A novel phosphorescence sensor for Co2+ ion based on Mn‐doped ZnS quantum dots

N‐acetyl‐l ‐cysteine‐capped Mn‐doped ZnS quantum dots (QDs) were prepared by hydrothermal methods. It could emit phosphorescence at 583 nm with the excitation wavelength at 315 nm. The phosphorescence intensity of QDs could be quenched dramatically by increasing the concentration of Co²⁺ ion. The novel phosphorescence sensor based on N‐acetyl‐l ‐cysteine‐capped QDs was developed for detecting Co²⁺ ion with a linear dynamic range of 1.25 × 10^–6–3.25 × 10^–5 m . The limit of detection and RSD were 6.0 × 10^–8 m and 2.3%, respectively. Interference experiments showed excellent selectivity over numerous cations such as alkali, alkaline earth and transitional metal ions. The possible quenching mechanism was also examined by phosphorescence decays. The proposed phosphorescence method was further applied to the trace determination of Co²⁺ ion in tap and pond water samples with recoveries of 97.75–103.32%. Copyright © 2013 John Wiley & Sons, Ltd.     

Synthesis and application of N‐[1‐(4‐(4‐fluorophenyl)‐2,6‐dioxocyclohexylidene)ethyl] (Fde)‐protected amino acids for optimization of solid‐phase peptide synthesis using gel‐phase 19F NMR spectroscopy

N‐[1‐(4‐(4‐fluorophenyl)‐2,6‐dioxocyclohexylidene)ethyl] (Fde) protected amino acids have been prepared and applied in solid‐phase peptide synthesis monitored by gel‐phase ¹⁹F NMR spectroscopy. The Fde protective group could be cleaved with 2% hydrazine or 5% hydroxylamine solution in DMF as determined with gel‐phase ¹⁹F NMR spectroscopy. The dipeptide Ac‐L ‐Val‐L ‐Val‐NH₂ 12 was constructed using Fde‐L ‐Val‐OH and no noticeable racemization took place during the amino acid coupling with N,N′‐diisopropylcarbodiimide and 1‐hydroxy‐7‐azabenzotriazole or Fde deblocking. To extend the scope of Fde protection, the hydrophobic nonapeptide LLLLTVLTV from the signal sequence of mucin MUC1 was successfully prepared using Fde‐L ‐Leu‐OH at diagnostic positions. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Escherichia coli W as a new platform strain for the enhanced production of L-valine by systems metabolic engineering

A less frequently employed Escherichia coli strain W, yet possessing useful metabolic characteristics such as less acetic acid production and high L ‐valine tolerance, was metabolically engineered for the production of L ‐valine. The ilvA gene was deleted to make more pyruvate, a key precursor for L ‐valine, available for enhanced L ‐valine biosynthesis. The lacI gene was deleted to allow constitutive expression of genes under the tac or trc promoter. The ilvBN^mut genes encoding feedback‐resistant acetohydroxy acid synthase (AHAS) I and the L ‐valine biosynthetic ilvCED genes encoding acetohydroxy acid isomeroreductase, dihydroxy acid dehydratase, and branched chain amino acid aminotransferase, respectively, were amplified by plasmid‐based overexpression. The global regulator Lrp and L ‐valine exporter YgaZH were also amplified by plasmid‐based overexpression. The engineered E. coli W (ΔlacI ΔilvA) strain overexpressing the ilvBN^mut, ilvCED, ygaZH, and lrp genes was able to produce an impressively high concentration of 60.7 g/L L ‐valine by fed‐batch culture in 29.5 h, resulting in a high volumetric productivity of 2.06 g/L/h. The most notable finding is that there was no other byproduct produced during L ‐valine production. The results obtained in this study suggest that E. coli W can be a good alternative to Corynebacterium glutamicum and E. coli K‐12, which have so far been the most efficient L ‐valine producer. Furthermore, it is expected that various bioproducts including other amino acids might be more efficiently produced by this revisited platform strain of E. coli. Bioeng. 2011; 108:1140–1147. © 2010 Wiley Periodicals, Inc.     

