Identification of RNA sequence isomer by isotope labeling and LC–MS/MS

Recently, we developed a method for modified ribonucleic acid (RNA) analysis based on the comparative analysis of RNA digests (CARD). Within this CARD approach, sequence or modification differences between two samples are identified through differential isotopic labeling of two samples. Components present in both samples will each be labeled, yielding doublets in the CARD mass spectrum. Components unique to only one sample should be detected as singlets. A limitation of the prior singlet identification strategy occurs when the two samples contain components of unique sequence but identical base composition. At the first stage of mass spectrometry, these sequence isomers cannot be differentiated and would appear as doublets rather than singlets. However, underlying sequence differences should be detectable by collision‐induced dissociation tandem mass spectrometry (CID MS/MS), as y‐type product ions will retain the original enzymatically incorporated isotope label. Here, we determine appropriate instrumental conditions that enable CID MS/MS of isotopically labeled ribonuclease T1 (RNase T1) digestion products such that the original isotope label is maintained in the product ion mass spectrum. Next, we demonstrate how y‐type product ions can be used to differentiate singlets and doublets from isomer sequences. We were then able to extend the utility of this approach by using CID MS/MS for the confirmation of an expected RNase T1 digestion product within the CARD analysis of an Escherichia coli mutant strain even in the presence of interfering and overlapping digestion products from other transfer RNAs. Copyright © 2014 John Wiley & Sons, Ltd.     

Stabilization and translation of immobilized mRNA on latex beads for cell-free protein synthesis system

The stability of immobilized mRNA against ribonucleases was investigated in a cell-free protein synthesis system. The plasmid-encoding protein A with the 20-mer poly(A) tail under the control of T7 promoter was constructed, and the corresponding mRNA was synthesized by T7 RNA polymerase reaction. The resulting mRNA was immobilized on oligo(dT)-immobilized latex beads by hybridization utilizing the poly(A) tail of mRNA at the 3'-terminus. The mRNA was stabilized against three types of nucleases (3'-OH exonuclease, 5'-OH exonuclease, and endonuclease) by immobilization. Translation of immobilized mRNA with a continuous-flow cell-free protein-synthesizing system from Saccharomyces cerevisiae was ascertained. Reusability of the immobilized mRNA as genetic information was also examined.     

Flow-injection spectrophotometric determination of cyanate in bioremediation processes by use of immobilised inducible cyanase

A new flow injection (FI) method for photometric monitoring of cyanate in bioremediation processes using immobilised native cyanase is described. The method is based on the catalytic reaction between cyanate and bicarbonate to produce ammonia and carbon dioxide in the presence of an inducible native cyanase, immobilised in a reactor packed with glass beads. Two degrees of purification of the biocatalyst were used-heated cell-free extract and purified extract of cyanase from Pseudomonas pseudoalcaligenes CECT 5344. The ammonia produced by the enzymatic reaction is finally monitored photometrically at 700 nm using a modification of the conventional Berthelot method. The method furnishes different calibration curves depending on the degree of purification of the cyanase, with linear ranges between 1.23 and 616.50 micromol L(-1) ( r(2)=0.9979, n=7) and between 1.07 and 308.25 micro mol L(-1) ( r(2)= 0.9992, n=7) for the heated cell-free extract and the purified cyanase extract, respectively. No statistically significant differences between the samples were found in the precision study evaluated at two cyanate concentration levels using one-way analysis of variance. A sampling frequency of 15 h(-1) was achieved. The method was used to monitor cyanate consumption in a cyanate bioremediation tank inoculated with Pseudomonas pseudoalcaligenes CECT 5344 strain. The correlation between cyanate degradation and ammonia production was tested using a conventional method. Finally, the method was applied to different samples collected from the bioremediation tank using the standard addition method; recoveries between 85.9 and 97.4% were obtained.     

