Engineering and mechanistic studies of the Arabidopsis FAE1 beta-ketoacyl-CoA synthase, FAE1 KCS.

The Arabidopsis FAE1 beta-ketoacyl-CoA synthase (FAE1 KCS) catalyzes the condensation of malonyl-CoA with long-chain acyl-CoAs. Sequence analysis of FAE1 KCS predicted that this condensing enzyme is anchored to a membrane by two adjacent N-terminal membrane-spanning domains. In order to characterize the FAE1 KCS and analyze its mechanism, FAE1 KCS and its mutants were engineered with a His6-tag at their N-terminus, and expressed in Saccharomyces cerevisiae. The membrane-bound enzyme was then solubilized and purified to near homogeneity on a metal affinity column. Wild-type recombinant FAE1 KCS was active with several acyl-CoA substrates, with highest activity towards saturated and monounsaturated C16 and C18. In the absence of an acyl-CoA substrate, FAE1 KCS was unable to carry out decarboxylation of [3-(14)C]malonyl-CoA, indicating that it requires binding of the acyl-CoA for decarboxylation activity. Site-directed mutagenesis was carried out on the FAE1 KCS to assess if this condensing enzyme was mechanistically related to the well characterized soluble condensing enzymes of fatty acid and flavonoid syntheses. A C223A mutant enzyme lacking the acylation site was unable to carry out decarboxylation of malonyl-CoA even when 18:1-CoA was present. Mutational analyses of the conserved Asn424 and His391 residues indicated the importance of these residues for FAE1-KCS activity. The results presented here provide the initial analysis of the reaction mechanism for a membrane-bound condensing enzyme from any source and provide evidence for a mechanism similar to the soluble condensing enzymes.     

Explaining density-dependent regulation in earthworm populations using life-history analysis

At present there is little knowledge about how density regulates population growth rate and to what extent this is determined by life-history patterns. We compared density dependent population consequences in the Nicholsonian sense based on experimental observations and life-history modeling for the earthworms Lumbricus terrestris and Eisenia fetida . Both species differ in their life-histories, L. terrestris being a relatively long-lived species with slow reproduction and occurring at low densities compared to E. fetida which has a more opportunistic strategy with a high reproductive output. E. fetida is able to colonise new habitats rapidly and may occur at relatively high population densities. Density dependency of population growth rate was estimated by incorporating density dependent effects on reproduction and growth using a modified Euler equation. The results point out that E. fetida was not as strongly impacted by density as compared to L. terrestris . Population growth rate in E. fetida was hardly affected at low and moderate density, being reduced only at high level, this compares to L. terrestris where even relatively small density effects resulted in a strong negative effect on population growth rate. Our findings indicate that density-dependent regulation in earthworms can be quantified using life-history analysis. The outcomes are in agreement with empirical field observations for populations (i.e. L. terrestris occurs ar low density, E. fetida at high density). Consideration of the potential importance of Nicholsonian density dependence for field populations of these two species in light of their known biology however produces counterintuitive conclusions. In E. fetida , although density tolerant, rapid population growth may mean this species may be subject to density dependeny regulation. In L. terrestris , although density sensitive, complex behavioural ecology (surface activity, territoriality) may limit of feedback influence on population size.     

Peptides and multiple antigen peptides from Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase: preparation, immunogenicity and immunoprotective capacity in C57BL/6 mice.

Four monoepitopic MAPs (MAP A, B, C and E) and one bis-diepitopic MAP B-E derived fromthe primary sequence of Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase, previously tested in BALB/c mice, were examined for their immunogenicity and protective capacity in C57BL/6 mice. Despite multimerization into MAPs, MAP Aand MAP C were poorly immunogenic. In contrast toBALB/c mice, MAP E was non-immunogenic in C57BL/6 mice. Peptide B in the form of MAP B orbis-diepitopic MAPB-E elicited immune responses in C57BL/6 mice that were associated with a significant decrease in worm burden. The MAPs were prepared by the stepwise solid-phase peptide synthesis using Boc/Bzl chemistry, successfully purified on the RP-HPLC column and characterized by RP-HPLC, HPCE and MALDI-TOF MS techniques. A general strategy for MAPs purification is discussed here and the purification of MAP Band MAP E is documented in detail.     

