Analysis of Petal Anthocyanins to Investigate Flower Coloration of Zhongyuan (Chinese) and Daikon Island (Japanese) Tree Peony Cultivars

Pn, Pg; Pn, Pg > Cy ; Pn, Cy and Pn, Cy > Pg groups. Each group consequently specified significant features among CIELAB color notation and petal pigmentation, being adequate to characterize tree peony flowers as similar between Zhongyuan and Daikon Island cultivars, thus the cultivars of the two areas are suggested to be related to one another. Received 25 April 2000/ Accepted in revised form 21 December 2000     

Development,Growth and Survivorship of Juvenile Calanoid Copepods on Diets of Cyanobacteria and Algae

Nauplii and immature copepodites of Boeckella triarticulata, B. hamata and B. dilatata were tested for their ability to survive and grow on monospecific diets of four species of filamentous cyanobacteria, Cyclotella (Cy) and Cryptomonas (Cr) at 2 mg dry weight 1⁻¹. Cr supported the development from nauplius to adult of all three calanoid species; complete development on other diets occurred only in B. dilatata on Cy and Anabaena oscillarioides (Ao). Differences between nauplii and copepodites of B. dilatata and B. hamata in survivorship and development on diets of Cy, Anabaena flos-aquae (Af), Ao and Nostoc sp. 2 (N2) imply ontogenetic shifts in resource utilization with developmental phase. Ranked in order of their ability to support the development of juvenile boeckellids the foods were Cr > Cy > Ao > N2 > Af > Nostoc calcicola = no food.     

The 8-oxoguanine DNA N-glycosylase 1 (hOGG1) Ser326Cys variant affects the susceptibility to Graves' disease

Oxidative DNA damage, caused by either endogenous or exogenous sources of reactive oxygen species (ROS), has been linked several diseases including Graves' disease (GD). 7,8‐Dihydro‐8‐oxoguanine (8‐oxoG) is a major lesion produced by ROS and is considered a key biomarker of oxidative DNA damage. In humans, 8‐oxoG is mainly repaired by 8‐oxoguanine DNA N‐glycosylase‐1 (hOGG1), which is an essential component of the base excision repair (BER) pathway. The functional studies showed that hOGG1 Ser326Cys polymorphism is associated with the reduced DNA repair activity and increased risk for some oxidative stress‐related diseases. In this study, we firstly investigated hOGG1 Ser326Cys polymorphism in GD. According to our results, Cys/Cys genotype frequency in the GD patients (23.4%) was significantly higher than the controls (9.2%). Cys/Cys genotype had an 3.5‐fold [95% CI (confidence interval): 2.10–6.01, p < 0.001] the Cys allele had 1.83‐fold (95% CI: 1.43–2.34, p < 0.001) increase in the risk for developing GD. Our results suggest that Ser326Cys polymorphism of the hOGG1 gene is associated with GD risk. Copyright © 2011 John Wiley & Sons, Ltd.     

Species Concepts in the Cylindrocladium floridanum and Cy. spathiphylli Complexes (Hypocreaceae) Based on Multi-allelic Sequence Data, Sexual Compatibility and Morphology

Much attention has recently been devoted to the delimitation of species units in Cylindrocladium(Cy.). In this regard the present study focuses on the taxa within the unresolved Cy. floridanum and Cy. spathiphylli species complexes. Maximum parsimony analyses of DNA sequences of ITS, β-tubulin and histone regions of rRNA genes, and mating experiments revealed a geographically isolated species of Cylindrocladium in the Cy. spathiphylli(teleomorph:Calonectria spathiphylli) species complex.Cy. pseudospathiphylli sp. nov. (teleomorph:Ca. pseudospathiphylli sp. nov.) is described as a new phylogenetic, biological and morphological species. It is distinguished from Cy. spathiphylli by being homothallic, having smaller macroconidia, and distinct DNA sequences of β-tubulin and histone genes. Similarly, parsimony analysis of a combined data set also indicated several phylogenetic species to exist within Cy. floridanum(teleomorph:Ca. kyotensis). Based on differences in vesicle morphology and conidium dimensions, the Canadian population of Cy. floridanum, formerly known as Cy. floridanum Group 2, is described as Cy. canadense sp. nov., while a further collection from Hawaii is described as Cy. pacificum sp. nov.     

