Molecular modeling study on the differential microtubule-stabilizing effect in singly- and doubly-bonded complexes with peloruside A and paclitaxel

Microtubules (MT) are dynamic cytoskeletal components that play a crucial role in cell division. Disrupting MT dynamics by MT stabilizers is a widely employed strategy to control cell proliferation in cancer therapy. Most MT stabilizers bind to the taxol (TX) site located at the luminal interface between protofilaments, except laulimalide and peloruside A (PLA), which bind to an interfacial pocket on outer MT surface. Cryo-electron microscopy MTs reconstructions have shown differential structural effects on the MT lattice in singly- and doubly-bonded complexes with PLA, TX, and PLA/TX, as PLA is able to revert the lattice heterogeneity induced by TX association leading to more regular MT assemblies. In this work, fully-atomistic molecular dynamics simulations were employed to examine the single and double association of MT stabilizers to reduced MT models in the search for structural and energetic evidence that could be related to the differential regularization and stabilization effects exerted by PLA and TX on the MT lattice. Our results revealed that the double association of PLA/TX (a) strengthens the lateral contact between tubulin dimers compared to singly-bonded complexes, (b) favors a more parallel arrangement between tubulin dimers, and (c) induces a larger restriction in the interdimeric conformational motion increasing the probability of finding structures consistent with 13-protofilaments arrangements. These results and are valuable to increase understanding about the molecular mechanism of action of MT stabilizers, and could account for an overstabilization of MTs in doubly-bonded complexes compared to singly-bonded systems.     

Antimicrobial activity of flavonoids extracted from bergamot (Citrus bergamia Risso) peel, a byproduct of the essential oil industry

AIMS: To evaluate the antimicrobial properties of flavonoid-rich fractions derived from bergamot peel, a byproduct from the Citrus fruit processing industry and the influence of enzymatic deglycosylation on their activity against different bacteria and yeast. METHODS AND RESULTS: Bergamot ethanolic fractions were tested against Gram-negative bacteria (Escherichia coli, Pseudomonas putida, Salmonella enterica), Gram-positive bacteria (Listeria innocua, Bacillus subtilis, Staphylococcus aureus, Lactococcus lactis) and the yeast Saccharomyces cerevisiae. Bergamot fractions were found to be active against all the Gram-negative bacteria tested, and their antimicrobial potency increased after enzymatic deglycosylation. The minimum inhibitory concentrations of the fractions and the pure flavonoids, neohesperidin, hesperetin (aglycone), neoeriocitrin, eriodictyol (aglycone), naringin and naringenin (aglycone), were found to be in the range 200 to 800 microg ml(-1). The interactions between three bergamot flavonoids were also evaluated. CONCLUSION: The enzyme preparation Pectinase 62L efficiently converted common glycosides into their aglycones from bergamot extracts, and this deglycosylation increased the antimicrobial potency of Citrus flavonoids. Pairwise combinations of eriodictyol, naringenin and hesperetin showed both synergistic and indifferent interactions that were dependent on the test indicator organism. SIGNIFICANCE AND IMPACT OF THE STUDY: Bergamot peel is a potential source of natural antimicrobials that are active against Gram-negative bacteria.     

Climate warming and agricultural stressors interact to determine stream macroinvertebrate community dynamics

Global climate change is likely to modify the ecological consequences of currently acting stressors, but potentially important interactions between climate warming and land‐use related stressors remain largely unknown. Agriculture affects streams and rivers worldwide, including via nutrient enrichment and increased fine sediment input. We manipulated nutrients (simulating agricultural run‐off) and deposited fine sediment (simulating agricultural erosion) (two levels each) and water temperature (eight levels, 0–6°C above ambient) simultaneously in 128 streamside mesocosms to determine the individual and combined effects of the three stressors on macroinvertebrate community dynamics (community composition and body size structure of benthic, drift and insect emergence assemblages). All three stressors had pervasive individual effects, but in combination often produced additive or antagonistic outcomes. Changes in benthic community composition showed a complex interplay among habitat quality (with or without sediment), resource availability (with or without nutrient enrichment) and the behavioural/physiological tendency to drift or emerge as temperature rose. The presence of sediment and raised temperature both resulted in a community of smaller organisms. Deposited fine sediment strongly increased the propensity to drift. Stressor effects were most prominent in the benthic assemblage, frequently reflected by opposite patterns in individuals quitting the benthos (in terms of their propensity to drift or emerge). Of particular importance is that community measures of stream health routinely used around the world (taxon richness, EPT richness and diversity) all showed complex three‐way interactions, with either a consistently stronger temperature response or a reversal of its direction when one or both agricultural stressors were also in operation. The negative effects of added fine sediment, which were often stronger at raised temperatures, suggest that streams already impacted by high sediment loads may be further degraded under a warming climate. However, the degree to which this will occur may also depend on in‐stream nutrient conditions.     

