Annexin 1 Is a Component of eATP-Induced Cytosolic Calcium Elevation in Arabidopsis thaliana Roots

Extracellular ATP (eATP) has long been established in animals as an important signalling molecule but this is less understood in plants. The identification of Arabidopsis thaliana DORN1 (Does Not Respond to Nucleotides) as the first plant eATP receptor has shown that it is fundamental to the elevation of cytosolic free Ca²⁺ ([Ca²⁺]_cyt) as a possible second messenger. eATP causes other downstream responses such as increase in reactive oxygen species (ROS) and nitric oxide, plus changes in gene expression. The plasma membrane Ca²⁺ influx channels involved in eATP-induced [Ca²⁺]_cyt increase remain unknown at the genetic level. Arabidopsis thaliana Annexin 1 has been found to mediate ROS-activated Ca²⁺ influx in root epidermis, consistent with its operating as a transport pathway. In this study, the loss of function Annexin 1 mutant was found to have impaired [Ca²⁺]_cyt elevation in roots in response to eATP or eADP. Additionally, this annexin was implicated in modulating eATP-induced intracellular ROS accumulation in roots as well as expression of eATP-responsive genes.     

Role of Methyl Salicylate on Oviposition Deterrence in Arabidopsis thaliana

Plants attacked by herbivores have evolved different strategies that fend off their enemies. Insect eggs deposited on leaves have been shown to inhibit further oviposition through visual or chemical cues. In some plant species, the volatile methyl salicylate (MeSA) repels gravid insects but whether it plays the same role in the model species Arabidopsis thaliana is currently unknown. Here we showed that Pieris brassicae butterflies laid fewer eggs on Arabidopsis plants that were next to a MeSA dispenser or on plants with constitutively high MeSA emission than on control plants. Surprisingly, the MeSA biosynthesis mutant bsmt1-1 treated with egg extract was still repellent to butterflies when compared to untreated bsmt1-1. Moreover, the expression of BSMT1 was not enhanced by egg extract treatment but was induced by herbivory. Altogether, these results provide evidence that the deterring activity of eggs on gravid butterflies is independent of MeSA emission in Arabidopsis, and that MeSA might rather serve as a deterrent in plants challenged by feeding larvae.     

Isolation and enzymatic characterization of the first reported hyaluronidase from Yak (Bos grunniens) testis

A novel hyaluronidase (BgHya1) from Yak Bos grunniens testis was isolated and shown to have comparatively high activity on sodium hyaluronate. However, surveys on BgHya1 are still limited. The enzyme was purified through gel filtration on Sephacryl S-100 and cation-exchange on SP Sepharose fast flow; the purity was confirmed by a reverse phase FPLC Shodex C₄ column. The specific activity of the purified BgHya1 was 20.4 U/mg assayed by the colorimetric method against 0.85 U/mg for the crude enzyme, representing a 24-fold purification. It was a monomeric protein of 55 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Sephacryl S-200. It exhibited maximum activity in the presence of 0.15 M NaCl at 37 °C, pH 3.8, and a specificity to sodium hyaluronate higher than that of chondroitin-4-sulfate, chondroitin-6-sulfate, and dermatan. The K _m value for BgHya1, using sodium hyaluronate as substrate, was 0.106 mg/mL. Activity of BgHya1 was inhibited mildly by Ca²⁺ and Fe²⁺, and significantly by Fe³⁺, Mg²⁺, EDTA, urea, heparin, and 0.5 M NaCl. It was not affected by Cu²⁺, Zn²⁺, Co²⁺, ascorbic acid, PMSF, DTT, glutathione (reduced), or L-cysteine. BgHya1 was shown to be heat unstable in the range of 4–45 °C. In terms of storage stability, 92% of the activity was retained after four weeks at 4 °C, and 58% at room temperature. In addition, adding BSA (1.0 mg/mL) to the enzyme sample prior to freezing resulted in complete retention of enzyme activity. This work yielded a high purity hyaluronidase, the first one isolated from Bos grunniens by-product.     

