Transcriptional responses of detoxification genes to four plant allelochemicals in Aphis gossypii.

Aphis gossypii Glover (Hemiptera: Aphididae) can damage a variety of agricultural crops, so it is very important for cotton aphids to evolve adaptive mechanisms to various allelochemicals from host plants. Our results aim to provide a fundamental and rich resource for exploring aphid functional genes in A. gossypii. A transcriptome data set and five expression profile data sets of A. gossypii samples were analyzed by Illumina sequencing platform. In total, 53,763,866 reads were assembled into 1,963,516 contigs and 28,555 unigenes. Compared with the control, 619 genes were significantly up- or downregulated in the treatment group by 2-tridecanone. There were 516, 509, and 717 of differential expression genes in tannic acid, quercetin, and gossypol treatment groups, respectively. Furthermore, there were 4 of 54 putative cytochrome P450 genes and 1 of 7 putative carboxylesterases downregulated in all treatment groups by four plant allelochemicals. When aphids fed on 2-tridecanone, tannic acid, and quercetin, only one P450 gene was upregulated. These results show that plant allelochemical stress can induce differential gene expression in A. gossypii. The differential response information of gene expression based on a large-scale sequence would be useful to reveal molecular mechanisms of adaptation for A. gossypii to plant allelochemicals.

Testing the limits of gradient sensing.

The ability to detect a chemical gradient is fundamental to many cellular processes. In multicellular organisms gradient sensing plays an important role in many physiological processes such as wound healing and development. Unicellular organisms use gradient sensing to move (chemotaxis) or grow (chemotropism) towards a favorable environment. Some cells are capable of detecting extremely shallow gradients, even in the presence of significant molecular-level noise. For example, yeast have been reported to detect pheromone gradients as shallow as 0.1 nM/µm. Noise reduction mechanisms, such as time-averaging and the internalization of pheromone molecules, have been proposed to explain how yeast cells filter fluctuations and detect shallow gradients. Here, we use a Particle-Based Reaction-Diffusion model of ligand-receptor dynamics to test the effectiveness of these mechanisms and to determine the limits of gradient sensing. In particular, we develop novel simulation methods for establishing chemical gradients that not only allow us to study gradient sensing under steady-state conditions, but also take into account transient effects as the gradient forms. Based on reported measurements of reaction rates, our results indicate neither time-averaging nor receptor endocytosis significantly improves the cell's accuracy in detecting gradients over time scales associated with the initiation of polarized growth. Additionally, our results demonstrate the physical barrier of the cell membrane sharpens chemical gradients across the cell. While our studies are motivated by the mating response of yeast, we believe our results and simulation methods will find applications in many different contexts.

A pharm-ecological perspective of terrestrial and aquatic plant-herbivore interactions.

We describe some recent themes in the nutritional and chemical ecology of herbivores and the importance of a broad pharmacological view of plant nutrients and chemical defenses that we integrate as "Pharm-ecology". The central role that dose, concentration, and response to plant components (nutrients and secondary metabolites) play in herbivore foraging behavior argues for broader application of approaches derived from pharmacology to both terrestrial and aquatic plant-herbivore systems. We describe how concepts of pharmacokinetics and pharmacodynamics are used to better understand the foraging phenotype of herbivores relative to nutrient and secondary metabolites in food. Implementing these concepts into the field remains a challenge, but new modeling approaches that emphasize tradeoffs and the properties of individual animals show promise. Throughout, we highlight similarities and differences between the historic and future applications of pharm-ecological concepts in understanding the ecology and evolution of terrestrial and aquatic interactions between herbivores and plants. We offer several pharm-ecology related questions and hypotheses that could strengthen our understanding of the nutritional and chemical factors that modulate foraging behavior of herbivores across terrestrial and aquatic systems.

Quantitative determination of daumone in rat plasma by liquid chromatography-mass spectrometry.

Daumone, 6-(3,5-dihydroxy-6-methyl-tetrahydro-pyran-2-yloxy)-heptanoic acid is a pheromone secreted by Caenorhabditis elegans, and has been known as a pivotal regulator of chemosensory processes in development and ageing. A quantification method using mass spectrometry was developed for the determination of daumone in rat plasma. After simple protein precipitation with acetonitrile including an internal standard, the analytes were chromatographed on a reversed-phase column and detected by liquid chromatography/tandem mass spectrometry with electrospray ionization. The accuracy and precision of the assay were in accordance with FDA regulations for validation of bioanalytical methods. This method was applied to measure the plasma daumone concentrations after a single intravenous administration of daumone in rats.