Predicting the response to simultaneous selection: genetic architecture and physiological constraints

A great deal is known about the evolutionary significance of body size and development time. They are determined by the nonlinear interaction of three physiological traits: two hormonal events and growth rate (GR). In this study we investigate how the genetic architecture of the underlying three physiological traits affects the simultaneous response to selection on the two life-history traits in the hawkmoth Manduca sexta. The genetic architecture suggests that when the two life-history traits are both selected in the same direction (to increase or decrease) the response to selection is primarily determined by the hormonal mechanism. When the life-history traits are selected in opposite directions (one to increase and one to decrease) the response to selection is primarily determined by factors that affect the GR. To determine how the physiological traits affect the response to selection of the life-history traits, we simulated the predicted response to 10 generations of selection. A total of 83% of our predictions were supported by the simulation. The main components of this physiological framework also exist in unicellular organisms, vertebrates, and plants and can thus provide a robust framework for understanding how underlying physiology can determine the simultaneous evolution of life-history traits.     

Exo-beta-D-glucosaminidase from Amycolatopsis orientalis: catalytic residues, sugar recognition specificity, kinetics, and synergism

Catalytic residues and the mode of action of the exo-beta-D-glucosaminidase (GlcNase) from Amycolatopsis orientalis were investigated using the wild-type and mutated enzymes. Mutations were introduced into the putative catalytic residues resulting in five mutated enzymes (D469A, D469E, E541D, E541Q, and S468N/D469E) that were successfully produced. The four single mutants were devoid of enzymatic activity, indicating that Asp469 and Glu541 are essential for catalysis as predicted by sequence alignments of enzymes belonging to GH-2 family. When mono-N-acetylated chitotetraose [(GlcN)3-GlcNAc] was hydrolyzed by the enzyme, the nonreducing-end glucosamine unit was produced together with the transglycosylation products. The rate of hydrolysis of the disaccharide, 2-amino-2-deoxy-D-glucopyranosyl 2-acetamido-2-deoxy-D-glucopyranose (GlcN-GlcNAc), was slightly lower than that of (GlcN)2, suggesting that N-acetyl group of the sugar residue located at (+1) site partly interferes with the catalytic reaction. The time-course of the enzymatic hydrolysis of the completely deacetylated chitotetraose [(GlcN)4] was quantitatively determined by high-performance liquid chromatography (HPLC) and used for in silico modeling of the enzymatic hydrolysis. The modeling study provided the values of binding free energy changes of +7.0, -2.9, -1.8, -0.9, -1.0, and -0.5 kcal/mol corresponding, respectively, to subsites (-2), (-1), (+1), (+2), (+3), and (+4). When chitosan polysaccharide was hydrolyzed by a binary enzyme system consisting of A. orientalis GlcNase and Streptomyces sp. N174 endochitosanase, the highest synergy in the rate of product formation was observed at the molar ratio 2:1. Thus, GlcNase would be an efficient tool for industrial production of glucosamine monosaccharide.     

Functional characterization of a bacterial expansin from Bacillus subtilis for enhanced enzymatic hydrolysis of cellulose

Expansin is a plant protein family that induces plant cell wall‐loosening and cellulose disruption without exerting cellulose‐hydrolytic activity. Expansin‐like proteins have also been found in other eukaryotes such as nematodes and fungi. While searching for an expansin produced by bacteria, we found that the BsEXLX1 protein from Bacillus subtilis had a structure that was similar to that of a β‐expansin produced by maize. Therefore, we cloned the BsEXLX1 gene and expressed it in Escherichia coli to evaluate its function. When incubated with filter paper as a cellulose substrate, the recombinant protein exhibited both cellulose‐binding and cellulose‐weakening activities, which are known functions of plant expansins. In addition, evaluation of the enzymatic hydrolysis of filter paper revealed that the recombinant protein also displayed a significant synergism when mixed with cellulase. By comparing the activity of a mixture of cellulase and the bacterial expansin to the additive activity of the individual proteins, the synergistic activity was found to be as high as 240% when filter paper was incubated with cellulase and BsEXLX1, which was 5.7‐fold greater than the activity of cellulase alone. However, this synergistic effect was observed when only a low dosage of cellulase was used. This is the first study to characterize the function of an expansin produced by a non‐eukaryotic source. Biotechnol. Bioeng. 2009;102: 1342–1353. © 2008 Wiley Periodicals, Inc.     