Asymmetrically simultaneous synthesis of L‐homophenylalanine and N6‐protected‐2‐oxo‐6‐amino‐hexanoic acid by engineered Escherichia coli aspartate aminotransferase

L ‐Homophenylalanine (L ‐HPA) and N⁶‐protected‐2‐oxo‐6‐amino‐hexanoic acid (N⁶‐protected‐OAHA) can be used as building blocks for the manufacture of angiotensin‐converting enzyme inhibitors. To synthesize L ‐HPA and N⁶‐protected‐OAHA simultaneously from 2‐oxo‐4‐phenylbutanoic acid (OPBA) and N⁶‐protected‐L ‐lysine, several variants of Escherichia coli aspartate aminotransferase (AAT) were developed by site‐directed mutagenesis and their catalytic activities were investigated. Three kinds of N⁶‐protected‐L ‐lysine were tested as potential amino donors for the bioconversion process. AAT variants of R292E/L18H and R292E/L18T exhibited specific activities of 0.70±0.01 U/mg protein and 0.67±0.02 U/mg protein to 2‐amino‐6‐tert‐butoxycarbonylamino‐hexanoic acid (BOC‐lysine) and 2‐amino‐6‐(2,2,2‐trifluoro‐acetylamino)‐hexanoic acid, respectively. E. coli cells expressing R292E/L18H variant were able to convert OPBA and BOC‐lysine to L ‐HPA and 2‐oxo‐6‐tert‐butoxycarbonylamino‐hexanoic acid (BOC‐OAHA) with 96.2% yield in 8 h. This is the first report demonstrating a process for the simultaneous production of two useful building blocks, L ‐HPA and BOC‐OAHA. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Determination of p‐Aminobenzenesulfonic acid based on the electrochemiluminescence quenching of tris (2,2’‐bipyridine)‐ ruthenium (II)

This study describes the quenching effects of p‐aminobenzenesulfonic acid (p‐ABSA) based on electrochemiluminescence (ECL) of the tris (2,2^’‐bipyridyl)‐ruthenium(II)(Ru(bpy)₃²⁺)/tri‐n‐propylamine (TPrA) system in aqueous solution. Quenching behaviours were observed with a 200‐fold excess of p‐ABSA over Ru(bpy)₃²⁺. In the presence of 0.1 M TPrA, the Stern‐Volmer constant (K_SV) of ECL quenching was as high as 1.39 × 10⁴ M^‐1 for p‐ABSA. The logarithmic plot of inhibited ECL versus concentration of p‐ABSA was linear over the range of 6.0 × 10^‐6 ‐3.0 × 10^‐4 mol/L. The corresponding limit of detection was 1.2 × 10^‐6 mol/L for p‐ABSA (S/N = 3). The mechanism of quenching is believed to involve an energy transfer from the excited‐state luminophore to a dimer of p‐ABSA and the adsorption of free radicals of p‐ABSA at the electrode surface that impeded the oxidation of the Ru(bpy)₃²⁺/TPrA system. Copyright © 2012 John Wiley & Sons, Ltd.     

Competition of Ethionine and Methionine for Aminoacyl-tRNA Formation in an In Vitro System from Ochromonas danica*

Aminoacyl-tRNA synthetase and tRNA were isolated from the chrysomonad Ochromonas danica. The mutual effect of methionine and ethionine, and the effect of other amino acids on methionyl- and ethionyl-tRNA formation, were tested in an in vitro system. The tRNA^Met had a similar accepting capacity for either methionine or ethionine. Ethionine and methionine, but none of the other amino acids tested, competed for the same aminoacyl-tRNA synthetase. The K_m of methionine was 0.88 × 10^–5 M, and that of ethionine 5 × 10^–4 M. Ethionine inhibited methionine binding; K_i 3.4 × 10^–4 M. The respective values in a similar system isolated from E. coli were 2.2 × 10^–5, 1.95 × 10^–3, and 1.95 × 10^–3.     

Enhanced direct ethanol production by cofactor optimization of cell surface‐displayed xylose isomerase in yeast