Safety assessment of 2-ethyl-3,(5 or 6) dimethylpyrazine as a food ingredient.

2-Ethyl-3,(5 or 6)-dimethylpyrazine (CAS No. 27043-05-6), a heterocyclic, nitrogen-containing compound, is used in the food industry as a flavor ingredient for its characteristic roasted odor and flavor, reminiscent of roasted cocoa or nuts. Pyrazines, including 2-ethyl-3,(5 or 6)-dimethylpyrazine, are widely distributed in foods and because of their natural unavoidable occurrence in cooked food; therefore, pyrazine compounds, including 2-ethyl-3,(5 or 6)-dimethylpyrazine, are commonly consumed in the daily diet. 2-Ethyl-3,(5 or 6)-dimethylpyrazine is oxidized in rats almost exclusively via its aliphatic side-chain to carboxylic acid derivatives. The LD(50) of 2-ethyl-3,(5 or 6)-dimethylpyrazine in rats was reported as 460 mg/kg and it is reported to be irritating to the skin, eyes and the upper respiratory tract. Two 90-day rat feeding studies have been conducted on 2-ethyl-3,(5 or 6)-dimethylpyrazine, with the one reporting a no effect level of 12.5mg/kg/day (both sexes) and a second study reporting a NOAEL of 2-ethyl-3,(5 or 6)-dimethylpyrazine 17 and 18 mg/kg/day for male and female rats, respectively. Although no genotoxicity studies were found on 2-ethyl-3,(5 or 6)-dimethylpyrazine, structurally similar pyrazine derivatives were reported as clastogenic in mammalian cells and non-mutagenic in bacterial assays. The relevance of the positive results in assays with Saccharomyces cerevisiae and Chinese hamster ovary cells in vitro is unclear. The data and information available, including a prolonged history of safe use, indicate that at the current level of intake, the food flavoring use of 2-ethyl-3,(5 or 6)-dimethylpyrazine is safe.

Receptores de feromonas de mamíferos: supervivencia y sexualidad / Mammalian pheromone receptors: survival and sexuality

En 1995 Catherine Dulac y Richard Axel publicaron la existencia de una nueva familia de genes que codificaban los posibles receptores de feromonas, pertenecientes a la amplia familia de los de siete hélices transmembranares y acoplados a proteínas G. Desde entonces se han clonado nuevos genes que han sido agrupados en dos familias, los receptores vomeronasales tipo 1 y 2, V1R y V2R, con diferente estructura y situados con diferente distribución en el órgano vomeronasal. La naturaleza química de las feromonas y de las proteínas que las asocian y transportan conocidas como lipocalinas es otro de los aspectos de los que se dispone de abundante información. Los mecanismos de transducción de la señal mediada por feromonas sobre los receptores V1R y V2R implican la activación de la fosfolipasa C tipo β2, PLCβ2, generando el fosfatidilinositol trifosfato y el diacilglicerol en la cara interna de la membrana neuronal. El diacilglicerol es un ligando endógeno, que permite la apertura del canal de la familia TRPC (Transient Receptor Potential Channel) denominado TRPC2 que se abre y deja pasar iones Ca2+ y Na+ al interior de la neurona sensorial, iniciando la despolarización de la membrana y originando el potencial de acción. La señal eléctrica es conducida al bulbo olfativo auxiliar por axones que llegan de modo disperso y establecen conexión con las células mitrales, las cuales envían sus prolongaciones hasta el sistema límbico y otras estructuras cerebrales, donde influencian o provocan las respuestas de supervivencia de la especie, entre ellas las de apareamiento y agresividad. Un aspecto relevante desde el punto de vista evolutivo es que en primates el gen TRPC2 es un pseudogen sin funcionalidad y por lo tanto el órgano vomeronasal es un vestigio carente de función. Recientes estudios indican que la captación de feromonas en primates se realiza a través del epitelio olfativo y el bulbo olfativo principal e incluso en otros mamíferos esta estructura parece mediar en algunas respuestas especie específicas