Effects of co-ion initial concentration ratio on removal of Pb<Superscript>2+</Superscript> from aqueous solution by modified sugarcane bagasse

A modified sugarcane bagasse (SCB) fixed bed column was used to remove Pb²⁺ from aqueous solution. To determine the optimal condition for Pb²⁺ separation, Ca²⁺ was chosen as the model interfering ion, and effects of Ca²⁺ and Pb²⁺ initial concentration ratio (C ₀ ^Ca : C ₀ ^Pb ) on the adsorption of Pb²⁺ were investigated. Results showed that adsorption amount ratio of Ca²⁺ and Pb²⁺ (q _e ^Ca : q _e ^Pb ) had a good linear relationship with C ₀ ^Ca : C ₀ ^Pb . Mass ratio of Pb²⁺ absorbed on the modified SCB was higher than 95% at C ₀ ^Ca : C ₀ ^Pb <1.95, illustrating that Pb²⁺ could be selectively removed from aqueous solution. To verify that, simulated waste water containing co-ions of K⁺, Na⁺, Cd²⁺ and Ca²⁺ was treated, and results showed that the equilibrium amount of Pb²⁺, K⁺, Na⁺, Cd²⁺ and Ca²⁺ adsorbed was 134.14, 0.083, 0.058, 1.28, and 1.28mg g^?1, respectively, demonstrating that the modified SCB could be used to remove Pb²⁺ from aqueous solution in the investigated range.     

Crystallographic studies on the site selectivity of Ca<Superscript>2+</Superscript>, K<Superscript>+</Superscript>, and Rb<Superscript>+</Superscript> ions within zeolite Y (Si/Al = 1.56)

The single-crystals of Ca²⁺, K⁺-exchanged zeolite Y, and Ca²⁺, Rb⁺-exchanged zeolite Y were prepared by using flow method with mixed ion-exchange solution, whose Ca(NO₃)₂:KNO₃ mole ratios were 1:1 (crystal 1) and 1:100 (crystal 2), and Ca(NO₃)₂:RbNO₃ mole ratios were 1:1 (crystal 3) and 1:100 (crystal 4), respectively, with a total concentration of 0.05 M. They were fully dehydrated by vacuum dehydration at 723 K and 1 × 10^?6 Torr for 2 days. Their crystals were determined by single-crystal synchrotron X-ray diffraction techniques in the cubic space group \(Fd \overline{3}\) m, respectively, and were refined to the final error indices R ₁/wR ₂ = 0.057/0.196, 0.073/0.223, 0.055/0.188, and 0.049/0.175 for crystals 1, 2, 3, and 4, respectively. In the structure of crystal 1 (|Ca₂₃K₂₉|[Si₁₁₇Al₇₅O₃₈₄]-FAU), 23 Ca²⁺ ions per unit cell occupy sites I, II′, and II; 29 K⁺ ions per unit cell are at sites II′, II, and III′. In the structure of crystal 2 (|Ca_18.5K₃₈|[Si₁₁₇Al₇₅O₃₈₄]-FAU), 18.5 Ca²⁺ ions per unit cell occupy sites I, I′, and II; 38 K⁺ ions are at sites I′, II, and III′. In the structure of crystal 3 (|Ca₂₇Rb₂₁|[Si₁₁₇Al₇₅O₃₈₄]-FAU), 27 Ca²⁺ ions per unit cell occupy sites I, II′, and II; 21 Rb⁺ ions per unit cell are at sites II′, II, and III. In the structure of crystal 4 (|Ca₁₈Rb₃₉|[Si₁₁₇Al₇₅O₃₈₄]-FAU), 18 Ca²⁺ ions per unit cell occupy sites I and II; 39 Rb⁺ ions per unit cell are at sites I′, II′, II, III, and III′. In the four crystals, the Ca²⁺ ion which has much smaller size and higher charge than other cations such as K⁺ and Rb⁺ energetically preferred at site I and so the first to be filled on it. Unlike Ca²⁺ ion, no K⁺ and Rb⁺ ions are found at site I, which are clearly less favorable for K⁺ and Rb⁺ ions.     

Specificity of Herbivore-Induced Hormonal Signaling and Defensive Traits in Five Closely Related Milkweeds (Asclepias spp.)