Disposable Immunosensor for Escherichia Coli O157:H7 Based on a Multi-Walled Carbon Nanotube-Sodium Alginate Nanocomposite Film Modified Screen-Printed Carbon Electrode

A disposable immunosensor for the detection of Escherichia coli O157:H7 based on a multiwalled carbon nanotube–sodium alginate nanocomposite film was constructed. The nanocomposite was placed on a screen-printed carbon electrode, and horseradish peroxidase-labeled antibodies were immobilized to E. coli O157:H7 on the modified electrode to construct the immunosensor. The modification procedure was characterized by atomic force microscopy and cyclic voltammetry. Under optimal conditions, the proposed immunosensor exhibited good electrochemical sensitivity to E. coli O157:H7 in a concentration range of 10³–10¹⁰ cfu/mL, with a relatively low detection limit of 2.94 × 10² cfu/mL (S/N = 3). This immunosensor exhibited satisfactory specificity, reproducibility, stability, and accuracy, making it a potential alternative tool for early assessment of E. coli O157:H7.     

Synthesis of n-alkylated quaternary ammonium chitosan and its long-term antibacterial finish for rabbit hair fabric

In this work, n-alkyl chitosan (N-CTS) was obtained by alkylation modification of chitosan with n-butylaldehyde using Schiff alkali method. The etherifying agent 3-chloro-2-hydroxypropyl triethylammonium chloride (CHPTAC-ethyl) was synthesized from triethylamine and epichlorohydrin. The N-CTS and CHPTAC-ethyl were etherized to finally synthesize n-alkyl quaternary ammonium chitosan (N-CCTS). Scanning electron microscope, X-ray diffractometer, Fourier transform infrared, X-ray photoelectron spectroscopy, and ¹³CNMR were used to characterize the surface morphology and chemical structure. Viscosity method and spectrophotometry were used to determine its physical and chemical properties. The etherification reaction mechanism was studied systematically and the influence of reaction conditions on the degree of substitution and solubility of N-CCTS was investigated. The minimum inhibitory concentration (MIC) of N-CCTS against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was both 0.15 g/L, which was superior to the MIC value of natural chitosan. N-CCTS was used to finish rabbit hair fabric with citric acid as cross-linking agent and sodium hypophosphite as catalyst. The antibacterial and washing resistance of the product were investigated. After 25 times of washing, the antibacterial rate of N-CCTS against E. coli and S. aureus remained stable at about 90%, and the antibacterial rate was higher than that of N-CTS and natural chitosan, and it was a kind of natural polymer long-acting antibacterial finishing agent for rabbit hair fabric.     

Cloning and expression of full-lengthTrichoderma reesi cellobiohydrolase I cDNAs inEscherichia coli

The process of converting lignocellulosic biomass to ethanol via fermentation depends on developing economic sources of cellulases.Trichoderma reesei cellobiohydrolase (CBH) I is a key enzyme in the fungal cellulase system; however, specific process application requirements make modification of the enzyme by site-directed mutagenesis (SDM) an attractive goal. To undertake SDM investigations, an efficient, cellulase-free host is required. To test the potential ofEscherichia coli as a host, T.reesei CBH I cDNA was expressed inE. coli strain GI 724 as a C-terminal fusion to thermostable thioredoxin protein. Full-length expression of CBH I was subsequently verified by molecular weight, Western blot analysis, and activity on soluble substrates.

     

High-level expression of soluble human β-defensin-2 fused with green fluorescent protein in Escherichia coli cell-free system

Human β-defensin-2 (hBD2), a small cationic peptide, exhibits a broad range of antimicrobial activity and does not acquire any microbial resistance. To produce this uneasily detectable, degradable, and toxic polypeptide efficiently, an alternative approach based on the Escherichia coli cell-free biosynthesis system was proposed. The approach implies that a polypeptide of interest is synthesized as a fusion protein linked to a green fluorescent protein (GFP) through a cleavable spacer. With batch-mode operation, a significant amount of hBD2 fused with GFP (0.25 mg/mL) can be expressed in this cell-free system. The productivity of the fusion protein can be improved up to 1.2 mg/mL by employing a continuous-exchange cell-free system. Furthermore, the GFP moiety provides directly visible and quantitative monitoring of the polypeptide synthesis, and the product is soluble and stable. This work will be helpful in allowing the rapid and visible expression of other similar defensins using an in vitro cell-free system.     