The chitin‐binding capability of Cy‐AMP1 from cycad is essential to antifungal activity

Antimicrobial peptides are important components of the host innate immune responses by exerting broad‐spectrum microbicidal activity against pathogenic microbes. Cy‐AMP1 found in the cycad (Cycas revoluta) seeds has chitin‐binding ability, and the chitin‐binding domain was conserved in knottin‐type and hevein‐type antimicrobial peptides. The recombinant Cy‐AMP1 was expressed in Escherichia coli and purified to study the role of chitin‐binding domain. The mutants of Cy‐AMP1 lost chitin‐binding ability completely, and its antifungal activity was markedly decreased in comparison with native Cy‐AMP1. However, the antimicrobial activities of the mutant peptides are nearly identical to that of native one. It was suggested that the chitin‐binding domain plays an essential role in antifungal, but not antimicrobial, activity of Cy‐AMP1. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Characterization of intermolecular interaction between cyanidin‐3‐glucoside and bovine serum albumin: Spectroscopic and molecular docking methods

The intermolecular interaction between cyanidin‐3‐glucoside (Cy‐3‐G) and bovine serum albumin (BSA) was investigated using fluorescence, circular dichroism and molecular docking methods. The experimental results revealed that the fluorescence quenching of BSA at 338 nm by Cy‐3‐G resulted from the formation of Cy‐3‐G–BSA complex. The number of binding sites (n) for Cy‐3‐G binding on BSA was approximately equal to 1. The experimental and molecular docking results revealed that after binding Cy‐3‐G to BSA, Cy‐3‐G is closer to the Tyr residue than the Trp residue, the secondary structure of BSA almost not change, the binding process of Cy‐3‐G with BSA is spontaneous, and Cy‐3‐G can be inserted into the hydrophobic cavity of BSA (site II′) in the binding process of Cy‐3‐G with BSA. Moreover, based on the sign and magnitude of the enthalpy and entropy changes (ΔH⁰ = – 29.64 kcal/mol and ΔS⁰ = – 69.51 cal/mol K) and the molecular docking results, it can be suggested that the main interaction forces of Cy‐3‐G with BSA are Van der Waals and hydrogen bonding interactions. Copyright © 2013 John Wiley & Sons, Ltd.     

Structural basis for the modulation of plant cytosolic triosephosphate isomerase activity by mimicry of redox‐based modifications

Reactive oxidative species (ROS) and S‐glutathionylation modulate the activity of plant cytosolic triosephosphate isomerases (cTPI). Arabidopsis thaliana cTPI (AtcTPI) is subject of redox regulation at two reactive cysteines that function as thiol switches. Here we investigate the role of these residues, AtcTPI‐Cys13 and At‐Cys218, by substituting them with aspartic acid that mimics the irreversible oxidation of cysteine to sulfinic acid and with amino acids that mimic thiol conjugation. Crystallographic studies show that mimicking AtcTPI‐Cys13 oxidation promotes the formation of inactive monomers by reposition residue Phe75 of the neighboring subunit, into a conformation that destabilizes the dimer interface. Mutations in residue AtcTPI‐Cys218 to Asp, Lys, or Tyr generate TPI variants with a decreased enzymatic activity by creating structural modifications in two loops (loop 7 and loop 6) whose integrity is necessary to assemble the active site. In contrast with mutations in residue AtcTPI‐Cys13, mutations in AtcTPI‐Cys218 do not alter the dimeric nature of AtcTPI. Therefore, modifications of residues AtcTPI‐Cys13 and AtcTPI‐Cys218 modulate AtcTPI activity by inducing the formation of inactive monomers and by altering the active site of the dimeric enzyme, respectively. The identity of residue AtcTPI‐Cys218 is conserved in the majority of plant cytosolic TPIs, this conservation and its solvent‐exposed localization make it the most probable target for TPI regulation upon oxidative damage by reactive oxygen species. Our data reveal the structural mechanisms by which S‐glutathionylation protects AtcTPI from irreversible chemical modifications and re‐routes carbon metabolism to the pentose phosphate pathway to decrease oxidative stress.     