Xylose isomerase (XylC) from Clostridium cellulovorans can simultaneously perform isomerization and fermentation of d ‐xylose, the main component of lignocellulosic biomass, and is an attractive candidate enzyme. In this study, we optimized a specified metal cation in a previously established Saccharomyces cerevisiae strain displaying XylC. We investigated the effect of each metal cation on the catalytic function of the XylC‐displaying S. cerevisiae. Results showed that the divalent cobalt cations (Co²⁺) especially enhanced the activity by 46‐fold. Co²⁺ also contributed to d ‐xylose fermentation, which resulted in improving ethanol yields and xylose consumption rates by 6.0‐ and 2.7‐fold, respectively. Utility of the extracellular xylose isomerization system was exhibited in the presence of mixed sugar. XylC‐displaying yeast showed the faster d ‐xylose uptake than the yeast producing XI intracellularly. Furthermore, direct xylan saccharification and fermentation was performed by unique yeast co‐culture system. A xylan‐degrading yeast strain was established by displaying two kinds of xylanases; endo‐1,4‐β‐xylanase (Xyn11B) from Saccharophagus degradans, and β‐xylosidase (XlnD) from Aspergillus niger. The yeast co‐culture system enabled fine‐tuning of the initial ratios of the displayed enzymes (Xyn11B:XlnD:XylC) by adjusting the inoculation ratios of Xylanases (Xyn11B and XlnD)‐displaying yeast and XylC‐displaying yeast. When the enzymes were inoculated at the ratio of 1:1:2 (1.39 × 10¹³: 1.39 × 10¹³: 2.78 × 10¹³ molecules), 6.0 g/L ethanol was produced from xylan. Thus, the cofactor optimization and the yeast co‐culture system developed in this study could expand the prospect of biofuels production from lignocellulosic biomass. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1068–1076, 2017     

CARBON SOURCE DEPENDENCE OF THE RATIO OF δ‐AMINOLEVULINIC ACID BIOSYNTHESIS VIA THE C5 AND SHEMIN PATHWAYS IN EUGLENA GRACILIS (EUGLENOPHYCEAE)1

The ratio of two biosynthetic pathways was estimated, the C5 and Shemin pathways, to δ‐aminolevulinic acid (ALA, a biosynthetic intermediate of tetrapyrrole) from the ¹³C‐enrichment ratios (¹³C‐ER) at the carbon atoms of chl a (after conversion to methyl pheophorbide a) biosynthesized by Euglena gracilis G. A. Klebs when l ‐[3‐¹³C]alanine was used as a carbon source. On the basis of these estimations, we confirmed that ALA was efficiently biosynthesized via both the C5 and Shemin pathways in the plastids of E. gracilis, and we determined that the ratio of ALA biosynthesis via the Shemin pathway was increased in the ratio of 14%–67%, compared with that in our previous d ‐[1‐¹³C]glucose feeding experiment ( Iida et al. 2002 ). This carbon source dependence of the contributions of the two biosynthetic pathways might be related to activation of gluconeogenesis by the amino acid substrate. The methoxy carbon of the methoxycarbonyl group at C‐13² of chl a was labeled with the ¹³C‐carbon of l ‐[methyl‐¹³C]methionine derived from l ‐[3‐¹³C]alanine via [2‐¹³C]acetyl coenzyme A (CoA), through the atypical tricarboxylic acid (TCA) cycle, gluconeogenesis, and l‐ [3‐¹³C]serine. The phytyl moiety of chl a was also labeled on C‐P2, C‐P3¹, C‐P4, C‐P6, C‐P7¹, C‐P8, C‐P10, C‐P11¹, C‐P12, C‐P14, C‐P15¹, and C‐P16 from ¹³C‐isoprene (2‐[1,2‐methyl,3‐¹³C₃]methyl‐1,3‐butadiene) generated from l ‐[3‐¹³C]alanine via [2‐¹³C]acetyl CoA.     

Chiral superstructures from homochiral Zn2+, Co2+, Fe2+‐2,6‐bis (aryl ethylimine)pyridine complexes

We report the hierarchical supramolecular organization of metallosupramolecular homochiral complexes 1 ‐Λ‐(S,S,S,S)‐M²⁺/ 1 ‐?‐(R,R,R,R)‐M²⁺ and 2 ‐ Λ‐(S,S,S,S)‐M²⁺/ 2 ‐?‐ (R,R,R,R)‐M²⁺ of M²⁺ = Co²⁺, Fe²⁺, Zn²⁺ metal ions with chiral pseudo‐terpyridine‐type ligands: 1‐ (S,S) or 1‐ (R,R) = 2,6‐bis (naphthyl ethylimine)pyridine and 2‐ (S,S) or 2‐ (R,R) = 2,6‐bis (phenyl‐ethylimine)pyridine. Circular dichroism measurements in solution were used to confirm the enantiomeric nature of all twelve complexes. For crystal structures of 1 ‐ Λ‐ (S,S,S,S)‐M²⁺ or 1 ‐?‐ (R,R,R,R)‐M²⁺ complexes, absolute configurations {? (or P), Λ (or M)} were confirmed by refinement of the Flack parameter x: ?0.007 ≤ x ≤ 0.11 for the single crystals of 1 ‐Λ‐(S,S,S,S)‐M²⁺/ 1 ‐?‐ (R,R,R,R)‐M²⁺, 2 ‐ Λ‐ (S,S,S,S)‐Fe²⁺, and 2 ‐?‐ (R,R,R,R)‐Co²⁺.     

