Effect of polyacetylenic acids from Prunella vulgaris on various plant pathogens

Aims: This study is aiming at characterizing antifungal substances from the methanol extract of Prunella vulgaris and at investigating those substances’ antifungal and antioomycete activities against various plant pathogens. Methods and Results: Two polyacetylenic acids were isolated from P. vulgaris as active principles and identified as octadeca‐9,11,13‐triynoic acid and trans‐octadec‐13‐ene‐9,11‐diynoic acid. These two compounds inhibited the growth of Magnaporthe oryzae, Rhizoctonia solani, Phytophthora infestans, Sclerotinia sclerotiorum, Fusarium oxysporum f. sp. raphani, and Phytophthora capsici. In addition, these two compounds and the wettable powder‐type formulation of an n‐hexane fraction of P. vulgaris significantly suppressed the development of rice blast, tomato late blight, wheat leaf rust, and red pepper anthracnose. Conclusions: These data show that the extract of P. vulgaris and two polyacetylenic acids possess antifungal and antioomycete activities against a broad spectrum of tested plant pathogens. Significance and Impact of the Study: This is the first report on the occurrence of octadeca‐9,11,13‐triynoic acid and trans‐octadec‐13‐ene‐9,11‐diynoic acid in P. vulgaris and their efficacy against plant diseases. The crude extract containing the two polyacetylenic acids can be used as a natural fungicide for the control of various plant diseases.     

Effect of glycosylation on the protein pattern in 2-D-gel electrophoresis

Single proteins, when analyzed with 2-D-PAGE, often show multiple spots due to PTMs. In gels of human body fluids, the spot patterns facilitate the assignment and identification of the proteins. We analyzed serums from patients with congenital disorders of glycosylation (CDG) in which glycoproteins are strongly impacted and exhibit highly distinguishable spot patterns compared to healthy controls. We detected a typical protein pattern for alpha1-acid glycoprotein (AGP) and transferrin (Trf) that are markers for CDG. AGP contains five glycosylation sites which results in a complex microheterogeneity of the glycoprotein. On the other hand, in Trf, a glycoprotein with only two glycosylation sites, mainly biantennary complex-type-N-linked glycans are bound. We used 2-D-PAGE, MALDI-TOF-MS, and ESI-MS for the analysis of these glycoproteins and their corresponding glycans. In AGP, the heterogenic glycosylation of the different glycosylation sites is responsible for the complex spot pattern. In contrast to AGP, the protein spots of Trf cannot be explained by glycosylation. We found strong evidence that oxidation of cysteine is responsible for the spot pattern. This study contradicts the commonly accepted assumption that the multiple protein spots of Trf observed in 2-D-PAGE are due, as in AGP, to the glycosylation of the protein.     

Enthalpy change on mixing a couple of S- and R-enantiomers of some chiral compounds at 298.15 K

Enthalpy change on the mixing of R- and S-enantiomers of chiral liquid compounds such as dimethyl malate (1), methyl 3-hydroxylbutanoate (2), 2-butanol (3), ethyl 4-chloro-3-hydroxylbutanoate (4), 1,3,3-trimethylbicycle-[2.2.1]heptan-2-one (5), 3,7-dimethyl-6-octenal (6), and 8-bromo-2,6-dimethyl-2-octene (7) is measured over the entire range of mole fractions at 298.15 K, albeit very small values. The mixing of chiral liquids of R-1 + S-1, R-2 + S-2, R-3 + S-3, R-6 + S-6, and R-7 + S-7 produces enthalpic destabilization over the entire range of mole fractions, while that of R-4 + S-4 and R-5 + S-5 shows enthalpic stabilization over entire compositions. Enthalpy change on mixing at an equimolar concentration and the intermolecular interaction obtained by the molecular mechanics calculations show a linear correlation, except for a few compounds measured.     