Characterization of S‐thiolation on secreted proteins from E. coli by mass spectrometry

S‐thiolation is a reversible post‐translational modification in which thiol metabolites of low molecular masses are linked to protein sulfhydryl groups through disulfide bonds. This modification is commonly observed in recombinant proteins secreted from E. coli cells. Since it can alter protein functions and introduce molecular heterogeneity, S‐thiolation is undesirable for recombinant protein production. To date, few published studies have characterized thiol modifiers or investigated the mechanism of S‐thiolation in recombinant proteins. In this work, reversed‐phase liquid chromatography and mass spectrometry were used to characterize four of the most abundant thiol modifiers on recombinant proteins secreted from E. coli BL21 (DE3) strain. These thiol modifiers have been identified as glutathione, 4‐phosphopantetheine, gluconoylated glutathione, and dephosphorylated coenzyme A. S‐thiolation by these thiol modifiers increases protein mass by 305, 356, 483, and 685 Da, respectively. These specific mass increases can be used as markers for identifying S‐thiolation in recombinant proteins. Copyright © 2009 John Wiley & Sons, Ltd.     

In vitro antimicrobial activity of extracts and compounds isolated from Cladonia uncialis

Heptane (Hep), diethyl ether (Et₂O), acetone (Me₂CO) and methanolic (MeOH) extracts, as well as ( ? )-usnic acid and squamatic acid, were obtained from thallus of Cladonia uncialis (Cladoniaceae). The antimicrobial activities of these extracts, ( ? )-usnic acid and squamatic acid, were tested against reference strains: Staphylococcus aureus, Escherichia coli and Candida albicans. In addition, Me₂CO extract was analysed against 10 strains of Methicillin-resistant S. aureus (MRSA) isolated from patients. All extracts exerted antibacterial activity against the reference strain S. aureus, comparably to chloramphenicol [minimum inhibitory concentration (MIC) = 5.0 μg/mL]. The Me₂CO extract exhibited the strongest activity against S. aureus (MIC = 0.5 μg/mL), higher than ( ? )-usnic acid, whereas squamatic acid proved inactive. The Me₂CO extract showed potent antimicrobial activity against MRSA (MIC 2.5–7.5 μg/mL). Also no activity of C. uncialis extracts against E. coli and C. albicans was observed.     

Cloning and Expression of Che a 1, the Major Allergen of <Emphasis Type="Italic">Chenopodium album</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Chenopodium album is a weedy annual plant in the genus Chenopodium. C. album pollen represents a predominant allergen source in Iran. The main C. album pollen allergens have been described as Che a 1, Che a 2, and Che a 3. The aim of this work was to clone the Che a 1 in Escherichia coli to establish a system for overproduction of the recombinant Che a 1 (rChe a 1). In order to clone this allergen, the pollens were subjected to RNA extraction. A full-length fragment encoding Che a 1 was prepared by polymerase chain reaction amplification of the first-strand cDNA synthesized from extracted RNA. Cloning was carried out by inserting the cDNA into the pET21b (+) vector, thereafter the construct was transformed into E. coli Top10 cells and expression of the protein was induced by IPTG. The rChe a 1 was purified using histidine tag in recombinant protein by means of Ni–NTA affinity chromatography. IgE immunoblotting, ELISA, and inhibition ELISA were done to evaluate IgE binding of the purified protein. In conclusion, the cDNA for the major allergen of the C. album pollen, Che a 1, was successfully cloned and rChe a 1 was purified. Inhibition assays demonstrated allergic subjects sera reacted with rChe a 1 similar to natural Che a 1 in crude extract of C. album pollen. This study is the first report of using the E. coli as a prokaryotic system for Che a 1 cloning and production of rChe a 1.     