Transformation of sensory organs by mutations of the cut locus of D. melanogaster

The identities of two types of sensory organs in the body wall of Drosophila, namely the external sensory organs and the chordotonal organs, are under genetic control. Embryonic lethal mutations in the cut gene complex transform the external sensory organs into chordotonal organs. The neurons, as well as the support cells forming the external sensory structures, change their morphological and antigenic characteristics to those of chordotonal organs, providing genetic evidence that these two types of sensory organs are homologous. Similar transformations of external sensory organs are observed in adult mosaic flies. Analysis of mosaic larvae and flies suggests that the cut gene function is required either in or near external sensory organs in order for them to acquire their correct identity.     

Attraction ofCarpoglyphus lactis (Acarina: Acaridae) to selected organic compounds

Various chemicals commonly found in food (twelve monosaccharides, nine sugar alcohols, twenty triglycerides, eleven unsaturated fatty acids and nine saturated fatty acids) were tested in different concentrations for their ability to attract and sustain feeding by the dried-fruit mite,Carpoglyphus lactis (L.). Oleic acid, -d-glucose and some triglycerides act as phagoincitants and phagostimulants, whiled-fucose and trilaurin are phagodeterrents.     

Moniliformin,deoxynivalenol, 3Acetyldeoxynivalenol and zearalenone — Mycotoxins associated with corn cob fusariosis in Poland

An isolated rat liver was perfused with deoxynivalenol (DON) at a dose of 3 mg in a recirculating perfusion system. To identify glucuronide conjugates equal amounts of bile samples, perfusate and liver homogenates were incubated with and without (control) a β-glucuronidase preparation and analyzed by thin layer chromatography and capillary gas liquid chromatography — chemical ionization mass spectrometry. A total of 40.4% of the administered dose of DON was found to be conjugated with glucuronic acid (perfusate 20.4%, bile 19.2%, liver 0.8%), while only 1.3% of the parent DON (perfusate 1.1%, bile 0.2%) was detected. The cleavage of DON-glucuronide was demonstrated by incubating DON-glucuronide containing bile samples with intestine contents under anaerobic conditions.     

Denitrification with methanol in the presence of high salt concentrations and at high pH levels

Summary In the combined ion exchange/biological denitrification process for nitrate removal from ground water, in which nitrate is removed by ion exchange, the resins are regenerated in a closed circuit by a biological denitrification reactor. This denitrification reactor eliminates nitrate from the regenerant. Methanol is used as electron donor for biological denitrification. To obtain sufficient regeneration of the resins within a reasonable time, high NaCl or NaHCO₃ concentrations (10–30 g/l) in the regenerant are necessary. High NaHCO₃ concentrations affected the biological denitrification in three ways: a) a slight decrease in denitrification capacity (30%) was observed; b) the yield coefficient and CH₃OH/NO₃ ^-–N ratio decreased. When high NaHCO₃ concentrations (above 10g NaHCO₃/l) were used, the yield coefficient was 0.10–0.13 g VSS/g NO₃ ^-–N and the CH₃OH/NO₃ ^-–N ratio was 2.00–2.03 g/g; c) high NaHCO₃ concentrations influenced nitrite production. Nitrite is an intermediate product of biological denitrification and with rising NaHCO₃ concentrations nitrite accumulation was suppressed. This was explained by the effect of high NaHCO₃ concentrations on the pH in the microenvironment of the denitrifying organisms. High NaCl concentrations also resulted in a slight decrease in denitrification capacity, but the second and third effects were not observed in the presence of high NaCl concentrations.Although the pH in the regenerant will rise as a result of biological denitrification, the capacity of a denitrification reactor did not decrease significantly when a pH of 8.8–9.2 was reached.     

Release of dormancy during after-ripening of Agrostemma githago seeds

Freshly harvested seeds of Agrostemma githago L. do not germinate when they are imbibed at 20°C. The block is located in the embryo and is relased by dry storage at 20°C (after-ripening). Freshly harvested seeds complete only a small part of the processes that occur in after-ripened seeds during the lag phase prior to germination (radicle protrusion). After-ripening removed the block on lag phase processes much faster than the block on germination. This was shown both by direct determinations of the completion of lag phase processes and by measurements of the rate of axial protein synthesis, which approximately doubles when seeds are progressing through the lag phase. It is concluded that the percentage germination does not adequately reflect the extent to which the dormancy mechanism has been overcome.