Effect of dexamethasone on neutrophil metabolism

The effect of dexamethasone on glucose and glutamine metabolism was investigated. The consumption and oxidation of glucose and glutamine, and the production of glutamate and lactate were determined in neutrophils cultured for 3 h in the presence of dexamethasone. The activities and expression of glucose-6-phosphate dehydrogenase (G6PDH) and phosphate-dependent glutaminase were also determined under the same conditions. Addition of dexamethasone to the culture medium caused a significant increase of glucose consumption at 0.5 microm (123.9%) and 1.0 microm (78.3%) concentrations. In spite of this, however, glucose oxidation remained unchanged. The glucocorticoid did not change glutamine consumption but caused a significant increase of glutamate production and did not alter glutamine oxidation. Dexamethasone-treated neutrophils had a significant decrease of G6PDH activity and expression in particular at 1.0 microm concentration. Phosphate- dependent glutaminase activity was also decreased (about 34%) by dexamethasone treatment. A similar effect was observed on glutaminase expression as indicated by RT-PCR analysis. Thus, the effect of dexamethasone on neutrophil metabolism was particularly noticeable with respect to G6PDH and glutaminase activities where a decrease in the respective mRNA levels was demonstrated.     

Optimized selective N-methylation of peptides on solid support.

Peptides containing N(alpha)-methylamino acids exhibit interesting therapeutic profiles and are increasingly recognized as potentially useful therapeutics. Unfortunately, their synthesis is hampered by the high price and nonavailability of many N(alpha)-methylamino acids. An efficient and practical three-step procedure for selective N-methylation of peptides on solid support is described. The procedure was based on the well known solid-phase N-methylation of N(alpha)-arylsulfonyl peptides, which was improved by using dimethylsulfate and the less expensive DBU as base. Every step of the procedure, amine activation by an o-nitrobenzenesulfonyl group, selective N-methylation and removal of the sulfonamide group, was optimized in respect of time and economy. The described optimized three-step procedure is performed in 35 min without solvent changes, instead of 3 h. Tripeptides (Fmoc-Phe-MeXaa-Leu-OH) containing N-methylated common amino acids were also prepared using the optimized procedure to demonstrate its compatibility with these amino acids. The described procedure allows an efficient synthesis of N(alpha)-methylamino acid containing peptides in a very short time using Fmoc solid-phase peptide synthesis.     

Optimization of multiple-sequence alignment based on multiple-structure alignment

Routinely used multiple-sequence alignment methods use only sequence information. Consequently, they may produce inaccurate alignments. Multiple-structure alignment methods, on the other hand, optimize structural alignment by ignoring sequence information. Here, we present an optimization method that unifies sequence and structure information. The alignment score is based on standard amino acid substitution probabilities combined with newly computed three-dimensional structure alignment probabilities. The advantage of our alignment scheme is in its ability to produce more accurate multiple alignments. We demonstrate the usefulness of the method in three applications: 1) computing more accurate multiple-sequence alignments, 2) analyzing protein conformational changes, and 3) computation of amino acid structure-sequence conservation with application to protein-protein docking prediction. The method is available at http://bioinfo3d.cs.tau.ac.il/staccato/.     

Dose-dependent effects of the caspase inhibitor Q-VD-OPh on different apoptosis-related processes

The effects of the pan-caspase inhibitor Q-VD-OPh on caspase activity, DNA fragmentation, PARP cleavage, 7A6 exposition, and cellular adhesivity to fibronectin were analyzed in detail in three different apoptotic systems involving two cell lines (JURL-MK1 and HL60) and two apoptosis inducers (imatinib mesylate and suberoylanilide hydroxamic acid). Q-VD-OPh fully inhibited caspase-3 and -7 activity at 0.05 μM concentration as indicated both by the measurement of the rate of Ac-DEVD-AFC cleavage and anti-caspase immunoblots. Caspase-8 was also inhibited at low Q-VD-OPh concentrations. On the other hand, significantly higher Q-VD-OPh dose (10 μM) was required to fully prevent the cleavage of PARP-1. DNA fragmentation and disruption of the cell membrane functionality (Trypan blue exclusion test) were both prevented at 2 μM Q-VD-OPh while 10 μM inhibitor was needed to inhibit the drug-induced loss of cellular adhesivity to fibronectin which was observed in JURL-MK1 cells. The exposition of the mitochondrial antigen 7A6 occurred independently of Q-VD-OPh addition and may serve to the detection of cumulative incidence of the cells which have initiated the apoptosis. Our results show that Q-VD-OPh efficiency in the inhibition of caspase-3 activity and DNA fragmentation in the whole-cell environment is about two orders of magnitude higher than that of z-VAD-fmk. This difference is not due to a slow permeability of the latter through the cytoplasmic membrane.     