A novel optical probe based on d‐penicillamine‐functionalized graphene quantum dots: Preparation and application as signal amplification element to minoring of ions in human biofluid

In this study, d ‐penicillamine‐functionalized graphene quantum dots (DPA‐GQD) has been synthesized, which significantly increases the fluorescence intensity of GQD. We used this simple fluorescent probe for metal ions detection in human plasma samples. Designed DPA‐GQD respond to Hg²⁺, Cu²⁺, Au²⁺, Ag⁺, Co²⁺, Zn²⁺, and Pb²⁺ with high sensitivity. The fluorescence intensity of this probe decreased significantly in the presence of metal ions such as, Hg²⁺, Cu²⁺, Au²⁺, Ag⁺, Co²⁺, Zn²⁺, and Pb²⁺. In this work, a promising probe for ions monitoring was introduced. Moreover, DPA‐GQD probe has been tested in plasma samples. The functionalized DPA‐GQDs exhibits great promise as an alternative to previous fluorescent probes for bio‐labeling, sensing, and other biomedical applications in aqueous solution.     

Purification and characterization of phenoloxidase from the hemolymph of healthy and diseased Antheraea assamensis Helfer (Lepidoptera: Saturniidae): Effects of certain biological components and chemical agents on enzyme activity

In the current study, a dimeric phenoloxidase (PO) from the hemolymph of healthy and diseased (pebrine infected) larvae of Antheraea assamensis Helfer was extracted and purified. The protein was subjected to purification using Sephacryl S‐100 and CM Sepharose chromatography. The enzyme comprised of two subunits of ~76.8 and 76 kDa that showed PO activity in 6 mM l ‐3,4‐dihydroxyphenylalanine (L ‐DOPA) and 8 mM catechol but not in hydroquinone. Optimum temperature for PO activity was 30°C in l ‐DOPA and 37°C in catechol. Optimum pH ranged from 6.8 to 7.0 in L ‐DOPA and 7.0–7.2 in catechol. Specific activity of the purified PO from healthy larvae was 53.9 µM/min per mg of protein per ml in L ‐DOPA and 50.77 µM/min per mg of protein per ml in catechol. Specific activity of PO from diseased larvae was 30.0 µM/min per mg of protein per ml in L ‐DOPA and 28.55 µM/min per mg of protein per ml in catechol. Purification fold was 3.27–4.21 for healthy and 2.38–2.56 for diseased fractions. The enzyme showed the Michaelis constant (K_m) of 2.46–2.85 mM for healthy and diseased fractions in L ‐DOPA. In catechol K_m of 9.23–17.71 mM was observed. Peptidoglycan was the best activator of purified PO from both healthy and diseased fractions. Interactions between controls and activators appeared statistically significant (F = 767.5; df = 3; P < 0.0001). Na⁺, K⁺, and Cu²⁺ increased, whereas Ca²⁺, Zn²⁺, Mg²⁺, and Co²⁺ decreased PO activity. The overall interactions appeared highly significant (F = 217.0; df = 27; P < 0.0001). Kojic acid, dithiothreitol, thiourea, phenylthiourea, carbendazim, N‐bromosuccinimide, N,N,N′,N′‐tetraacetic acid, and diethyldithiocarbamate inhibited PO activity.     

A novel fluorescent probe based on rhodamine hydrazone derivatives bearing a thiophene group for Al3+

In the present work, a novel 5‐methyl‐thiophene‐carbaldehyde‐functionalized rhodamine 6G Schiff base (RA) was designed and easily prepared as an Al³⁺ fluorescent and colorimetric probe, which could selectively and sensitively detect Al³⁺ by showing enhanced fluorescence emission. Meanwhile distinct color variation from colorless to pink also provided ‘naked eye’ detection of Al³⁺, due to the ring spirolactam opening of the rhodamine derivative. Other metal ions (including K⁺, Mg²⁺, Na⁺, Ba²⁺, Mn²⁺, Cd²⁺, Fe²⁺, Ni²⁺, Pb²⁺, Zn²⁺, Hg²⁺, Co²⁺, Li⁺, Sr²⁺ and Cu²⁺) could only induce limited interference. The detection limit of the fluorescent probe was estimated to be 4.17 × 10^?6 M, the binding constant of the RA–Al³⁺ complex was 1.4 × 10⁶ M^?1. Moreover, this fluorescent probe RA possessed high reversibility. As aluminum is a ubiquitous metal in nature and plays vital roles in many biological processes, this chemosensor could be explored for biological study applications. Copyright © 2015 John Wiley & Sons, Ltd.     

