 L fermentor after 32 h of cultivation. In addition, E. coli PHBTY4 maintained high genetic stability during the cultivation processes. These results revealed a practical method to construct genetically stable strains harboring extra NAD kinase gene to enhance NADP(H)-dependent bio-reactions.     

NMR Solution Structure of Phospholamban

Phospholamban (PLN), an amphipathic intrinsic membrane protein of 52 amino acids, is the modulator of the Ca²⁺ pump of cardiac, slow‐twitch, and smooth‐muscle sarcoplasmic reticulum. In response to β‐adrenergic stimulation, it becomes phosphorylated at Ser¹⁶ and/or Thr¹⁷, and dissociates from the pump, which, in turn, achieves its full activity. Here we present the three‐dimensional structure of chemically synthesized, monomeric PLN in an organic solvent. Monomerization (PLN normally forms homopentamers) was obtained by replacing Cys⁴¹ with phenylalanine (Phe=F), a modification that did not affect biological activity. The structure was determined by high‐resolution NMR in CHCl₃/MeOH of the unphosphorylated state of [F⁴¹]PLN (C41F). Of the hydrophilic cytoplasmic parts IA (Met¹ to Pro²¹) and IB (Gln²² to Asn³⁰) and the membrane‐spanning hydrophobic domain II (Leu³¹ to Leu⁵²) of PLN, domain IA, which contains the two phosphorylation sites Ser¹⁶ and Thr¹⁷, and domain II have been suggested to be helical and connected through the less‐structured hinge‐region IB. In the structural study presented here, [F⁴¹]PLN is composed of two α‐helical regions connected by a β‐turn (type III). The residues of the β‐turn (type III) are Thr¹⁷, Ile¹⁸, Glu¹⁹, and Met²⁰, the first being one of the two phosphorylation sites (Ser¹⁶ and Thr¹⁷). The hinge region is located at the C‐terminal end of domain IA, and domain IB is part of a second helix. The two α‐helices comprising amino acids 4 – 16 and 21 – 49 are well‐defined (the root‐mean‐square deviations for the backbone atoms, calculated for a family of the structures, are 0.58 and 0.92 Å, resp.). Pro²¹ is at the beginning of the C‐terminal helix and in the trans conformation.     

Sequence and three-dimensional structure of cycloreticulins A and B, new cyclooctapeptides from the seeds of Annona reticulata

Two cyclooctapeptides, cycloreticulin A, cyclo(Pro¹-Gly²-Asp³-Ile⁴-Ser⁵-Ile⁶-Tyr⁷-Tyr⁸) (1) and cycloreticulin B, cyclo(Pro¹-Mso²-Tyr³-Gly⁴-Thr⁵-Val⁶-Ala⁷-Val⁸) (2), have been isolated from the methanol extract of the seeds of Annona reticulata L. The sequences were elucidated on the basis of the MS/MS fragmentation using a QTOF mass spectrometer equipped with an ESI source, chemical degradation and extensive 2D-NMR. The solid state conformation of cycloreticulin A, carried out by X-ray study, is characterised by the presence of two β-turns (types II and III) and an inversed γ-turn. Its solution structure appeared quite similar to the crystal one. The cyclic backbone solution structure of cycloreticulin B, close to that of the cyclooctapeptide squamin A, from which its sequence only differs by a Val⁸/Ile⁸ substitution, involves three β-turns, two of type I and one of type III, being similar to the crystal structure of squamin A.     

Detection of nisin expression by Lactococcus lactis using two susceptible bacteria to associate the effects of nisin with EDTA

Nisin, a bacteriocin produced during the exponential growth phase of Lactococcus lactis ATCC 11454, inhibits the growth of a broad range of Grampositive bacteria. Gram-negative bacteria can also be inhibited by nisin with EDTA. In this study, nisin production was assayed by the agar diffusion method using Lactobacillus sake ATCC 15521 and a recombinant Escherichia coli DH5-α expressing the recombinant green fluorescent protein as the nisin-susceptible test organisms. The titers of nisin expressed and released in culture media were quantified and expressed in arbitrary units (AU/mL of medium) and converted to standard nisin concentration (Nisaplin^®, 25 mg of pure nisin with an activity of 1×10⁶ AU/mL). The expression and release of nisin by L. lactis in skimmed milk (9.09% total solids) with Man Rugosa Shepeer-Bacto Lactobacilli broth (1∶1) was monitored in a 5 L New Brunswick fermentor. Combining EDTA with nisin increased the bactericidal effect of nisin on the bacteria examined. The presence of EDTA was necessary to inhibit E. coli growth with nisin. L. sake was shown to be a good indicator for the evaluation of nisin release in the culture media, including with the addition of EDTA.