Chemical constituents,in vitro antioxidant and antimicrobial properties of ethyl acetate extract obtained from Cytisus triflorus l’Her

Abstract

The present study investigates the phenolic composition, antioxidant and antimicrobial activities of an ethyl acetate extract from C. triflorus L’Her. The phytochemical study on the aerial parts of C. triflorus belonging to the Fabaceae family led to the isolation and structural elucidation of 5-Hydroxy-7-O-glucosylflavone (P1), 5-Hydroxy-7-O-galactosylflavone (P2), 5,7-Dihydroxy-flavone (P3), 5,7,3’-Trihydroxy,4’-methoxy-flavone (P4). These compounds were identified by 1D and 2D NMR combined analysis as well as by UV.

Ethyl acetate extract of C. triflorus showed a significant and dose-dependent antioxidant activity in vitro, related to the presence of phenolics (180.33?±?12.22?µg GAE/mg) and flavonoids (16.78?± 1.54?µg QE/mg). The antimicrobial activity was evaluated in vitro against Staphylococcus aureus CECT 240 and Escherichia coli CECT 4099, by agar-diffusion method. The most active antibacterial activity was expressed by ethyl acetate extract of C. triflorus against Gram-positive bacteria S. aureus. The gathered results suggest that C. triflorus polyphenols and flavonoids are closely associated to its antioxidant and antimicrobial properties.     

Molecular analysis and heterologous expression of the gene encoding methylmalonyl—coenzyme a mutase from rifamycin SV-producing strain Amycolatopsis mediterranei U32

The conversion of succinyl-coenzyme A (CoA) into methylmalonyl-CoA, catalyzed by adenosylcobalamin-dependent methylmalonyl-CoA mutase (MCM), represents an important source of building blocks for rifamycin SV biosynthesis. The structural gene for MCM from rifamycin SV—producing strain Amycolatopsis mediterranei U32 was isolated by using a heterologous gene probe encoding the MCM of Streptomyces cinnamonensis. A 7.8-kbp fragment was sequenced and four complete open reading frames (ORFs) and two incomplete ORFs were found. Two central ORFs, ORF3 and ORF4, overlap by four nucleotides and were found to encode MCM small (602 residues) and large (721 residues) subunits, respectively. Comparison showed that the MCM gene of A. mediterranei U32 was quite similar to those from other sources. The functionally unknown ORF5, immediately downstream of the mut AB gene, was quite similar to the ORFs downstream of mut AB from S. cinnamonensis and Mycobacterium tuberculosis. Such a striking cross-species conservation of gene order suggested that ORF5 could also be involved in the metabolism of methylmalonyl-CoA. MCM gene was overexpressed in Escherichia coli under T7 promoter, and MCM activity could be detected in the recombinant E. coli clone harboring MCM gene after the addition of coenzyme B₁₂. A purification procedure based on the B₁₂ affinity column was established to purify the MCM from E. coli. The molecular weight of purified MCM from E. coli was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, which corresponds to that calculated from the MCM protein sequence and is also the same size as that of the enzyme purified directly from A. mediterranei U32. MCM gene was overexpressed in polyketide monensin producing S. cinnamonensis, and the total monensin production was increased by 32%. Both authors contributed equally to this paper.     

Green Synthesized Silver Nanoparticles Immobilized on Activated Carbon Nanoparticles: Antibacterial Activity Enhancement Study and Its Application on Textiles Fabrics

