Biochemical Investigations on Microbial Prenyltransferases in the Presence of DMAPP Analogues

Plants, bacteria and fungi provide a plethora of diverse structures derived from the primary as well as the secondary metabolism. Representative substances from secondary metabolite pathways are flavonoids, coumarins, xanthones and indole alkaloids. Although not necessary for the growth and reproduction of the respective organisms, nevertheless these compounds give them an advantage over other organisms in the form of, e.g. attractants or protection agents against natural enemies as well as competitors. The attachment of isoprene units (n  C-5) such as dimethylallyl, geranyl or farnesyl moieties to aromatic secondary metabolites as backbones is a further step in the broad diversification of these compounds. Enzymes of the class of prenyltransferases accomplish this prenyl transfer reaction in nature. The prenylated natural products often exhibit strong pharmacological activities in contrast to their substrates. Therefore, prenylated compounds represent interesting targets for drug development. Investigations on alternative alkyl residues transferable via prenyltransferases, i.e. prenyl diphosphate analogues, could have the potential to play an important role in the understanding of the catalytic mechanism of these enzymes as well as in the finding process of new drugs. As the first project in this thesis, the acceptance of four chemically synthesized alkyl and allyl diphosphates in the presence of three L-tryptophan prenyltransferases (FgaPT2, 5 DMATS, 7 DMATS) was elucidated. Retaining the double bond in β position and performing alterations on the methyl groups of the allyl residue led to regular alkylated products and thereby showed successful utilization of DMAPP analogues. However, depending on the DMAPP analogue, the regiospecificity of the applied prenyltransferases was shifted partially or completely. Furthermore, the enzymes did not catalyze the transfer reaction of alkyl diphosphates onto the L tryptophan scaffold, if no double bond was present or if it was relocated to  position compared to DMAPP. Subsequently, the behavior of cyclic dipeptide prenyltransferases (AnaPT, CdpNPT, CdpC3PT, FtmPT1, BrePT) towards the unnatural alkyl and allyl donors was examined. The cyclic dipeptide prenyltransferases also used the unsaturated DMAPP analogues resulting in the formation of multiple products. Regardless of the prenylation position and orientation for the prenyl attachment (C 2 or C 3; regular or reverse) in the presence of DMAPP, the enzymatic reactions with both DMAPP analogues, i.e. MAPP and 2-pentenyl-PP, yielded a mixture of C2 reverse as well as C3 reverse alkylated diastereomers in different ratios depending on the donor and used enzyme. After the successful alkylation of L tryptophan and tryptophan-containing cyclic dipeptides utilizing simple unnatural allyl DMAPP analogues, the acceptance of the structurally more complex DMAPP analogue benzyl diphosphate was tested. Preliminary investigations showed the successful usage of this benzyl donor by several L tryptophan and cyclic dipeptide prenyltransferases, whereas FgaPT2 showed the highest activity. FgaPT2 also displayed a remarkable promiscuity using tryptophan analogues as substrates and catalyzing a highly regiospecific C5 benzylation. Consequently, the usage of benzyl diphosphate instead of dimethylallyl diphosphate results in a complete shift of the prenylation position from C 4 to C 5. To complete our findings regarding the acceptance of unnatural alkyl and benzyl analogues by prenyltransferases, several tryptophan C5-, C6- and C7-prenylating enzymes of fungal and bacterial origin have been assayed with the three analogues. Depending on the enzyme used, one to four products could be identified from the incubation mixtures. The predominant products were regular C6-alkylated or C6-benzylated derivatives in all cases. Therefore, for the tested tryptophan C5-, C6- and C7-prenylating enzymes, C-6 seemed to be the preferred position for attachment of the alkyl or benzyl moiety. The results obtained during this thesis show that allyl as well as benzyl analogues of DMAPP are potential alternatives for chemoenzymatic FriedelCrafts alkylations of simple indole derivatives and tryptophan-containing cyclic dipeptides and could be used for the production of alkylated compounds

A Needs Assessment of the Perceptions and Opportunities to Enhance Nature Exploration at Publicly Funded Preschools

Extensive research supports the benefits of nature exploration in children’s lives. Research also suggests, however, that low-income families and other historically marginalized groups experience multiple barriers to accessing green spaces. In an attempt to counteract this inequality, a needs assessment was performed to understand the challenges and barriers public preschool providers face in regularly leading their children in nature exploration. The target audience of this evaluation was Head Start and Early Childhood Education and Assistance Program [ECEAP] teachers and parents. Along with measuring the challenges and barriers at these centers in regard to nature exploration, this evaluation assessed the resources and assistance needed to support the Head Start and ECEAP community’s environmental education interests. Informed by common worlds framework and theory supporting nature and child development, a thematic analysis of interviews with Head Start and ECEAP teachers and parents suggests a need to increase the amount of daily outdoor play and nature exploration within Head Start and ECEAP centers. Results of this evaluation also suggest a need to address safety and liability concerns, lack of weather appropriate outdoor clothing, limited play yard features, and access, or perceived access, to natural areas. Future efforts to encourage opportunities for nature exploration in public preschools should incorporate professional development, parent outreach, and culturally sustaining pedagogies. The findings of this evaluation may be used to guide program improvement and development that supports and incorporates routine nature exploration into the Head Start and ECEAP curriculum