苦豆子提取物及其7种生物碱单体对埃玛菌素的增效作用

以苦豆子（Sophora alopecuroids L．）种子的甲醇提取物、水提取物及其7种生物碱单体和小菜蛾（Plutella xylostella）幼虫为试材,对生物源杀虫剂埃玛菌素进行增效作用研究。结果表明,7种生物碱单体对埃玛菌素增效比值为1．013～1．565。其中,苦豆碱和槐胺碱的增效作用最为明显,增效比值分别为1．565和1．413;而苦参碱和槐定碱对埃玛菌素的增效作用较差,增效比值仅为1．138和1．013,这2种生物碱单体对埃玛菌素仅表现出联合作用。苦豆子甲醇提取物和水提取物对埃玛菌素增效活性大于各生物碱单体,增效比值分别为3．443和2．027。     

新疆北部马铃薯甲虫幼虫对常规杀虫剂的抗药性（英文）

为了了解并掌握马铃薯甲虫Leptinotarsa decemlineata (Say)在新疆的抗性水平及动态, 为科学用药提供理论依据, 利用点滴法测定了新疆北部马铃薯甲虫6个田间种群(乌鲁木齐、 阜康、 昌吉、 阿勒泰、 尼勒克和特克斯) 的4龄幼虫对14种常规药剂的敏感性, 并测试了磷酸三苯酯（TPP）、 顺丁烯二酸二乙酯（DEM）和氧化胡椒基丁醚（PBO）的增效作用。结果表明: 与已报道的基线相比, 特克斯种群对几种药剂都敏感, 因此确定特克斯田间种群为敏感品系。与特克斯敏感品系相比, 昌吉种群对三氟氯氰菊酯、 溴氰菊酯、 克百威及丁硫克百威的抗性倍数分别为108.8, 30, 17.6和24.7, 而尼勒克种群对这4种药剂的抗性倍数分别为29.4, 42.5, 10.1和89.9。此外, 昌吉和阜康种群对高效氯氰菊酯也分别产生8.9和13.0倍的中等抗性。增效测定结果显示: 对于尼勒克种群, PBO对三氟氯氰菊酯有明显增效作用; 对于昌吉种群, PBO, TPP和DEM对克百威有明显增效作用。因此推断解毒酶活性增强可能是新疆马铃薯甲虫对三氟氯氰菊酯和克百威的抗性机制之一。由于增效作用不能完全消除抗性, 因此新疆马铃薯甲虫的抗药性还可能涉及到其他机制。     

Synergistic responses of the chorda tympani to mixtures of umami and sweet substances in rats

It has been known that umami substances such as monosodium L-glutamate (MSG) and 5'-inosine monophosphate (IMP) elicit a unique taste called 'umami' in humans. One of the characteristics of the umami taste is synergism: the synergistic enhancement of the magnitude of response produced by the addition of 5'-ribonucleotides to MSG. In addition to this well-documented synergism, we report here for the first time on another type of synergism between a glutamate receptor agonist, L-AP4, and sweet substances, by analyzing the chorda tympani responses in rats. The results are as follows: (i) when L-AP4 was mixed with one of the sweet substances, such as sucrose, glucose, fructose and maltose, large synergistic responses were observed. (ii) These synergistic responses, except to L-AP4 + sucrose, were not suppressed by sweet taste suppressants, gurmarin and pronase E. (iii) These synergistic responses were not suppressed by either metabotropic or ionotropic glutamate receptor antagonists. (iv) Fibers that responded well to the binary mixtures of L-AP4 and sweet substances also responded well to NaCl and HCl, but very weakly to sucrose. These findings are different from the characteristics of synergism between glutamate and IMP. The multiple transduction mechanisms for the umami taste in rat taste cells are discussed.