Enantioseparation of racemic 4-aryl-3,4-dihydro-2(1H)-pyrimidones on chiral stationary phases based on 3,5-dimethylanilides of N-(4-alkylamino-3,5-dinitro)benzoyl L-alpha-amino acids

Three novel chiral packing materials for high-performance liquid chromatography were prepared by covalently binding of (2S)-N-(3,5-dimethylphenyl)-2-[(4-chloro-3,5-dinitrophenyl)carbonylamino]propan-amide (7), (2S)-N-(3,5-dimethylphenyl)-2-[(4-chloro-3,5-dinitrophenyl)carbonylamino]-4-methylpentanamide (8), and (2S)-N-(3,5-dimethylphenyl)-2-[(4-chloro-3,5-dinitrophenyl)carbonyl-amino]-2-phenylacetamide (9) to aminopropyl silica. The resulting chiral stationary phases (CSPs 1-3) proved effective for the resolution of racemic 4-aryl-3,4-dihydro-2(1H)-pyrimidone derivatives (TR 1-14). The mechanism of their enantioselection, supported by the elution order of (S)-TR 13 and (R)-TR 13 and molecular modeling of the complex of the slower running (S)-TR 13 with CSP 1 is discussed.     

Structural investigation on WlaRG from Campylobacter jejuni: A sugar aminotransferase

Campylobacter jejuni is a Gram‐negative bacterium that represents a leading cause of human gastroenteritis worldwide. Of particular concern is the link between C. jejuni infections and the subsequent development of Guillain‐Barré syndrome, an acquired autoimmune disorder leading to paralysis. All Gram‐negative bacteria contain complex glycoconjugates anchored to their outer membranes, but in most strains of C. jejuni, this lipoglycan lacks the O‐antigen repeating units. Recent mass spectrometry analyses indicate that the C. jejuni 81116 (Penner serotype HS:6) lipoglycan contains two dideoxyhexosamine residues, and enzymological assay data show that this bacterial strain can synthesize both dTDP‐3‐acetamido‐3,6‐dideoxy‐d ‐glucose and dTDP‐3‐acetamido‐3,6‐dideoxy‐d ‐galactose. The focus of this investigation is on WlaRG from C. jejuni, which plays a key role in the production of these unusual sugars by functioning as a pyridoxal 5′‐phosphate dependent aminotransferase. Here, we describe the first three‐dimensional structures of the enzyme in various complexes determined to resolutions of 1.7 Å or higher. Of particular significance are the external aldimine structures of WlaRG solved in the presence of either dTDP‐3‐amino‐3,6‐dideoxy‐d ‐galactose or dTDP‐3‐amino‐3,6‐dideoxy‐d ‐glucose. These models highlight the manner in which WlaRG can accommodate sugars with differing stereochemistries about their C‐4′ carbon positions. In addition, we present a corrected structure of WbpE, a related sugar aminotransferase from Pseudomonas aeruginosa, solved to 1.3 Å resolution.     

Studies on the antioxidant activities of some new chromone compounds

Recent reviews evidence that the naturally occurring compounds containing the chromone skeleton exhibit antiradical activities, providing protection against oxidative stress. The antioxidant activities of 13 new synthesized chromonyl‐2,4‐thiazolidinediones, chromonyl‐2,4‐imidazolidinediones and chromonyl‐2‐thioxoimidzolidine‐4‐ones were evaluated using in vitro antioxidant assays, including superoxide anion radical (), hydroxyl radical (), 2,2‐diphenyl‐1‐picryl‐hydrazyl free radical (DPPH^?) scavenging capacity and total antioxidant capacity ferric ion reducing activity. Superoxide anion radical was produced using potassium superoxide/18‐crown‐6‐ether dissolved in dimethylsulfoxide, and the Fenton‐like reaction (Fe(II) + H₂O₂) was a generator of hydroxyl radicals. Chemiluminescence, spectrophotometry, electron paramagnetic resonance (EPR) and 5,5‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO) as the spin trap were the measurement techniques. The results showed that the majority of the chromone derivatives tested showed a strong scavenging effect towards free radicals, similar to the chemiluminescence reaction with superoxide anion radical with a high activity, inhibition of the DMPO‐OOH radical EPR signal (24–58%), the DMPO‐OH radical EPR signal (4–75%) and DPPH radical EPR signal (6–100%) at 1 mmol/L. Several of the examined compounds exhibited the high reduction potentials. The results obtained show that the new synthesized chromone derivatives may directly scavenger reactive oxygen species and thus may play a protective role against oxidative damage. Copyright © 2014 John Wiley & Sons, Ltd.     