This research aimed to enhance the antibacterial activity of silver nanoparticles (AgNPs) synthesized from silver nitrate (AgNO₃) using aloe vera extract. It was performed by means of incorporating AgNPs on an activated carbon nanoparticle (ACNPs) under ultrasonic agitation (40 kHz, 2 × 50 watt) for 30 min in an aqueous colloidal medium. The successful AgNPs synthesis was clarified with both Ultraviolet-Visible (UV-Vis) and Fourier Transform Infrared (FTIR) spectrophotometers. The successful AgNPs–ACNPs incorporation and its particle size analysis was performed using Transmission Electron Microscope (TEM). The brown color suspension generation and UV-Vis’s spectra maximum wavelength at around 480 nm confirmed the existence of AgNPs. The particle sizes of the produced AgNPs were about 5 to 10 nm in the majority number, which collectively surrounded the aloe vera extract secondary metabolites formed core-shell like nanostructure of 8.20 ± 2.05 nm in average size, while ACNPs themselves were about 20.10 ± 1.52 nm in average size formed particles cluster, and 48.00 ± 8.37 nm in average size as stacking of other particles. The antibacterial activity of the synthesized AgNPs and AgNPs-immobilized ACNPs was 57.58% and 63.64%, respectively (for E. coli); 61.25%, and 93.49%, respectively (for S. aureus). In addition, when the AgNPs-immobilized ACNPs material was coated on the cotton and polyester fabrics, the antibacterial activity of the materials changed, becoming 19.23% (cotton; E. coli), 31.73% (polyester; E. coli), 13.36% (cotton; S. aureus), 21.15% (polyester; S. aureus).     

PHOTOBIOLOGY OF RNA BACTERIOPHAGES—I. ULTRAVIOLET INACTIVATION AND PHOTOREACTIVATION STUDIES*

Abstract— Biologically active f2-RNA, Obtained from bacteriophage f2, was inactivated by ultraviolet (u.v) light (2537 Å) with a quantum yield of 3.3 ± 0.3 times 10^-3 when assayed in the dark with protoplasts of an F- strain of E. coli k12. Assay under “black light” gave a quantum yield of 2.7 ± 0.5 times 10^-3 which was just enough lower to suggest that 17 per cent photorecovery of the u.v. lesions has taken place. Intact phage f2 was inactivated by u.v. radiation with a quantum yield of 0.7 ± 0.12 times 10^-3, Thus the whole phage is much less sensitive than the free RNA. No evidence of photorecovery was found in u.v.-irradiated RNA phage 7S assayed in its host Pseudomonas aeruginosa.     

Carba-LNA-5MeC/A/G/T modified oligos show nucleobase-specific modulation of 3'-exonuclease activity, thermodynamic stability, RNA selectivity, and RNase H elicitation: synthesis and biochemistry

Using the intramolecular 5-exo-5-hexenyl radical as a key cyclization step, we previously reported an unambiguous synthesis of carba-LNA thymine (cLNA-T), which we subsequently incorporated in antisense oligonucleotides (AON) and investigated their biochemical properties [J. Am. Chem. Soc.2007, 129 (26), 8362-8379]. These cLNA-T incorporated oligos showed specific RNA affinity of +3.5-5 °C/modification for AON:RNA heteroduplexes, which is comparable to what is found for those of LNAs (Locked Nucleic Acids). These modified oligos however showed significantly enhanced nuclease stability (ca. 100 times more) in the blood serum compared to those of the LNA modified counterparts without compromising any RNase H recruitment capability. We herein report the synthesis of 5-methylcytosine-1-yl ((Me)C), 9-adeninyl (A), and 9-guaninyl (G) derivatives of cLNA and their oligonucleotides and report their biochemical properties as potential RNA-directed inhibitors. In a series of isosequential carba-LNA modified AONs, we herein show that all the cLNA modified AONs are found to be RNA-selective, but the magnitude of RNA-selectivity of 7'-R-Me-cLNA-G (cLNA-G) (ΔT(m) = 2.9 °C/modification) and intractable isomeric mixtures of 7'-(S/R)-Me-cLNA-T (cLNA-T, ΔT(m) = 2.2 °C/modification) was found to be better than diastereomeric mixtures of 7'-(S/R)-Me-cLNA-(Me)C with trace of cENA-(Me)C (cLNA-(Me)C, ΔT(m) = 1.8 °C/modification) and 7'-R-Me-cLNA-A (cLNA-A, ΔT(m) = 0.9 °C/modification). cLNA-(Me)C modified AONs however exhibited the best nuclease stability, which is 4-, 7-, and 20-fold better, respectively, than cLNA-T, cLNA-A, and cLNA-G modified counterparts, which in turn was more than 100 times stable than that of the native. When the modification sites are appropriately chosen in the AONs, the cLNA-A, -G, and -(Me)C modified sites in the AON:RNA hybrids can be easily recognized by RNase H, and the RNA strand of the hybrid is degraded in a specific manner, which is important for the design of oligos for therapeutic purposes. The cLNA-(Me)C modified AON/RNA, however, has been found to be degraded 4 times faster than cLNA-A and G modified counterparts. By appropriately choosing the carba-LNA modification sites in AON strands, the digestion of AON:RNA can be either totally repressed or be limited to cleavage at specific sites or at a single site only (similar to that of catalytic RNAzyme or DNAzyme). Considering all physico- and biochemical aspects of cLNA modified oligos, the work suggests that the cLNA modified antisense oligos have the potential of being a promising therapeutic candidate due to their (i) higher nucleobase-specific RNA affinity and RNA selectivity, (ii) greatly improved nuclease stability, and (iii) efficient RNase H recruitment capability, which can induce target RNA cleavage in a very specific manner at multiple or at a single site, in a designed manner.     