Pertussis toxin modulation of sodium channels in the central neurons of cyhalothrin-resistant and cyhalothrin-susceptible cotton bollworm, Helicoverpa armigera

Pertussis toxin （FIX） inhibits the activation of the α-subunit of the inhibitory heterotrimeric G-proteins （Cαi/o） and modulates voltage-gated sodium channels, which may be one of the primary targets of pyrethroids. To investigate the potential mechanisms of agricultural pests resistance to pyrethroid insecticides, we examined the modulations by PTX on sodium channels in the central neurons of the 3rd-4th instar larvae of cyhalothrin-resistant （Cy-R） and cyhaiothrin-susceptible （Cy-S） Helicoverpa armigera by the whole-cell patch-clamp technique. The isolated neurons were cultured for 12-16 h in an improved L15 insect culture medium with or without PTX （400 ng/mL）. The results showed that both the Cy-R and Cy-S sodium channels exhibited fast kinetics and tetrodotoxin （TTX） sensitivity. The Cy-R sodium channels exhibited not only altered gating properties, including a 8.88-mV right shift in voltage-dependent activation （V0.5act） and a 6.54-mV right shift in voltage-dependent inactivation （V0.5inact）, but also a reduced peak in sodium channel density （Ⅰdensity） （55.2% of that in Cy-S neurons）. Cy-R sodium channels also showed low excitability, as evidenced by right shift of activation potential （Ⅴacti） by 5-10 mV and peak potential （Ⅴpcak） by 20 mV. FIX exerted significant effects on Cy-S sodium channels, reducing sodium channel density by 70.04%, right shifting V0.5act by 14.41 mV and V0.5inact by 9. 38 mV. It did not cause any significant changes of the parameters mentioned above in the Cy-R sodium channels. The activation time （Tpeak） from latency to peak at peak voltage and the fast inactivation time constant （τinact） in both Cy-S and Cy-R neurons were not affected. The results suggest that cotton bollworm resistant to pyrethroid insecticides involves not only mutations and allosteric alterations of voltage-gated sodium channels, but also might implicate perturbation of PTX-sensitive Gαi/o-COupled signaling Wansduction pathways.     

Computational investigation of the histidine ammonia-lyase reaction: a modified loop conformation and the role of the zinc(II) ion

Possible reaction intermediates of the histidine ammonia-lyase (HAL) reaction were investigated within the tightly closed active site of HAL from Pseudomonas putida (PpHAL). The closed structure of PpHAL was derived from the crystal structure of PpHAL inhibited with l-cysteine, in which the 39–80 loop including the catalytically essential Tyr53 was replaced. This modified loop with closed conformation was modeled using the structure of phenylalanine ammonia-lyase from Anabaena variabilis (AvPAL) with a tightly closed active site as a template. Three hypothetical structures of the covalently bound intermediate in the PpHAL active site were investigated by conformational analysis. The distances between the acidic pro-S β-hydrogen of the ligand and the appropriate oxygen atoms of Tyr53, Ty280 and Glu414 − which may act as enzymic bases − in the conformations of the three hypothetical intermediate structures were analyzed together with the substrate and product arrangements. The calculations indicated that the most plausible HAL reaction pathway involves the N-MIO intermediate structure in which the L-histidine substrate is covalently bound to the N-3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) prosthetic group of the apoenzyme via the amino group. Density functional theory (DFT) calculations − on a truncated model of the N-MIO intermediate containing a Zn²⁺ ion coordinated to the imidazole ring of the ligand and to His83, Met382 and a water molecule − indicated that Zn-complex formation plays a role in the reactivity and substrate specificity of HAL.     