Effect of bis-triazoles on a ribose-based fluorescent sensor

We synthesized two ribosyl-based fluorescent sensors. Both sensors have an anthracene as the fluorophore, but they differ in the recognition site for metal ions. One (3) has two ribosyl esters, and the other (6) has two triazole groups linked to two ribosyl esters. Among the metal ions examined in MeOH, compound 3 displayed a large chelation-enhanced fluorescence (CHEF) effect with Hg(2+) and Cu(2+) ions, and compound 6 displayed a large chelation-quenched fluorescence (CHQF) effect with Cu(2+) and Ni(2+) ions. The results demonstrated that the absence (sensor 3) and presence (sensor 6) of an incorporated bis-triazole group in a ribosyl-based fluorescent sensor conferred different preferences and distinct binding modes for metal ions.     

Liquid chromatographic resolution of 3-amino-1,4-benzodiazepin-2-ones on crown ether-based chiral stationary phases

3-Amino-5-phenyl (or 5-methyl)-1,4-benzodiazepin-2-ones, which are chiral precursors of anti-respiratory syncytial virus active agents, were resolved on three different chiral stationary phases (CSPs) based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid or (3,3'-diphenyl-1,1'-binaphthyl)-20-crown-6. Among the three CSPs, the CSP that is based on (3,3'-diphenyl-1,1'-binaphthyl)-20-crown-6 and containing residual silanol group-protecting n-octyl groups on the silica surface was found to be most effective with the use of 80% ethanol in water containing perchloric acid (10 mM) and ammonium acetate (1.0 mM) as a mobile phase. The separation factors (α) and resolutions (R(S) ) were in the range of 1.90-3.21 and 2.79-5.96, respectively. From the relationship between the analyte structure and the chromatographic resolution behavior, the chiral recognition mechanism on the CSP based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid was proposed to be different from that on the CSP based on (3,3'-diphenyl-1,1'-binaphthyl)-20-crown-6. In addition, the chromatographic resolution behavior of the most effective CSP was investigated as a function of the composition of aqueous mobile phase containing organic and acidic modifier and ammonium acetate.     

Systematical evaluation of the effects of sample collection procedures on low-molecular-weight serum/plasma proteome profiling

Blood is an ideal source for biomarker discovery. However, little has been done to address the effects of sampling, handling and storage procedures on serum/plasma proteomes. We used magnetic bead-based MALDI-TOF MS to systematically evaluate the influence of each procedure on low-molecular-weight serum/plasma proteome profiling on the basis of the whole spectra. We found that sampling procedures, including the selection of blood collection tubes and anticoagulants, variations in clotting time or time lag before centrifugation, and hemolysis, displayed significant effects on the proteomes. Moreover, serum and plasma were mutually incompatible for proteome comparison. By contrast, overnight fasting, handling procedures, including centrifugation speeds (1500 x g vs. 3000 x g) or time (15 min vs. 30 min), and storage conditions, such as at 4 degrees C or 25 degrees C for up to 24 h or at -80 degrees C for up to 3 months, and repeated freeze/thaw of up to ten cycles, had relatively minor effects on the proteomes based upon our analysis of about 100 peaks. We concluded that low-molecular-weight serum/plasma proteomes were diversely affected by sampling, handling and storage with most change from variations of sampling procedures. We therefore suggest the necessity of standardizing sampling procedure for proteome comparison and biomarker discovery.     

