Tricyclic cyanoguanidines: synthesis, site of action and insecticidal activity of a novel class of reversible acetylcholinesterase inhibitors

Bridged-tricyclic cyanoguanidines 1 were found to be active as insecticides. The preparation and structure-activity relationships of oxacyclic (X=O) and carbocyclic (X=CH(2)) analogues of 1 is described. Compounds 1 were found to inhibit acetylcholinesterase with IC(50) values comparable to the organophosphate Paraoxon. Unlike organophosphates, cyanoguanidines 1 were shown to reversibly bind acetylcholinesterase. This mode of action is shared by the structurally-related natural product Huperzine A.     

Response of isolated thylakoid membranes with altered fluidity to short term heat stress

The effect of alterations of lipid phase order of thylakoid membranes on the thermosensitivity of photosystem I (PS I) and photosystem II (PS II) was studied. Plant sterols stigmasterol and cholesterol were applied to decrease the fluidity in isolated membranes. After sterol treatment, a decrease of the temperature of 50 % inhibition of PSII activity was observed. Heat stress-induced stimulation of PSI-mediated electron transport rate was registered for control, but not for sterol-treated membranes. Effect of altered lipid order on oxygen evolving complex was evaluated by means of flash oxygen yields revealing changes in the stoichiometry of PSII_α and PSII_β centers. The effect of sterol incorporation on the changes in the thermotropic behavior of the main pigment-protein complexes was studied by differential scanning calorimetry (DSC). DSC traces of control thylakoids in the temperature range 20–98 °C exhibited several irreversible endothermic transitions. Incorporation of cholesterol and stigmasterol results in superimposition of the transitions and only two main bands could be resolved. While high temperature band peaks at the same temperature after treatment with both sterols, the band that combines low temperature transitions shows different melting temperature (T_m): 70 °C for stigmasterol- and 65 °C for cholesterol-treated membranes. The data presented here emphasise the crucial role of lipid order for the response of thylakoids to high temperatures, mediated not only by changes in the fluidity of bulk lipid phase as result of sterol incorporation but also by changes in the thermotropic properties of pigment-protein complexes.Key words: Cholesterol, Fluidity, Heat stress, Oxygen flash yields, Thylakoid membrane, Stigmasterol     

Diploidy and the selective advantage for sexual reproduction in unicellular organisms

This article develops mathematical models describing the evolutionary dynamics of both asexually and sexually reproducing populations of diploid unicellular organisms. The asexual and sexual life cycles are based on the asexual and sexual life cycles in Saccharomyces cerevisiae, Baker’s yeast, which normally reproduces by asexual budding, but switches to sexual reproduction when stressed. The mathematical models consider three reproduction pathways: (1) Asexual reproduction, (2) self-fertilization, and (3) sexual reproduction. We also consider two forms of genome organization. In the first case, we assume that the genome consists of two multi-gene chromosomes, whereas in the second case, we consider the opposite extreme and assume that each gene defines a separate chromosome, which we call the multi-chromosome genome. These two cases are considered to explore the role that recombination has on the mutation-selection balance and the selective advantage of the various reproduction strategies. We assume that the purpose of diploidy is to provide redundancy, so that damage to a gene may be repaired using the other, presumably undamaged copy (a process known as homologous recombination repair). As a result, we assume that the fitness of the organism only depends on the number of homologous gene pairs that contain at least one functional copy of a given gene. If the organism has at least one functional copy of every gene in the genome, we assume a fitness of 1. In general, if the organism has l homologous pairs that lack a functional copy of the given gene, then the fitness of the organism is κ _l . The κ _l are assumed to be monotonically decreasing, so that κ₀ = 1 > κ₁ > κ₂ > ⋯ > κ_∞ = 0. For nearly all of the reproduction strategies we consider, we find, in the limit of large N, that the mean fitness at mutation-selection balance is max{2 e^-m-1, 0} ,\hbox{max}\{2 e^{-\mu}-1, 0\} , where N is the number of genes in the haploid set of the genome, ε is the probability that a given DNA template strand of a given gene produces a mutated daughter during replication, and μ = Nε. The only exception is the sexual reproduction pathway for the multi-chromosomed genome. Assuming a multiplicative fitness landscape where κ _l  = α ^l for α ∈ (0, 1), this strategy is found to have a mean fitness that exceeds the mean fitness of all the other strategies. Furthermore, while other reproduction strategies experience a total loss of viability due to the steady accumulation of deleterious mutations once μ exceeds ln2 ,\ln 2 , no such transition occurs in the sexual pathway. Indeed, in the limit as α → 1 for the multiplicative landscape, we can show that the mean fitness for the sexual pathway with the multi-chromosomed genome converges to e ^−2μ, which is always positive. We explicitly allow for mitotic recombination in this study, which, in contrast to previous studies using different models, does not have any advantage over other asexual reproduction strategies. The results of this article provide a basis for understanding the selective advantage of the specific meiotic pathway that is employed by sexually reproducing organisms. The results of this article also suggest an explanation for why unicellular organisms such as Saccharomyces cerevisiae (Baker’s yeast) switch to a sexual mode of reproduction when stressed. While the results of this article are based on modeling mutation-propagation in unicellular organisms, they nevertheless suggest that, in more complex organisms with significantly larger genomes, sex is necessary to prevent the loss of viability of a population due to genetic drift. Finally, and perhaps most importantly, the results of this article demonstrate a selective advantage for sexual reproduction with fewer and much less restrictive assumptions than those of previous studies.     