Lupane triterpenoids,antioxidant potential and antimicrobial activity of Myrciaria floribunda (H. West ex Willd.) O. Berg.

Chemical composition of the methanol extract of Myrciaria floribunda leaves was investigated. The nor-lupane triterpenoids platanic acid and messagenic I acid were identified, along with other known triterpenoids (betulinic aldehyde, ursolic acid acetate and betulinic acid), a new lupane triterpenoid (2α,6α,30-trihydroxybetulinic acid) and the flavonoids catechin, quercetrin and mirycitrin. The structures were determined by spectroscopic methods (NMR, LC-MS, GC-MS). The major isolated compound was betulinic acid. The methanol extract and 2α,6α,30-trihydroxybetulinic acid were evaluated for their DPPH scavenging potential. The tested triterpenoid was one hundred times more active than betulinic acid, but less active than the extract. Screening for antimicrobial activity showed that the methanol extract was active against Staphylococcus aureus and Escherichia coli, but inactive against Candida albicans and Candida krusei, while 2α,6α,30-trihydroxybetulinic acid was inactive to all tested microorganisms.     

Macrocyclic Pyrrolizidine Alkaloids and Silphiperfolanol Angelate Esters from Solanecio mannii

Three new compounds, the silphiperfolanol angelate ester umutagarananol ( 1 ), the macrocyclic pyrrolizidine alkaloids umutagarinine A and B ( 2 – 3 ), and five known secondary metabolites ( 4 – 8 ) were isolated from the CH₂Cl₂−MeOH (1 : 1) extract of the roots and the stem bark of Solanecio mannii (Hook. f.) (Asteraceae). The isolated compounds were characterized by NMR and IR spectroscopic, and mass spectrometric analyses, whereas the relative stereochemistry of 4 was established by NAMFIS-based combined computational and solution NMR analysis. Synthetic modification of 5 provided two new compounds, 2-angeloyloxy-4,8-epoxypresilphiperfolane ( 9 ) and 2-angeloyloxy-4,8-epoxypresilphi-perfolane ( 10 ). The crude extracts and the isolated constituents showed weak antibacterial activities (EC₅₀ 0.7–13.3 mM) against the Gram-negative Escherichia coli and the Gram-positive Bacillus subtilis. Compounds 2 , 3 and 4 exhibited strong cytotoxicity against MCF-7 human breast cancer cells, with EC₅₀ values of 35.6, 21.7 and 12.5 μM, respectively.     

Accelerated hydrolysis of esters catalyzed by receptors, II. Degradation of RNA by polyvinylpyrrolidone

The nonenzymatic hydrolysis of RNA by polyvinylpyrrolidone (PVP) has been investigated. A bell-shaped kinetics was observed when the first order rate constant of the reaction was ploted as a function of PVP concentration, which means the kinetic feature of PVP as a degradative receptor distinct from those of degradative enzymes (e.g., ribonuclease).