Regulation of integrin affinity on cell surfaces

Lymphocyte activation triggers adhesiveness of lymphocyte function-associated antigen-1 (LFA-1; integrin α(L)β(2)) for intercellular adhesion molecules (ICAMs) on endothelia or antigen-presenting cells. Whether the activation signal, after transmission through multiple domains to the ligand-binding αI domain, results in affinity changes for ligand has been hotly debated. Here, we present the first comprehensive measurements of LFA-1 affinities on T lymphocytes for ICAM-1 under a broad array of activating conditions. Only a modest increase in affinity for soluble ligand was detected after activation by chemokine or T-cell receptor ligation, conditions that primed LFA-1 and robustly induced lymphocyte adhesion to ICAM-1 substrates. By stabilizing well-defined LFA-1 conformations by Fab, we demonstrate the absolute requirement of the open LFA-1 headpiece for adhesiveness and high affinity. Interaction of primed LFA-1 with immobilized but not soluble ICAM-1 triggers energy-dependent affinity maturation of LFA-1 to an adhesive, high affinity state. Our results lend support to the traction or translational motion dependence of integrin activation.     

Comparative proteomics of oxidative stress response of Lactobacillus acidophilus NCFM reveals effects on DNA repair and cysteine de novo synthesis

Probiotic cultures encounter oxidative conditions during manufacturing, yet protein abundance changes induced by such stress have not been characterized for some of the most common probiotics and starters. This comparative proteomics investigation focuses on the response by Lactobacillus acidophilus NCFM to H₂O_2, simulating an oxidative environment. Bacterial growth was monitored by BioScreen and batch cultures were harvested at exponential phase for protein profiling of stress responses by 2D gel based comparative proteomics. Proteins identified in 19 of 21 spots changing in abundance due to H₂O₂ were typically related to carbohydrate and energy metabolism, cysteine biosynthesis, and stress. In particular, increased cysteine synthase activity may accumulate a cysteine pool relevant for protein stability, enzyme catalysis, and the disulfide‐reducing pathway. The stress response further included elevated abundance of biomolecules reducing damage such as enzymes from DNA repair pathways and metabolic enzymes with active site cysteine residues. By contrast, a protein‐refolding chaperone showed reduced abundance, possibly reflecting severe oxidative protein destruction that was not overcome by refolding. The proteome analysis provides novel insight into resistance mechanisms in lactic acid bacteria against reactive oxygen species and constitutes a valuable starting point for improving industrial processes, food design, or strain engineering preserving microorganism viability.     

Effect of the canopy complexity of trees on the rate of predation of insects

The effect of the complexity of tree canopies on the effectiveness of biological control of tree‐dwelling insect herbivores by predators has been neglected. A complex canopy provides a greater variety of resources than a simple canopy, which may result in an increase in the abundance of both herbivores and predators in complex canopies and a higher predation rate. On the other hand, it may be more difficult to locate prey in trees with a complex canopy, which may lower the predation rate. The main aim of this study is to determine the relationship between predation rate and canopy complexity. We have used a system consisting of lime trees, larvae of blowflies as prey and naturally occurring predators (mainly birds and ants). The complexity of the canopy of 12 of the lime trees was reduced by pruning, and the predation rate was determined by recording the fate of prey pinned on each of the trees for 30 min several times during the course of a year. The predation rate was negatively associated with canopy complexity. We compare these results with those reported in the literature and contrary to the widely held view conclude that a negative relationship between predation rate and canopy complexity is a common phenomenon and can be expected to be reported in studies not only on ambush but also on predators that actively search for prey.     

Effect of short-range forces on the length distribution of fibrous cytoskeletal proteins

The length distribution of cytoskeletal filaments is an important physical parameter, which can modulate physiological cell functions. In both eukaryotic and prokaryotic cells various biological cytoskeletal polymers form supramolecular structures due to short-range forces induced mainly by molecular crowding or cross linking proteins, but their in vivo length distribution remains difficult to measure. In general, based on experimental evidence and mathematical modeling of actin filaments in aqueous solutions, the steady state length distribution of fibrous proteins is believed to be exponential. We performed in vitro TIRF- and electron-microscopy to demonstrate that in the presence of short-range forces, which are an integral part of any living cell, the steady state length distributions of the eukaryotic cytoskeletal biopolymer actin, its prokaryotic homolog ParM and microtubule homolog FtsZ deviate from the classical exponential and are either double-exponential or Gaussian, as recent theoretical modeling predicts. Double exponential or Gaussian distributions opposed to exponential can change for example the visco-elastic properties of actin networks within the cell, influence cell motility by decreasing the amount of free ends at the leading edge of the cell or effect the assembly of FtsZ into the bacterial Z-ring thus modulating membrane constriction.     