Multimode selective detection of mercury by chiroptical fluorescent sensors based on methionine/cysteine

Two multimode Hg(II) sensors, L‐MethBQA and L‐CysBQA, were obtained by fusing methionine or S‐methyl cysteine, into a bis‐quinolyl amine‐based chiral podand scaffold. Quinolyl groups serve as the fluorophore and possess nitrogen lone pairs capable of chelating metal ions. On exposure to Hg²⁺ or Zn²⁺, these sensors show signal enhancement in fluorescence. However, Cu²⁺ quenches their fluorescence in 30:70 acetontrile/water. L‐CysBQA complexes with Hg²⁺, producing an exciton‐coupled circular dichroism spectrum with the opposite sign to the one that is produced by Cu²⁺ or Zn²⁺ complexation. L‐CysBQA binds Hg²⁺ more strongly than Zn²⁺ and is shown to differentiate Hg²⁺ from other metal ions, such as Zn²⁺, Cu²⁺, Ni²⁺, and Pb²⁺, exceptionally well. The synergistic use of relatively soft sulfur, quinoline‐based chiral ligands and chiroptically enhanced fluorescence detection results in high sensitivity and selectivity for Hg²⁺. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

Synthesis of a novel fluorescent probe based on 7‐nitrobenzo‐2‐oxa‐1,3‐diazole skeleton for the rapid determination of vitamin B12 in pharmaceuticals

A new fluorescent probe, 4‐N,N‐di(2‐hydroxyethyl)imino‐7‐nitrobenzo‐2‐oxa‐1,3‐diazole (HINBD) was synthesized in a single step with reasonably good yield. The water‐soluble HINBD emits strongly in the visible region (λ_ex = 479 nm, λ_em = 545 nm) and is stable over a wide range of pH values. It was found that vitamin B₁₂ (VB₁₂) had the ability to quench the fluorescence of HINBD, and the quenched fluorescence intensity was proportional to the concentration of VB₁₂. A method for VB₁₂ determination based on the quenching fluorescence of HINBD was thus established. Interference effects of various substances, including sugars, vitamins, amino acids, inorganic cations and some organic substances have been studied. Under optimal conditions, the linear range is 0.0–2.4 × 10^–5 mol/L. The determination limit is 8.3 × 10^–8 mol/L. The method was applied to measure VB₁₂ in pharmaceutical preparations with satisfactory results. Copyright © 2013 John Wiley & Sons, Ltd.     

Chimeras of P-type ATPases and their transcriptional regulators: contributions of a cytosolic amino-terminal domain to metal specificity

Zn²⁺‐responsive repressor ZiaR and Co²⁺‐responsive activator CoaR modulate production of P₁‐type Zn²⁺‐ (ZiaA) and Co²⁺‐ (CoaT) ATPases respectively. What dictates metal selectivity? We show that Δ ziaΔcoa double mutants had similar Zn²⁺ resistance to Δzia single mutants and similar Co²⁺ resistance to Δcoa single mutants. Controlling either ziaA or coaT with opposing regulators restored no resistance to metals sensed by the regulators, but coincident replacement of the deduced cytosolic amino‐terminal domain CoaT_N with ZiaA_N (in ziaR‐_p ziaA‐ziaA_NcoaT) conferred Zn²⁺ resistance to ΔziaΔcoa, Zn²⁺ content was lowered and residual Co²⁺ resistance lost. Metal‐dependent molar absorptivity under anaerobic conditions revealed that purified ZiaA_N binds Co²⁺ in a pseudotetrahedral two‐thiol site, and Co²⁺ was displaced by Zn²⁺. Thus, the amino‐terminal domain of ZiaA inverts the metals exported by zinc‐regulated CoaT from Co²⁺ to Zn²⁺, and this correlates simplistically with metal‐binding preferences; K_ZiaAN Zn²⁺ tighter than Co²⁺. However, Zn²⁺ did not bleach Cu⁺‐ZiaA_N, and only Cu⁺ co‐migrated with ZiaA_N after competitive binding versus Zn²⁺. Bacterial two‐hybrid assays that detected interaction between the Cu⁺‐metallochaperone Atx1 and the amino‐terminal domain of Cu⁺‐transporter PacS_N detected no interaction with the analogous, deduced, ferredoxin‐fold subdomain of ZiaA_N. Provided that there is no freely exchangeable cytosolic Cu⁺, restricted contact with the Cu⁺‐metallochaperone can impose a barrier impairing the formation of otherwise favoured Cu⁺–ZiaA_N complexes.     