Alien Species, Agents of Global Change : Ecology and management of the Gypsy Moth in North America as a case History

USDA Forest ServiceScedule:17-18 March 2003, Vemue: Kanazawa, Japan, Kanazawa Citymonde Hotel, Project Leader : Hayakawa, Kazuichi, Symposium Secretariat: XO kamata, Naoto, Edited by:Kamata, Naoto

Effects of Gypsy Moth Outbreaks on North American Woodpeckers

We examined the effects of the introduced gypsy moth (Lymantria dispar) on seven species of North American woodpeckers by matching spatially explicit data on gypsy moth outbreaks with data on breeding and wintering populations. In general, we detected modest effects during outbreaks: during the breeding season one species, the Red-headed Woodpecker (Melanerpes erythrocephalus), increased over pre-outbreak levels, while during the winter one species, the Yellow-bellied Sapsucker (Sphyrapicus varius), increased and one, the Downy Woodpecker (Picoides pubescens), decreased from pre-outbreak levels. Responses following outbreaks were similarly variable, and in general we were unsuccessful at predicting population responses to outbreaks from a priori knowledge of woodpecker ecology and behavior. We did, however, find evidence that the response of at least half of the species changed over the 34-year period covered by the study: except for the Northern Flicker (Colaptes auratus), whose response to outbreaks during the winter decreased, populations generally responded more positively to outbreaks with time. This temporal response suggests that North American woodpeckers may be taking greater advantage of the resource pulse and/or habitat changes caused by outbreaks of this exotic pest now than previously, so in the future the effects of gypsy moth outbreaks on these species may increase

Emergent Fungal Entomopathogen Does Not Alter Density Dependence in a Viral Competitor

Population cycles in forest Lepidoptera often result from recurring density-dependent epizootics of entomopathogens. While these systems are typically dominated by a single pathogen species, insects are often infected by multiple pathogens, yet little is known how pathogens interact to affect host dynamics. The apparent invasion of northeastern North America by the fungal entomopathogen Entomophaga maimaiga some time prior to 1989 provides a unique opportunity to evaluate such interactions. Prior to the arrival of E. maimaga, the oscillatory dynamics of host gypsy moth, Lymantria dispar, populations were apparently driven by epizootics of a nucleopolyhedrovirus. Subsequent to its emergence, E. maimaiga has caused extensive mortality in host populations, but little is known about how it has altered multigenerational dynamics of the gypsy moth and its virus. Here we compared demographic data collected in gypsy moth populations prior to vs. after E. maimaiga\u27s invasion. We found that the recently invading fungal pathogen virtually always causes greater levels of mortality in hosts than does the virus, but fungal mortality is largely density independent. Moreover, the presence of the fungus has apparently not altered the gypsy moth–virus density-dependent interactions that were shown to drive periodic oscillations in hosts before the arrival of the fungus

Collateral damage: military invasions beget biological invasions

Biological invasions are frequently and closely associated with armed conflict. As a key element of human history, war involves the invasion of (often distant) enemy territories, during which time species can be translocated, intentionally or unintentionally. Large-scale conflicts such as World War I and II, in which thousands of soldiers and supplies (including foodstuffs) were transported within and between continents, resulted in the spread of non-native taxa. However, smaller scale military actions may also involve rapid movements of troops between geographical areas, potentially facilitating the accidental introduction of species into previously unoccupied areas. Furthermore, invasive pests have occasionally been used by armies as weapons to weaken and disrupt opposing forces or nations. The introduction of invasive species during war could be considered relatively minor collateral damage, but many biological invasions in conflict zones have long-lasting effects. Regulation of military practices to minimize or prevent biological invasions through existing international conventions has so far been unsuccessful, necessitating the development of additional measure

European gypsy moth (Lymantria dispar dispar L.) completes development and defoliates exotic radiata pine plantations in Spain

Background Like most pines, radiata pine (Pinus radiata D.Don) is conventionally considered resistant to defoliation by European gypsy moth, i.e. it is only consumed by late larval stages, or when susceptible species are not available or are scarce. The ability of this moth to complete development on this host in field conditions has never been reported. Findings A gypsy moth outbreak in a pure radiata pine plantation was observed in north-western Spain, where this insect is endemic. During the 2 years of outbreak culmination (2012 and 2013), approximately 46 ha of radiata pine were severely defoliated (>75 % of leaf area removed) and no defoliation was evident in nearby stands of susceptible species. Large numbers of egg masses were present on stems of pine trees. Larvae were observed feeding exclusively on radiata pine needles beginning with the first instar and continuing to pupation. Nearly 100 % tree mortality occurred in stands severely defoliated. Conclusions The occurrence of a European gypsy moth outbreak in a pure radiata pine plantation contradicts previous observations that larvae of this species cannot complete development in stands comprised entirely of pines. These results suggest that European strains of the gypsy moth hold great potential for damage to commercial radiata pine plantations where this species is utilised in plantation forestry, such as in New Zealand or AustraliaFunding for this research was provided by the Local Goberment of Cubillos del Sil by the contract “Seguimiento y bases para la gestión de las masas forestales afectadas por defoliación de Lymantria dispar en el municipio de Cubillos del Sil”S