Despite the recognition that phytohormonal signaling mediates induced responses to herbivory, we still have little understanding of how such signaling varies among closely related species and may generate herbivore-specific induced responses. We studied closely related milkweeds (Asclepias) to link: 1) plant damage by two specialist chewing herbivores (milkweed leaf beetles Labidomera clivicolis and monarch caterpillars Danaus plexippus); 2) production of the phytohormones jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA); 3) induction of defensive cardenolides and latex; and 4) impacts on Danaus caterpillars. We first show that A. syriaca exhibits induced resistance following monarch herbivory (i.e., reduced monarch growth on previously damaged plants), while the defensively dissimilar A. tuberosa does not. We next worked with a broader group of five Asclepias, including these two species, that are highly divergent in defensive traits yet from the same clade. Three of the five species showed herbivore-induced changes in cardenolides, while induced latex was found in four species. Among the phytohormones, JA and ABA showed specific responses (although they generally increased) to insect species and among the plant species. In contrast, SA responses were consistent among plant and herbivore species, showing a decline following herbivore attack. Jasmonic acid showed a positive quantitative relationship only with latex, and this was strongest in plants damaged by D. plexippus. Although phytohormones showed qualitative tradeoffs (i.e., treatments that enhanced JA reduced SA), the few significant individual plant-level correlations among hormones were positive, and these were strongest between JA and ABA in monarch damaged plants. We conclude that: 1) latex exudation is positively associated with endogenous JA levels, even among low-latex species; 2) correlations among milkweed hormones are generally positive, although herbivore damage induces a divergence (tradeoff) between JA and SA; 3) induction of cardenolides and latex are not necessarily physiologically linked; and 4) even very closely related species show highly divergent induction, with some species showing strong defenses, hormonally-mediated induction, and impacts on herbivores, while other milkweed species apparently use alternative strategies to cope with insect attack.     

Complete mineralization of the antibiotic amoxicillin by electro-Fenton with a BDD anode

On April 30, 2014, the World Health Organization’s first global report on the presence of antibiotics in waters focused on their negative consequences, which may include the growth of microorganisms with antimicrobial resistance. The β-lactam antibiotic amoxicillin (AMX) is widely used in human and veterinary medicine, and it has been recently detected in sewage treatment plants and effluents. In this paper, the degradation of acidic aqueous solutions of AMX by electro-Fenton process has been studied at constant current. Experiments have been performed in an undivided cell equipped with a carbon-felt cathode and a Pt or boron-doped diamond (BDD) anode. In such systems, the organic molecules are mainly oxidized by hydroxyl radical (^?OH) simultaneously formed on the anode surface from water oxidation as well as in the bulk from Fenton’s reaction between Fe²⁺ catalyst and electrogenerated H₂O₂. The decay and mineralization of AMX was monitored by means of high performance liquid chromatography (HPLC) and TOC measurements. The evolution of the concentration of the final aliphatic carboxylic acids and inorganic ions like ammonium, nitrate and sulfate was assessed by HPLC and ion chromatography, respectively. The effect of the anode material, initial AMX concentration and current density was thoroughly studied. The AMX decay always followed a pseudo-first-order kinetics using either Pt or BDD, and the apparent rate constant increased with applied current. A quicker mineralization was reached with BDD because of the larger production of active ^?OH. The absolute rate constant between hydroxyl radical and AMX determined by the competition kinetics method using p-hydroxybenzoic acid as the reference compound was found to be (2.02 ± 0.01) × 10⁹ M^?1 s^?1.     

(R,R)-salen/salan-based polymer fluorescence sensors for Zn detection

(R,R)-salen-based polymer fluorescence sensor P-1 could be synthesized by the polymerization of 5,5′-(isoquinoline-5,8-diylbis(ethyne-2,1-diyl))-bis(3-tert-butyl-2-hydroxybenzaldehyde) (M-1) with (R,R)-1,2-diaminocyclohexane (M-2) via nucleophilic addition-elimination reaction, and (R,R)-salan-based polymer sensor P-2 could be obtained by the reduction reaction of P-1 with NaBH₄. The fluorescence response behaviors of two chiral polymers P-1 and P-2 on Zn²⁺ were investigated by fluorescence spectra. The fluorescence intensities of P-1 and P-2 can exhibit gradual enhancement upon addition of Zn²⁺. Compared with other cations, such as Na⁺, K⁺, Mg²⁺, Ca²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Ag⁺, Cd²⁺, Cr³⁺ and Pb²⁺, Zn²⁺ can lead to the pronounced fluorescence enhancement as high as 22.8-fold for P-1 and 3.75-fold for P-2, respectively. The results show that P-1 and P-2 incorporating (R,R)-salen/salan moieties as receptors in the polymer main chain backbone can exhibit high sensitivity and selectivity for Zn²⁺ detection.     