Enhanced chemiluminescence of peroxomonosulfate–cobalt (II) system in the presence of dicarboxylic acids

In the present work, the effects of molecular mass aliphatic dicarboxylic acids on the HSO₅^‐–Co²⁺ chemiluminescence (CL) system were investigated. It was found that the aliphatic dicarboxylic acids could enhance the CL of the HSO₅^‐–Co²⁺ system. Moreover, the CL intensities improved regularly with increasing carbon chain length of the dicarboxylic acids. To investigate the CL enhancement mechanism, dynamic profiles, CL spectroscopy, ESR spectrum and the effects of various free radical scavengers on the CL system were employed. The results indicated that the enhancement of the CL should be attributed to the formation of peroxo‐diacid, which finally decomposed to the original dicarboxylic acid and singlet oxygen. The mechanism of the HSO₅^‐–Co²⁺‐dicarboxylic acid CL system was then proposed. Copyright © 2010 John Wiley & Sons, Ltd. Copyright © 2010 John Wiley & Sons, Ltd.     

Differential effects of mineral and organic N sources,and of ectomycorrhizal infection by Hebeloma cylindrosporum,on growth and N utilization in Pinus pinaster

The effect of ectomycorrhizal association of Pinus pinaster with Hebeloma cylindrosporum was investigated in relation to the nitrogen source supplied as mineral (NH₄⁺ or NO₃^?) or organic N (L ‐glutamate) and at 5 mol m^?3. Plants were grown for 14 and 16 weeks with mineral and organic N, respectively, and samples were collected during the last 6 weeks of culture. Total fungal biomass was estimated using glucosamine amount and its viability was assessed using the glucosamine to ergosterol ratio. Non‐mycorrhizal plants grew better with NH₄⁺ than with NO₃^? and grew very slowly when supplied with L ‐glutamate. The presence of the fungus decreased the growth of the host plant with mineral N whereas it increased it with L ‐glutamate. Whatever the N source, most of the living fungal biomass was associated with the roots, whereas the main part of the total biomass was assayed outside the root. The form of mineral N did not significantly affect N accumulation rates over the 42 d in control plants. In mycorrhizal plants grown on either N source, the fungal tissues developing outside of the root were always the main N sink. The ectomycorrhizal association did not change ¹⁵NH₄⁺ uptake rate by roots, suggesting that the growth decrease of the host‐plant was related to the carbon cost for fungal growth and N assimilation rather than to a direct effect on NH₄⁺ acquisition. In contrast, in NO₃^?‐grown plants, in addition to draining carbon for NO₃^? reduction the fungus competed with the root for NO₃^? uptake. With NH₄⁺ or NO₃^? feeding, although mycorrhizal association improved N accumulation in shoots, we concluded that it was unlikely that the fungus had supplied the plant with N. In L ‐glutamate‐grown plants, the presence of the fungus increased the proportion of glutamine in the xylem sap and improved both N nutrition and the growth rate of the host plant.     