In 1995 Catherine Dulac and Richard Axel discovered a new gene family corresponding to the pheromone receptors. They were members of the seven transmembrane helix coupled to G proteins. Since then, new genes have been clonned and grouped according their sequence homology in two main families of vomeronasal receptors the V1R and the V2R. They exhibit different distribution pattern at the vomeronasal epithelium, where they are coupled to different G proteins. The chemical nature of the mammalian pheromones is very diverse and can associate with proteins called lipocalins to reach the vomeronasal organ. The transduction mechanisms of pheromone receptors, V1R and V2R, require respectively a Gi and a Go proteins, to further activate a phospholipase C, the PLCβ2. This enzyme hydrolyses the phosphatidyl inositol located at the plasma membrane originating phosphatidylinositol triphosphate and diacylglycerol. Diacylglycerol is an endogenous ligand that opens the TRPC2 channel (Transient Receptor Potential Channel), allowing the entrance of cations, mostly Ca2+ y Na+. The membrane depolarisation at the vomeronasal neuron originates the action potential that is sent to the accessory olfactory bulb by the axon, which in a different way as those from the main olfactory epithelia, do not organise the axonal prolongations and reach the mitral neurones in a disperse way, without forming a glomerular structure, afterwards the mitral cells send their axons to the limbic system and other cerebral structures related to aggressive behaviour and mating. It is relevant to underline that in monkeys from the old world and primates including humans, the vomeronasal organ is only a vestigial structure without function. The reason relies on the TRPC2 gene, which is a pseudo gene, without physiological function. Recent experimental approaches have demonstrated that the sensing of some pheromonal signals in these species, and also in mammals with a functional vomeronasal organ, can be carried out by the main olfactory epithelia through the main olfactory bulb. This structure being also connected to the hypothalamus, where neurones releasing LHRH can control sexual behaviour. These data confirm the broad possibilities of signalling through pheromones and that much effort is still required to fully understand their possibilities

Enhanced oral availability/pheromonotropic activity of peptidase-resistant topical amphiphilic analogs of pyrokinin/PBAN insect neuropeptides.

The peptide bond between active core residues Pro and Arg is the primary site of susceptibility for the pyrokinin/PBAN neuropeptides to insect tissue-bound peptidases, and incorporation of modified Pro residues can enhance resistance to peptidase hydrolysis. An Hyp-containing amphiphilic analog (Hex-FT[Hyp]RLa) is shown to operate as a topically active tissue-bound peptidase-resistant analog of the pyrokinin/PBAN class of insect neuropeptides in adult Heliothis virescens moths. An Oic amphiphilic analog (Hex-FT[Oic]RLa) is ineffective topically, but proves to be a superior tissue-bound, peptidase-resistant pyrokinin/PBAN analog for oral administration; outperforming both the Hyp analog and the orally inactive natural hormone PBAN in the moths. The Oic analog is effective in penetrating an isolated, ligated foregut preparation, but less successful in transmigrating an isolated midgut preparation; whereas the opposite behavior is observed for the Hyp analog. The success of the Oic analog via oral administration may be related to its ability to effectively penetrate the foregut, thereby bypassing the hostile environment of the midgut region.

Peptide pheromone-induced transfer of plasmid pCF10 in Enterococcus faecalis: probing the genetic and molecular basis for specificity of the pheromone response.

The tetracycline resistance plasmid pCF10 represents a class of unique mobile genetic elements of the bacterial genus Enterococcus, whose conjugative transfer functions are inducible by peptide sex pheromones excreted by potential recipient cells. These plasmids play a significant role in the dissemination of virulence and antibiotic resistance genes among the enterococci, which have become major nosocomial pathogens. Pheromone response by plasmid-carrying donor cells involves specific import of the peptide signal molecule, and subsequent interaction of the signal with one or more intracellular regulatory gene products. The pheromones are chromosomally encoded hydrophobic octa- or hepta-peptides, and different families of homologous plasmids encode the ability to respond to each pheromone. Among the four pheromone-responsive plasmids that have been characterized in some detail, there is considerable conservation in the genes encoding pheromone sensing and regulatory functions, and the peptides themselves show considerable similarity. In spite of this, there is extremely high specificity of response to each peptide, with virtually no "cross-induction" of transfer of non-cognate pheromone plasmids by the pheromones. This communication reviews the evidence for this specificity and discusses current molecular and genetic approaches to defining the basis for specificity.

Humoral pathway for local transfer of the priming pheromone androstenol from the nasal cavity to the brain and hypophysis in anaesthetized gilts.