Synthesis and investigation of Ag-Cs-<Emphasis Type="Italic">X</Emphasis> (<Emphasis Type="Italic">X</Emphasis> = I,Br, Cl) glasses doped with Er<Superscript>3+</Superscript>

The glass formation and crystallization of ErI₃-doped melts in the Ag-Cs-Pb-X (X = I, Br, Cl) are investigated in the section corresponding to the cation ratio Ag: Cs: Pb = 59: 39: 2 with ErI₃ contents of 0.2–7.5 mol %. The glass transition, crystallization, and melting temperatures are determined for a number of compositions. The primary crystalline phases that precipitate from melts upon crystallization are identified. It is demonstrated that the iodide, bromide, and chloride glasses containing up to 0.5 mol % ErI₃ have the lowest crystallization ability. An increase in the ErI₃ content leads to the precipitation of unidentified erbium-containing phases from melts upon cooling and to an increase in the crystallization ability of glasses. The spectral and luminescence properties of the glass of the composition 52Ag · 17AgCl · 39CsBr · 2PbCl₂ + 0.5 mol % ErI₃ are studied in the range of the ⁴ I _13/2 → ⁴ I _15/2 transition of the Er³⁺ ion. It is found that the lifetime of the ⁴ I _13/2 → ⁴ I _15/2 level of the Er³⁺ ion is equal to 10 ms.     

Synthesis,characterization and in vitro antibacterial assessments of a novel modified poly[maleic anhydride-alt-acrylic acid]/acriflavine conjugate

Water-soluble maleic anhydride-containing poly[maleic anhydride-alt-acrylic acid] (poly(MA-alt-AA) or MAAA) copolymer was synthesized by free-radical chain polymerization reaction, in 1,4-dioxane in the presence of benzoyl peroxide, 0.1 %, as the radical initiator at 70 °C under a nitrogen atmosphere. The purified copolymer was then modified with an anti-external fungal and anti-cancer active agent, acriflavine (AF). The modification reaction was performed 48 h at 70 °C in dimethylformamide organic media, using triethylamine (TEA or Et₃N) as the catalyst. The modified or conjugated copolymer/drug couple was named as MAAA/AF. Detailed structural characterization of the copolymer (MAAA) and modified product (MAAA/AF) was carried out by Fourier Transform Infrared (FTIR) and Nuclear Magnetic Resonance (¹H-NMR and ¹³C-NMR). The obtained FTIR, ¹H-NMR, and ¹³C-NMR spectra confirmed that AF was successfully bound to the MAAA copolymer backbone by ring-opening reaction. Antimicrobial susceptibility of the MAAA, AF, and MAAA/AF was evaluated by Kirby Bauer Disc Diffusion method on Mueller–Hinton Agar using Enterecoccus faecium, Enterohaemorrhagic Escherichia coli (EHEC), Staphylococcus aureus and Listeria monocytogenes. Results obtained indicated that MAAA/AF had antibacterial activity on EHEC and S. aureus at 50, 40, and 30 µg. A mechanism for MAAA and AF was then also suggested for the conjugation reaction.     