Divalent Heavy Metal Cations Block the TRPV1 Ca2+ Channel

Transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel involved in pain sensation and in a wide range of non-pain-related physiological and pathological conditions. The aim of the present study was to explore the effects of selected heavy metal cations on the function of TRPV1. The cations ranked in the following sequence of pore-blocking activity: Co²⁺ [half-maximal inhibitory concentration (IC₅₀)?=?13 μM]?>?Cd²⁺ (IC₅₀?=?38 μM)?>?Ni²⁺ (IC₅₀?=?62 μM)?>?Cu²⁺?(IC₅₀?=?200 μM). Zn²⁺ proved to be a weak (IC₅₀?=?27 μM) and only partial inhibitor of the channel function, whereas Mg²⁺, Mn²⁺ and La³⁺ did not exhibit any substantial effect. Co²⁺, the most potent channel blocker, was able not only to compete with Ca²⁺ but also to pass with it through the open channel of TRPV1. In response to heat activation or vanilloid treatment, Co²⁺ accumulation was verified in TRPV1-transfected cell lines and in the TRPV1+ dorsal root ganglion neurons. The inhibitory effect was also demonstrated in vivo. Co²⁺ applied together with vanilloid agonists attenuated the nocifensive eye wipe response in mice. Different rat TRPV1 pore point mutants (Y627W, N628W, D646N and E651W) were created that can validate the binding site of previously used channel blockers in agonist-evoked ⁴⁵Ca²⁺ influx assays in cells expressing TRPV1. The IC₅₀ of Co²⁺ on these point mutants were determined to be reasonably comparable to those on the wild type, which suggests that divalent cations passing through the TRPV1 channel use the same negatively charged amino acids as Ca²⁺.     

Comparative kinetics of fatty acid-amino acid conjugate elicitor biosynthesis by midgut tissue microsomes of Lepidopterous caterpillar larvae

N‐Linolenoyl‐L ‐glutamine is one of several structurally similar fatty acid–amino acid conjugate (FAC) elicitors found in the oral secretions of Lepidopterous caterpillars and its biosynthesis is catalyzed by membrane‐associated alimentary tissue enzyme(s). FAC elicitors comprise 17‐hydroxylated or non‐hydroxylated linolenic acid coupled with L ‐glutamine or L ‐glutamate by an amide bond. We demonstrate in vitro biosynthesis of N‐linolenoyl‐L ‐glutamine by Manduca sexta, Heliothis virescens, and Helicoverpa zea tissue microsomes. Comparison of N‐linolenoyl‐L ‐glutamine biosynthesis kinetics for these species suggests that concurrent biosynthesis and hydrolysis contribute to proportions of FAC elicitors found in their oral secretions. The apparent K_m values for coupling of sodium linolenate were 8.75±0.79, 14.3±3.7 and 20.7±3.4 mM and V_max values were 2.92±0.14, 6.81±1.2 and 4.95±0.55 nmol/min/mg protein for H. zea, H. virescens and M. sexta, respectively. The K_m values for coupling of L ‐glutamine were 10.5±0.26, 22.3±2.0 and 18.9±2.4 mM and V_max values were 1.78±0.21, 3.71±0.50 and 2.49±0.41 nmol/min/mg of protein for H. zea, H. virescens and M. sexta, respectively. © 2010 Wiley Periodicals, Inc.     

Cloning and comparison of phylogenetically related chitinases from Listeria monocytogenes EGD and Enterococcus faecalis V583

Aims: To compare enzymatic activities of two related chitinases, ChiA and EF0361, encoded by Listeria monocytogenes and Enterococcus faecalis, respectively. Methods and Results: The chiA and EF0361 genes were amplified by PCR, cloned and expressed with histidine tags, allowing easy purification of the gene products. ChiA had a molecular weight as predicted from the amino acid sequence, whereas EF0361 was 1840 Da lower than expected because of C‐terminal truncation. The ChiA and EF0361 enzymes showed activity towards 4‐nitrophenyl N,N′‐diacetyl‐β‐d ‐chitobioside with K_m values of 1·6 and 2·1 mmol l^?1, respectively, and k_cat values of 21·6 and 6·5 s^?1. The enzymes also showed activity towards 4‐nitrophenyl β‐d ‐N, N′, N″‐triacetylchitotriose and carboxy‐methyl‐chitin‐Remazol Brilliant Violet but not towards 4‐nitrophenyl N‐acetyl‐β‐d ‐glucosaminide. Chitinolytic specificities of the enzymes were supported by their inactivity towards the substrates 4‐nitrophenyl β‐d ‐cellobioside and peptidoglycan. The pH and temperature profiles for catalytic activities were relatively similar for both the enzymes. Conclusion: The ChiA and EF0361 enzymes show a high degree of similarity in their catalytic activities although their hosts share environmental preferences only to some extent. Significance and Impact of the Study: This study contributes to an understanding of the chitinolytic activities by L. monocytogenes and Ent. faecalis. Detailed information on their chitinolytic systems will help define potential reservoirs in the natural environment and possible transmission routes into food‐manufacturing plants.