It is generally accepted that pheromones act by stimulating of the dendritic receptors of the olfactory neurones massed in the olfactory epithelium. This study was designed to ascertain whether it is possible for the boar pheromone androstenol (5alpha-androst-16-en-3-ol) to be transported from the nasal cavity of anaesthetized gilts to the brain and hypophysis via local transfer from the blood in the perihypophyseal vascular complex. The experiment was performed on days 18-21 of the porcine oestrous cycle (crossbred gilts, n = 6). Tritiated androstenol (3H-A; total amount 10(8) d.p.m. (758 ng)) was applied for 1 min onto the respiratory part of the nasal mucosa, 4-6 cm from the opening of the nares. Arterial blood samples from the aorta and from the carotid rete were collected every 2 min during the 60 min period following administration of the steroid. Total radioactive venous effluent from the head was removed and an adequate volume of homologous blood was transfused into the heart through the carotid external vein. At the end of the experiment gilts were killed and tissue samples of the hypophysis and some brain structures were collected to measure radioactivity. In addition, corresponding control tissues were collected from three untreated gilts and from three heads of gilts 60 min after 3H-A was applied post mortem into the nasal cavity. The concentration of 3H-A was significantly higher (P < 0.0001) in the arterial blood of the carotid rete than that of aorta. The mean rate of 3H-A counter current transfer from venous to arterial blood in the perihypophyseal vascular complex, expressed as the ratio of the 3H-A concentration in arterial blood of the carotid rete to the 3H-A concentration in blood sampled simultaneously from the aorta, was 1.96 +/- 0.1. The concentration of 3H-A in plasma from the venous effluent from the head ranged from 1.3 to 1.8 pg x ml(-1). During the 60 min period of the experiment, 0.68% of the total applied dose of 3H-A was resorbed from the nasal cavity into the venous blood. Moreover, we found that 3H-A was present in the olfactory bulb (P <0.01), amygdala, septum, hypothalamus, adenohypophysis, neurohypophysis (P > 0.05) and perihypophyseal vascular complex (P < 0.01). These results demonstrate that, in anaesthetized gilts, the boar pheromone androstenol may be resorbed from the nasal mucosa, transferred in the perihypophyseal vascular complex into arterial blood supplying the brain and hypophysis, and then arrested in the hypophysis and certain brain structures. We suggest that in addition to the standard neural pathway for signalling pheromones, another pathway exists whereby androstenol, as a priming pheromone, may be resorbed from the nasal cavity into the bloodstream and then pass locally from the perihypophyseal vascular complex into the arterial blood supplying the brain and hypophysis, thus avoiding the first passage metabolism in the liver.

Humoral pathway for transfer of the boar pheromone, androstenol, from the nasal mucosa to the brain and hypophysis of gilts.

Signaling and priming pheromones play an important role in intraspecies behavioral and sexual interactions and in the control of reproduction. It is generally accepted that pheromones act by stimulating the dendritic receptors in the mucus-imbedded cilia of olfactory neurons massed in the olfactory epithelium. The boar pheromone androstenol, known to induce sexual behavior in pigs, is 1 of 2 pheromones that have been chemically defined, tritiated and thus made available for use in studies. In Experiment 1, sexually mature cyclic gilts at Days 16 to 21 of the estrous cycle were humanely killed and the heads separated from the bodies. The heads were attached to a perfusion system using heated, oxygenated, heparinized, autologous blood. A total amount of 10(8) dpm (758 ng) of 3H-5 alpha-androstenol (3HA) was either infused into the angularis oculi veins that drain the nasal cavities (n = 7) over a 5-min period or applied through intranasal catheters onto the mucose surface (n = 16) for 2 min. In both groups frequent blood samples were collected from the carotid rete and from venous effluent. Concentration of 3HA in the arterial blood of the carotid rete after direct (into angularis oculi veins) or indirect (onto the nasal mucosa) administration of 3HA into veins draining the nasal cavities was significantly higher than background radioactivity before 3HA administration (P < 0.0001 and P < 0.05, respectively). The 3HA was selectively accumulated (compared with the respective control tissue) in the neurohypophysis (P < 0.001), adenohypophysis (P < 0.01), ventromedial hypothalamus (P < 0.05), corpus mammillare (P < 0.01), and perihypophyseal vascular complex (P < 0.001). In a second in vitro experiment, active uptake of 3HA into the nasal mucosa of the proximal, respiratory segment of the nasal cavity was observed. These results demonstrate a humoral pathway for the transfer of pheromones from the nasal cavity to the hypophysis and brain. Androstenol was taken up by the respiratory part of the nasal mucosa, resorbed into blood, transported to the cavernous sinus and transferred into the arterial blood of the carotid rete (supplying the hypophysis and brain), and then selectively accumulated in the hypophysis and certain brain structures.