Efficient nitroso group transfer from N-nitrosoindoles to nucleotides and 2'-deoxyguanosine at physiological pH. A new pathway for N-nitrosocompounds to exert genotoxicity.

The endogenous formation of N-nitrosoindoles is of concern since humans are exposed to a variety of naturally occurring and synthetic indolic compounds. As part of a study to evaluate the genotoxicity of N-nitrosoindoles, the reactions of three model compounds with purine nucleotides and 2'-deoxyguanosine at physiological pH were investigated. The profiles of reaction products were identical for each of the N-nitrosoindoles and three distinct pathways of reaction could be discerned. These pathways were: (i) depurination to the corresponding purine bases, (ii) deamination, coupled with depurination, to give hypoxanthine and xanthine, and (iii) formation of the novel nucleotide 2'-deoxyoxanosine monophosphate and its corresponding depurination product oxanine in reactions with 2'-deoxyguanosine monophosphate. 2'-Deoxyoxanosine and oxanine were observed in reactions with 2'-deoxyguanosine. Further studies showed that formation of all of these products could be rationalized by an initial transnitrosation step. These results suggest that, in contrast to many other genotoxic N-nitrosocompounds which are known to alkylate DNA, the genotoxicity of N-nitrosoindoles is likely to arise through transfer of the nitroso group to nucleophilic sites on the purine bases. All of the products resulting from transnitrosation by N-nitrosoindoles are potentially mutagenic. These findings reveal a new pathway for N-nitrosocompounds to exert genotoxicity.     

Potent oxadiazole CGRP receptor antagonists for the potential treatment of migraine

A pharmacophore model was built, based on known CGRP receptor antagonists, and this was used to aid the identification of novel leads. Analogues were designed, modelled and synthesised which incorporated alternative ‘LHS’ fragments linked via either an amide or urea to a privileged ‘RHS’ fragment commonly found in CGRP receptor antagonists. As a result a novel series of oxadiazole CGRP receptor antagonists has been identified and the subsequent optimisation to enhance both potency and bioavailability is presented.     

Expression, purification, and refolding of mouse islet neogenesis associated protein-related protein for NMR studies

Islet neogenesis associated protein-related protein (INGAPrP) is thought to be involved in the differentiation of non-insulin-producing cells to insulin-secreting cells. INGAPrP is a mouse gene product that has a 72% identical amino acid sequence to a known islet-generating factor, hamster islet-neogenesis-associated protein (INGAP), which acts by differentiating pancreatic ductal cells into beta-cells. The three-dimensional structure of these proteins is unknown. The structure would provide information about the conformation of the active portion of INGAP, the so-called INGAP pentadecapeptide, leading to a well-defined target for rational drug design. An efficient procedure for the production of INGAPrP would facilitate the process of structure determination. We have successfully produced and isolated (15)N-labeled INGAPrP by expression in Escherichia coli Rosetta (DE3) cells in Spectra-9 media followed by a two-step purification and refolding protocol. The hexahistidine tag engineered at the N-terminus of the protein is used in the first step for standard immobilized-metal affinity chromatography purification under denaturing conditions. The secondary purification step utilizes a gel permeation chromatography column, producing homogeneous INGAPrP as well as refolding the protein. To verify that the protein was folded, we performed a (1)H-(15)N HSQC NMR experiment that showed excellent dispersion of signals, indicative of a folded protein. We also performed circular dichroism experiments, which demonstrated the presence of secondary structure. In summary, we report the first expression and isolation of INGAPrP, as well as demonstrate that our method produces a folded protein, which is necessary for structure determination.     

Wasp sex ratios when females on a patch are related

Optimality theory of sex allocation in structured populations has proved remarkably successful in explaining patterns of facultative sex ratio behaviour in numerous species. Extensions to the basic theory have included more specific aspects of species biology, including the relatedness of interacting individuals. We considered the sex ratio decisions made by female Nasonia vitripennis wasps when they were ovipositing on a patch with either relatives or nonrelatives. Theory predicts that females should produce more female-biased sex ratios when ovipositing with relatives, for example sisters, than with unrelated females. This is because related females should limit the level of local mate competition between their sons for female partners. Contrary to theory, two experiments showed that female sex ratio behaviour was unaffected by the relatedness of their oviposition partner, and was also unrelated to an environmental cue that could signal relatedness, i.e. whether females responded differently to sisters emerging from the same or a different host. Instead, in both experiments, we found that only wasp strain significantly influenced sex ratio. A meta-analysis of studies conducted on a range of species on the effects of the relatedness of oviposition partners on sex ratio failed to show the predicted pattern. We discuss why females appear to behave in a maladaptive way when allocating sex under these conditions, and suggest that weak selection and/or conflict between females over optimal sex ratios may influence the evolution of kin discrimination.     