Identification of Cellular Proteome Modifications in Response to West Nile Virus Infection

Flaviviruses are positive-stranded RNA viruses that are a public health problem because of their widespread distribution and their ability to cause a variety of diseases in humans. West Nile virus is a mosquito-borne member of this genus and is the etiologic agent of West Nile encephalitis. Clinical manifestations of West Nile virus infection are diverse, and their pathogenic mechanisms depend on complex virus-cell interactions. In the present work, we used proteomics technology to analyze early Vero cell response to West Nile infection. The differential proteomes were resolved 24 h postinfection using two-dimensional DIGE followed by mass spectrometry identification. Quantitative analysis (at least 2-fold quantitative alteration, p < 0.05) revealed 127 differentially expressed proteins with 68 up-regulated proteins and 59 down-regulated proteins of which 93 were successfully identified. The implication for mammalian cellular responses to this neurotropic flavivirus infection was analyzed and made possible more comprehensive characterization of the virus-host interactions involved in pathogenesis. The present study thus provides large scale protein-related information that should be useful for understanding how the host metabolism is modified by West Nile infection and for identifying new potential targets for antiviral therapy.West Nile virus (WNV)¹ is a mosquito-borne flavivirus belonging to the Japanese encephalitis virus (JEV) serocomplex. The virus is maintained in nature in enzootic cycles in which it is transmitted between ornithophilic mosquitoes and avian hosts. In mammals, including humans, WNV is an encephalitic flavivirus and can cause natural infections of the central nervous system (CNS) with a neuropathogenesis involving neuroinvasiveness (ability to enter the CNS) and neurovirulence (replication within the CNS) (1). To date, no pharmacological treatment exists for WNV, and a vaccine is only available for horses.First isolated in 1937, WNV has become endemic in Africa, the Middle East, and parts of Asia and Europe (2, 3). Phylogenetics analysis groups WNV strains into two distinct lineages. Viruses in lineage 2 are found only in Africa, whereas viruses in lineage 1 are present both in Africa and in other areas, particularly Asia and Europe. Since 1999, WNV from lineage 1 (NY99) has reached North America where, in 2002, it caused the largest arboviral meningoencephalitis outbreak ever recorded in this area (4).It is known that flavivirus replication can cause extensive rearrangement of host cell cytoskeletal and membrane compartments leading to a “cytopathic effect” in various cell cultures of human, primate, rodent, and insect origin (5). Recent studies have revealed specific effects of viruses on cellular processes. It has been demonstrated that flaviviruses can induce cell death directly through viral replication and the production of proapoptotic proteins (6–11), but the mechanism of pathogenesis has not been elucidated.Although neurons are regarded as the major target of WNV in vivo (2), WNV infection has been shown to induce apoptosis in different cell lines in a similar manner in vitro (12, 13). This includes a wide range of different cell types with, in particular, the African green monkey kidney continuous cell line (Vero) recommended by the World Health Organization Collaborating Center for systematic research and isolation of arboviruses as well as a substrate to develop live attenuated and inactivated vaccines. Acute infection of Vero cells by WNV produces a lytic infection with a characteristic rounding cytopathic effect and the production of a large number of infectious particles in the culture fluid within 3 days postinfection (14). Although this permissive mammalian cell system is widely used for flavivirus isolation, propagation, and titration, to date no studies have focused on identifying Vero cellular proteins whose expression has been altered by WNV infection. We considered that Vero cells could be a good model for in vitro identification of cell protein alterations with possible implication in certain pathogenic mechanisms.In the present work, fluorescent 2D DIGE technology combined with MS analysis was used to examine the consequences of Vero cell infection by WNV. To evaluate early mammalian cell response after infection and to avoid the effect of cell death and protein degradation, the culture conditions (e.g. infectious dose and incubation time) were optimized. A total of 93 differentially expressed protein spots were identified (over ±2-fold, p < 0.05) and confirmed by fluorescent Western blot analysis. The implication for cellular responses to this flavivirus infection as well as the potential roles of certain altered identified proteins are discussed to characterize the pathophysiologic processes. This study can also provide useful clues for antiviral research.     