Stereoselective effect of kynurenine enantiomers on the excretion of serotonin and its metabolite in rat urine

A solution of optically pure kynurenine (KYN), i.e., D ‐KYN or L ‐KYN, was administered intravenously to male Sprague‐Dawley rats (10 mg kg^?1 ml^?1). The time‐course of changes in the concentrations of urinary monoamines and their metabolites such as 5‐hydroxytryptamine (5‐HT), 5‐hydroxyindole acetic acid (5‐HIAA), dopamine, and 3‐methoxytyramine were investigated by reversed‐phase high‐performance liquid chromatography with electrochemical detection after precolumn derivatization with (2R)‐2,5‐dioxopyrrolidin‐1‐yl‐2,5,7,8‐tetramethyl‐6‐(tetrahydro‐2H‐pyran‐2‐yloxy)chroman‐2‐carboxylate (NPCA). We observed a stereoselective difference in the effects of the KYN enantiomers. Only D ‐KYN, not L ‐KYN, caused a significant increase in urinary 5‐HT levels within 30 min after its administration. With regard to the metabolites, urinary 3‐MT level was increased by D ‐KYN administration. On the other hand, no significant change in the DA level was observed after administration of either D ‐KYN or L ‐KYN. These results suggest that D ‐KYN could affect the activity of neuroactive amines, especially 5‐HT, in vivo. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Role of histamine in short- and long-term effects of methamphetamine on the developing mouse brain

With the rise in methamphetamine (MA) use among women of childbearing age, the potential consequences of MA exposure to the developing brain for cognition in adulthood is a major concern. Histamine might mediate these MA effects. Following MA administration in neonatal mice, histamine levels in brain were elevated and the hypothalamic-pituitary-adrenal axis was activated. Co-administration of MA with the H3 receptor agonist immepip antagonized these effects. The effects of MA on histamine levels and on hypothalamic-pituitary-adrenal axis activation at P20 were more pronounced in female than male mice. These sex differences could have contributed to the increased susceptibility of female mice to the detrimental long-term cognitive effects of MA and the H3/H4 antagonist thioperamide. Following behavioral testing, mice neonatally treated with MA or thioperamide showed reduced levels of the dendritic marker microtubule-associated protein 2 in the CA3 region of the hippocampus and the enthorhinal cortex. This was not seen in mice neonatally treated with immepip and MA who did not show cognitive impairments, suggesting that these brain areas might be particularly important for the long-term effects of MA on cognitive function. These data support a role for histamine in the effects of MA on the developing brain.     

Effect of operating variables on the yield of recombinant trypsinogen for a pulse-fed dilution-refolding reactor.

The inclusion body process route for manufacturing proteins offers distinct process advantages in terms of expression levels and the ease of initial inclusion body recovery. The efficiency of the refolding unit operation, however, does determine the overall economic feasibility of a process. Dilution refolding is the simplest and most extensively used refolding operation, although significant yield losses often occur due mainly to aggregation. Operating variables may have a significant effect on the degree of aggregation, but a systematic study has not been reported. This study investigates the effect of operating variables on the dilution refolding of solubilized r-trypsinogen inclusion bodies in a pulse-fed stirred reactor. Variables investigated were inclusion body washing, stirring speed, feed rate, concentration of solubilized r-trypsinogen, and concentration of urea during solubilization of the inclusion bodies. Additionally, the effect of baffles in the reactor was investigated. The yield of renatured r-trypsinogen varied between 12 +/- 0.2% and 21 +/- 1.0% depending on the specific combination of operating variables employed. It is clear that a suboptimal operating strategy can significantly reduce protein yield. In particular, we note that an increased intensity of mixing adversely affected yield in contrast to previous reports indicating that enhanced dispersion increases yield. We conclude that yield is determined not only by the efficiency of dispersion, but also by the local chemical environment of the protein as it folds, and the rate of change of this environment. This will be controlled by micromixing effects, and hence the intensity of agitation, in a complex manner requiring further characterization.