Comparative trophic impacts of two globally invasive cyprinid fishes reveal species‐specific invasion consequences for a threatened native fish

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Purification, characterization, and sequencing of antimicrobial peptides, Cy-AMP1, Cy-AMP2, and Cy-AMP3, from the Cycad (Cycas revoluta) seeds

Novel antimicrobial peptides (AMP), designated Cy-AMP1, Cy-AMP2, and Cy-AMP3, were purified from seeds of the cycad (Cycas revoluta) by a CM cellulofine column, ion-exchange HPLC on SP COSMOGEL, and reverse-phase HPLC. They had molecular masses of 4583.2 Da, 4568.9 Da and 9275.8 Da, respectively, by MALDI–TOF MS analysis. Half of the amino acid residues of Cy-AMP1 and Cy-AMP2 were cysteine, glycine and proline, and their sequences were similar. The sequence of Cy-AMP3 showed high homology to various lipid transfer proteins. For Cy-AMP1 and Cy-AMP2, the concentrations of peptides required for 50% inhibition (IC₅₀) of the growth of plant pathogenic fungi, Gram-positive and Gram-negative bacteria were 7.0–8.9 μg/ml. The Cy-AMP3 had weak antimicrobial activity. The structural and antimicrobial characteristics of Cy-AMP1 and Cy-AMP2 indicated that they are a novel type of antimicrobial peptide belonging to a plant defensin family.     

Uniform Band Intensities in Fluorescent Dye Terminator Sequencing

Abstract

The use of Cyanine dye (Cy^TM 5 and Cy5.5) labeled dideoxy terminators with Thermo Sequenase^TM DNA polymerase in DNA sequencing provides uniform band intensity, improved sequence read-length, and accuracy. It also greatly improves the ability to detect single base heterozygotes with dye-terminator sequencing method.     

Morphological and Ribosomal DNA‐based Characterization of Six Antarctic Ciliate Morphospecies from the Amundsen Sea with Phylogenetic Analyses

We characterized six tintinnid ciliates from Antarctic waters using molecular markers and morphological traits: Amphorellopsis quinquealata, Codonellopsis gaussi, Cymatocylis convallaria, Cy. calyciformis, Cy. drygalskii, and Laackmanniella prolongata. The 100% similarity in SSU‐ITS1‐5.8S rDNA‐ITS2‐partial LSU rDNA sequences among Cy. convallaria, Cy. calyciformis, and Cy. drygalskii is supportive of synonymy. Codonellopsis gaussi and L. prolongata also showed high levels of similarity in SSU rDNA (99.83%) and the D2 domain of LSU rDNA (95.77%), suggesting that they are closely related. Phylogenetic analysis placed Cymatocylis in the Rhabdonellidae, Amphorellopsis in the Tintinnidae and L. prolongata/Co. gaussi within the Dictyocystidae.     

High‐throughput profiling of N‐myristoylation substrate specificity across species including pathogens

One of the most critical modifications affecting the N‐terminus of proteins is N‐myristoylation. This irreversible modification affects the membrane‐binding properties of crucial proteins involved in signal transduction cascades. This cotranslational modification, catalyzed by N‐myristoyl transferase, occurs both in lower and higher eukaryotes and is a validated therapeutic target for several pathologies. However, this lipidation proves very difficult to be evidenced in vivo even with state‐of‐the‐art proteomics approaches or bioinformatics tools. A large part of N‐myristoylated proteins remains to be discovered and the rules of substrate specificity need to be established in each organism. Because the peptide substrate recognition occurs around the first eight residues, short peptides are used for modeling the reaction in vitro. Here, we provide a novel approach including a dedicated peptide array for high‐throughput profiling protein N‐myristoylation specificity. We show that myristoylation predictive tools need to be fine‐tuned to organisms and that their poor accuracy should be significantly enhanced. This should lead to strongly improved knowledge of the number and function of myristoylated proteins occurring in any proteome.     