Enhanced Nematicidal Activity of Organic and Inorganic Ammonia-Releasing Amendments by Azadirachta indica Extracts

The nematicidal activities of ammonium sulfate, chicken litter and chitin, alone or in combination with neem (Azadirachta indica) extracts were tested against Meloidogyne javanica. Soil application of these amendments or the neem extracts alone did not reduce the root galling index of tomato plants or did so only slightly, but application of the amendments in combination with the neem extracts reduced root galling significantly. Soil analysis indicated that the neem extract inhibited the nitrification of the ammonium released from the amendments and extended the persistence of the ammonium concentrations in the soil. In microplot experiments, tomato plants were grown in pots filled with soils from the treated microplots. The galling indices of tomato plants grown in soil treated with ammonium sulfate or chicken litter in combination with the neem extract or a chemical nitrification inhibitor were far lower than those of plants grown in the control soil or in soil treated with chicken litter, neem extract or nitrification inhibitor alone. However, plants grown in the microplots showed only slight reductions in galling, probably because the soil amendments were inadequately mixed compared to their application in the pot experiments. The extended exposure of nematodes to ammonia as a result of nitrification inhibition by the neem extracts appeared to be the cause of the enhanced nematicidal activity of the ammonia-releasing amendments.     

Beyond sex allocation: the role of mating systems in sexual selection in parasitoid wasps

Despite the diverse array of mating systems and life histories which characterise the parasitic Hymenoptera, sexual selection and sexual conflict in this taxon have been somewhat overlooked. For instance, parasitoid mating systems have typically been studied in terms of how mating structure affects sex allocation. In the past decade, however, some studies have sought to address sexual selection in the parasitoid wasps more explicitly and found that, despite the lack of obvious secondary sexual traits, sexual selection has the potential to shape a range of aspects of parasitoid reproductive behaviour and ecology. Moreover, various characteristics fundamental to the parasitoid way of life may provide innovative new ways to investigate different processes of sexual selection. The overall aim of this review therefore is to re‐examine parasitoid biology with sexual selection in mind, for both parasitoid biologists and also researchers interested in sexual selection and the evolution of mating systems more generally. We will consider aspects of particular relevance that have already been well studied including local mating structure, sex allocation and sperm depletion. We go on to review what we already know about sexual selection in the parasitoid wasps and highlight areas which may prove fruitful for further investigation. In particular, sperm depletion and the costs of inbreeding under chromosomal sex determination provide novel opportunities for testing the role of direct and indirect benefits for the evolution of mate choice.     

The costs and benefits of multiple mating in a mostly monandrous wasp

The taxonomically widespread nature of polyandry remains a puzzle. Much of the empirical work regarding the costs and benefits of multiple mating to females has, for obvious reasons, relied on species that are already highly polyandrous. However, this makes it difficult to separate the processes that maintain the current level of polyandry from the processes that facilitate its expression and initiated its evolution. Here we consider the costs and benefits of polyandry in Nasonia vitripennis, a species of parasitoid wasp that is “mostly monandrous” in the wild, but which evolves polyandry under laboratory culture conditions. In a series of six experiments, we show that females gain a direct fecundity and longevity benefit from mating multiply with virgin males. Conversely, mating multiply with previously mated males actually results in a fecundity cost. Sexual harassment may also represent a significant cost of reproduction. Harassment was, however, only costly during oviposition, resulting in reduced fecundity, longevity, and disrupted sex allocation. Our results show that ecological changes, in our case associated with differences in the local mating structure in the laboratory can alter the costs and benefits of mating and harassment and potentially lead to shifts in mating patterns.     

Identification of the RT-RH/IN cleavage site of HTLV-I

Human T-cell leukemia virus type 1 (HTLV-1) is a type C human retrovirus and is the causative agent of adult T-cell leukemia and other diseases. The enzymatic and structural proteins of HTLV-I are synthesized as part of a Gag-Pro-Pol precursor polyprotein, and the mature proteins are released by proteolytic processing catalyzed by HTLV-I protease. The locations of most of the proteolytic cleavage sites are known, however, the site that creates the N-terminus of HTLV-1 integrase has not been previously identified. A 15 residue peptide corresponding to junction of the C-terminus of RNaseH and N-terminus of integrase (DALLITPVLQLSPAF-OH) was incubated with HTLV-1 protease. Analysis of the cleavage products by LC-MS revealed fragments Ac-DALLITPVLQL-OH and H(2)N-SPAF-OH were produced, indicating cleavage between the leucine and serine. This is the first physical identification of the N-terminal amino acid sequence of the integrase of HTLV-1.     

Sex ratios under asymmetrical local mate competition: theory and a test with parasitoid wasps

Sex ratio theory allows unparalleled opportunities for testing how well animal behavior can be predicted by evolutionary theory. For example, Hamilton's theory of local mate competition (LMC) is well understood and can explain variation in sex allocation across numerous species. This allows more specific predictions to be developed and tested. Here we extend LMC theory to a situation that will be common in a range of species: asymmetrical LMC. Asymmetrical LMC occurs when females lay eggs on a patch asynchronously and male offspring do not disperse, leading to relatively weaker LMC for males emerging from later broods. Varying levels of LMC then lead to varying optimal sex ratios for females, depending on when and where they oviposit. We confirm the assumptions of our theory using the wasp Nasonia vitripennis and then test our predictions. We show that females adjust their offspring sex ratios in the directions predicted, laying different sex ratios on different hosts within a patch. Specifically, there was a less female-biased sex ratio when ovipositing on an unparasitized host if another host on the patch had previously been parasitized and a less female-biased sex ratio on parasitized hosts if females also oviposited on an unparasitized host.