Emergence of new variants of antibiotic resistance genomic islands among multidrug-resistant Salmonella enterica in poultry

Non-typhoidal Salmonella enterica (NTS) are diverse and important bacterial pathogens consisting of more than 2600 different serovars, with varying host-specificity. Here, we characterized the poultry-associated serovars in Israel, analysed their resistome and illuminated the molecular mechanisms underlying common multidrug resistance (MDR) patterns. We show that at least four serovars including Infantis, Muenchen, Newport and Virchow present a strong epidemiological association between their temporal trends in poultry and humans. Worrisomely, 60% from all of the poultry isolates tested (n = 188) were multidrug resistant, mediated by chromosomal SNPs and different mobile genetics elements. A novel streptomycin-azithromycin resistance island and previously uncharacterized versions of the mobilized Salmonella genomic island 1 (SGI1) were identified and characterized in S. Blockley and S. Kentucky isolates respectively. Moreover, we demonstrate that the acquisition of SGI1 does not impose fitness cost during growth under nutrient-limited conditions or in the context of Salmonella infection in the mouse model. Overall, our data emphasize the role of the poultry production as a pool of specific epidemic MDR strains and autonomous genetic elements, which confer resistance to heavy metals and medically relevant antibiotics. These are likely to disseminate to humans via the food chain and fuel the increasing global antibiotic resistance crisis.     

<Emphasis Type="Italic">Beauveria bassiana</Emphasis> as an endophyte: a critical review on associated methodology and biocontrol potential

In the last decade there has been increased focus on the potential of endophytic Beauveria bassiana for the biocontrol of insect herbivores. Generally, detection of endophytes is acknowledged to be problematic and recovery method-dependent. Herein, we critically analyse the methodology reported for the detection of B. bassiana as endophytes following experimental inoculation. In light of the methodology, we further review the effects of endophytic B. bassiana on insect herbivores. Our review indicated the need for stringent protocols for surface sterilisation including thorough experimental controls. For molecular detection protocols by PCR, residual DNA from surface inocula must also be considered. The biocontrol potential of B. bassiana endophytes appears promising although both negative and neutral effects on insect herbivores were reported and there remains ambiguity with respect to the location and mode of action of the fungus in planta. We recommend that future studies adopt multiple techniques, including culture dependent and independent techniques for endophyte detection and elucidate the mechanisms involved against insect herbivores.     

Effect of depletion of monocytes/macrophages on early aortic valve lesion in experimental hyperlipidemia

Monocytes/macrophages are key players throughout atheroma development. The aim of this study was to determine the role of macrophages in lesion formation in heart valves in hyperlipidemia. We examined whether systemic depletion of monocytes/macrophages had a beneficial or adverse effect on the development of lesions in hyperlipemic hamsters injected twice weekly (for 2 months) with clodronate-encapsulated liposomes (H+Lclod), a treatment that selectively induces significant monocyte apoptosis. Hyperlipemic hamsters were employed as controls, as were hyperlipemic hamsters treated with plain liposomes. We assayed serum cholesterol (CH) and triglycerides (TG), the lipid and collagen contents and the size of the valve lesions, the matrix metalloproteinases (MMPs) in the serum and vessel wall, apolipoprotein E (ApoE), interleukin-1β (IL-1β), and superoxide anion production. In comparison with controls, H+Lclod hamsters exhibited: (1) increased lipid and collagen accumulation within the lesions, (2) decreased activity of MMP-9 and MMP-2 in sera and aortic homogenates, (3) decreased serum CH and TG and decreased expression of ApoE in sera and liver, (4) reduced expression of IL-1β in aorta and liver homogenates, and (5) no change in the level of superoxide anion in the aorta. Thus, initially, the presence of the macrophages is beneficial in valvular lesion formation. Depletion of monocytes/macrophages is a two-edged sword having a beneficial effect by decreasing the expression of IL-1β and MMP activities but an adverse effect by inducing a significant increase in the lipid and collagen content and expansion of valvular lesions. This work was supported by the Romanian Academy and a grant from the Romanian Ministry of Education and Research, National Program VIASAN (grant no. 330).     

The yeast par-1 homologs kin1 and kin2 show genetic and physical interactions with components of the exocytic machinery

Kin1 and Kin2 are Saccharomyces cerevisiae counterparts of Par-1, the Caenorhabditis elegans kinase essential for the establishment of polarity in the one cell embryo. Here, we present evidence for a novel link between Kin1, Kin2, and the secretory machinery of the budding yeast. We isolated KIN1 and KIN2 as suppressors of a mutant form of Rho3, a Rho-GTPase acting in polarized trafficking. Genetic analysis suggests that KIN1 and KIN2 act downstream of the Rab-GTPase Sec4, its exchange factor Sec2, and several components of the vesicle tethering complex, the Exocyst. We show that Kin1 and Kin2 physically interact with the t-SNARE Sec9 and the Lgl homologue Sro7, proteins acting at the final stage of exocytosis. Structural analysis of Kin2 reveals that its catalytic activity is essential for its function in the secretory pathway and implicates the conserved 42-amino acid tail at the carboxy terminal of the kinase in autoinhibition. Finally, we find that Kin1 and Kin2 induce phosphorylation of t-SNARE Sec9 in vivo and stimulate its release from the plasma membrane. In summary, we report the finding that yeast Par-1 counterparts are associated with and regulate the function of the exocytic apparatus via phosphorylation of Sec9.