Genetic evidence of a relationship between two maize transposable element systems: Cy and Mutator

Summary The Cy transposable element system is composed of two genetically defined elements: an rcy receptor element inserted at the Bronze-1 locus; and an independently segregating regulatory element, Cy. The Cy system is not functionally homologous to any of the non-Mutator transposable element systems. Evidence is presented that supports a relationship between the Cy system and the family of Mu1-homologous transposable elements that are responsible for the Mutator phenomenon. Although related, Cy elements and the Mu1-homologous elements are not identical; Cy is inherited in a near-Mendelian fashion, in contrast to the non-Mendelian inheritance of the Mu1-homologous elements.     

A Comparison of GFP‐Tagged Clathrin Light Chains with Fluorochromated Light Chains In Vivo and In Vitro

Clathrin triskelia consist of three heavy chains and three light chains (LCs). Green fluorescent protein (GFP)‐tagged LCs are widely utilized to follow the dynamics of clathrin in living cells, but whether they reflect faithfully the behavior of clathrin triskelia in cells has not been investigated yet thoroughly. As an alternative approach, we labeled purified LCs either with Alexa 488 or Cy3 dye and compared them with GFP‐tagged LC variants. Cy3‐labeled light chains (Cy3‐LCs) were microinjected into HeLa cells either directly or in association with heavy chains. Within 1–2 min the Cy3‐LC heavy chain complexes entered clathrin‐coated structures, whereas uncomplexed Cy3‐LC did not within 2 h. These findings show that no significant exchange of LCs occurs over the time–course of an endocytic cycle. To explore whether GFP‐tagged LCs behave functionally like endogenous LCs, we characterized them biochemically. Unlike wild‐type LCs, recombinant LCs with a GFP attached to either end did not efficiently inhibit clathrin assembly in vitro, whereas Cy3‐ and Alexa 488‐labeled LC behaved similar to wild‐type LCs in vitro and in vivo. Thus, fluorochromated LCs are a valuable tool for investigating the complex behavior of clathrin in living cells.     

Novel monoclonal antibodies broadly reactive to human recombinant sapovirus‐like particles

Sapovirus (SaV), a member of the family Caliciviridae, is an important cause of acute epidemic gastroenteritis in humans. Human SaV is genetically and antigenically diverse and can be classified into four genogroups (GI, GII, GIV, and GV) and 16 genotypes (7 GI [GI.1–7], 7 GII, [GII.1–7], 1 GIV and 1 GV), based on capsid sequence similarities. Monoclonal antibodies (MAbs) are powerful tools for examining viruses and proteins. PAI myeloma cells were fused with spleen cells from mice immunized with a single type of recombinant human SaV virus‐like particles (VLPs) (GI.1, GI.5, GI.6, GII.3, GIV, or GV). Sixty‐five hybrid clones producing MAbs were obtained. Twenty‐four MAbs were characterized by ELISA, according to their cross‐reactivity to each VLP (GI.1, GI.5, GI.6, GII.2, GII.3, GII.4, GII.7, GIV, and GV). The MAbs were classified by this method into: (i) MAbs broadly cross‐reactive to all GI, GII, GIV and GV strains; (ii) those reactive in a genogroup‐specific; and (iii) those reactive in a genotype‐specific manner. Further analysis of three broadly cross‐reactive MAbs with a competitive ELISA demonstrated that at least two different common epitopes are located on the capsid protein of human SaVs in the four genogroups. The MAbs generated and characterized in this study will be useful tools for further study of the antigenic and structural topography of the human SaV virion and for developing new diagnostic assays for human SaV.