Messages from the Other Side: Parasites Receive Damage Cues from their Host Plants

As sessile organisms, plants rely on their environment for cues indicating imminent herbivory. These cues can originate from tissues on the same plant or from different individuals. Since parasitic plants form vascular connections with their host, parasites have the potential to receive cues from hosts that allow them to adjust defenses against future herbivory. However, the role of plant communication between hosts and parasites for herbivore defense remains poorly investigated. Here, we examined the effects of damage to lupine hosts (Lupinus texensis) on responses of the attached hemiparasite (Castilleja indivisa), and indirectly, on a specialist herbivore of the parasite, buckeyes (Junonia coenia). Lupines produce alkaloids that act as defenses against herbivores that can be taken up by the parasite. We found that damage to lupine host plants by beet armyworm (Spodoptera exigua) significantly increased jasmonic acid (JA) levels in both the lupine host and parasite, suggesting uptake of phytohormones or priming of parasite defenses by using host cues. However, lupine host damage did not induce changes in alkaloid levels in the hosts or parasites. Interestingly, the parasite had substantially higher concentrations of JA and alkaloids compared to lupine host plants. Buckeye herbivores consumed more parasite tissue when attached to damaged compared to undamaged hosts. We hypothesize that increased JA due to lupine host damage induced higher iridoid glycosides in the parasite, which are feeding stimulants for this specialist herbivore. Our results demonstrate that damage to hosts may affect both parasites and associated herbivores, indicating cascading effects of host damage on multiple trophic levels.     

When Isolated at Full Receptivity,in Vitro Fertilized Wheat (Triticum aestivum,L.) Egg Cells Reveal [Ca2+]cyt Oscillation of Intracellular Origin

During in vitro fertilization of wheat (Triticum aestivum, L.) in egg cells isolated at various developmental stages, changes in cytosolic free calcium ([Ca²⁺]_cyt) were observed. The dynamics of [Ca²⁺]_cyt elevation varied, reflecting the difference in the developmental stage of the eggs used. [Ca²⁺]_cyt oscillation was exclusively observed in fertile, mature egg cells fused with the sperm cell. To determine how [Ca²⁺]_cyt oscillation in mature egg cells is generated, egg cells were incubated in thapsigargin, which proved to be a specific inhibitor of the endoplasmic reticulum (ER) Ca²⁺-ATPase in wheat egg cells. In unfertilized egg cells, the addition of thapsigargin caused an abrupt transient increase in [Ca²⁺]_cyt in the absence of extracellular Ca²⁺, suggesting that an influx pathway for Ca²⁺ is activated by thapsigargin. The [Ca²⁺]_cyt oscillation seemed to require the filling of an intracellular calcium store for the onset of which, calcium influx through the plasma membrane appeared essential. This was demonstrated by omitting extracellular calcium from (or adding GdCl₃ to) the fusion medium, which prevented [Ca²⁺]_cyt oscillation in mature egg cells fused with the sperm. Combined, these data permit the hypothesis that the first sperm-induced transient increase in [Ca²⁺]_cyt depletes an intracellular Ca²⁺ store, triggering an increase in plasma membrane Ca²⁺ permeability, and this enhanced Ca²⁺ influx results in [Ca²⁺]_cyt oscillation.     

Synthesis and crystal structure of a new oxohalide CdPb<Subscript>2</Subscript>O<Subscript>2</Subscript>Cl<Subscript>2</Subscript>

A new compound, CdPb₂O₂Cl₂, is synthesized by the method of solid-phase reactions. The compound has monoclinic symmetry, space group C2/m, a = 12.392(8) Å, b = 3.8040(14) Å, c = 7.658(5) Å, β = 122.64(5)°, and V = 304.0(3) ų. The structure contains one symmetrically independent position of the Pb²⁺ cation coordinated by three O^2? anions (Pb²⁺-O^2? = 2.29–2.34 Å) and five Cl^? anions (Pb²⁺-Cl^? = 3.35–3.57 Å). The Cd²⁺ cation has a symmetric coordination with the formation of two bonds Cd-O = 2.15 Å and four bonds Cd-Cl = 2.73 Å. The oxygen atom is tetrahedrally coordinated by three Pb²⁺ cations and one Cd²⁺ cation, which leads to the formation of oxo-centered heterometallic OPb₃Cd tetrahedra. The tetrahedra are linked together into chains through common Pb atoms and into layered complexes due to sharing of the equatorial Cd atoms. The chlorine atoms are located above the cavities of the oxo-centered layer.     

Composition and pH dependence on aggregation of SiO2–PVA suspension for the synthesis of porous SiO2–PVA nanocomposite

Porous monolithic SiO₂–poly(vinyl alcohol) (PVA) nanocomposites were fabricated by drying an SiO₂–PVA suspension. Depending on the amount of added PVA and pH value of the suspension, the Brunauer–Emmett–Teller surface areas, total pore volumes, and mean pore radii of the (100 ? x)SiO₂–xPVA (x = 0, 10, 20, 30 wt%) nanocomposites were 102–313 m² g^?1, 0.61–1.42 cm³ g^?1, and 8.1–14.7 nm, respectively. Some cracks were observed in the monolithic SiO₂–PVA nanocomposite, affected by the pore size. To elucidate crack generation, the correlation between the dispersion/aggregation in the SiO₂–PVA suspension and the pore size distribution of the nanocomposite was evaluated in terms of the added PVA amount and pH value. At x = 20 and pH 3, the SiO₂ particles and PVA aggregated in the suspension. The preparation of crack-free monolithic SiO₂–PVA nanocomposites was possible using the aggregated suspension owing to the low capillary force during drying because of the relatively large pores.     

Oxidizable Phenolic Concentrations Do Not Affect Development and Survival of <Emphasis Type="Italic">Paropsis Atomaria</Emphasis> Larvae Eating <Emphasis Type="Italic">Eucalyptus</Emphasis> Foliage

Insect folivores can cause extensive damage to plants. However, different plant species, and even individuals within species, can differ in their susceptibility to insect attack. Polyphenols that readily oxidize have recently gained attention as potential defenses against insect folivores. We tested the hypothesis that variation in oxidizable phenolic concentrations in Eucalyptus foliage influences feeding and survival of Paropsis atomaria (Eucalyptus leaf beetle) larvae. First we demonstrated that oxidizable phenolic concentrations vary both within and between Eucalyptus species, ranging from 0 to 61 mg.g^?1 DM (0 to 81% of total phenolics), in 175 samples representing 13 Eucalyptus species. Foliage from six individuals from each of ten species of Eucalyptus were then offered to batches of newly hatched P. atomaria larvae, and feeding, instar progression and mortality of the first and second instar larvae were recorded. Although feeding and survival parameters differed dramatically between individual plants, they were not influenced by the oxidizable phenolic concentration of leaves, suggesting that P. atomaria larvae may have effective mechanisms to deal with oxidizable phenolics. Larvae feeding on plants with higher nitrogen (N) concentrations had higher survival rates and reached third instar earlier, but N concentrations did not explain most of the variation in feeding and survival. The cause of variation in eucalypt herbivory by P. atomaria larvae is therefore still unknown, although oxidizable phenolics could potentially defend eucalypt foliage against other insect herbivores.     

Computational Analyses of the AtTPC1 (Arabidopsis Two-Pore Channel 1) Permeation Pathway

Two Pore Channels (TPCs) are cation-selective voltage- and ligand-gated ion channels in membranes of intracellular organelles of eukaryotic cells. In plants, the TPC1 subtype forms the slowly activating vacuolar (SV) channel, the most dominant ion channel in the vacuolar membrane. Controversial reports about the permeability properties of plant SV channels fueled speculations about the physiological roles of this channel type. TPC1 is thought to have high Ca²⁺ permeability, a conclusion derived from relative permeability analyses using the Goldman–Hodgkin–Katz (GHK) equation. Here, we investigated in computational analyses the properties of the permeation pathway of TPC1 from Arabidopsis thaliana. Using the crystal structure of AtTPC1, protein modeling, molecular dynamics (MD) simulations, and free energy calculations, we identified a free energy minimum for Ca²⁺, but not for K⁺, at the luminal side next to the selectivity filter. Residues D269 and E637 coordinate in particular Ca²⁺ as demonstrated in in silico mutagenesis experiments. Such a Ca²⁺-specific coordination site in the pore explains contradicting data for the relative Ca²⁺/K⁺ permeability and strongly suggests that the Ca²⁺ permeability of SV channels is largely overestimated from relative permeability analyses. This conclusion was further supported by in silico electrophysiological studies showing a remarkable permeation of K⁺ but not Ca²⁺ through the open channel.