Chemiluminescent acridinium esters (AEs) permit the development of high sensitivity ligand binding assays due to a combination of high intensity light emission and very low backgrounds. Here these advantages are exploited for use in homogeneous nucleic acid hybridisation assays using quenched chemiluminescence. AE chemiluminescence is conventionally initiated at highly alkaline pH. Novel "active" AEs were designed that permit initiation under conditions compatible with maintenance of nucleic acid hybrids (i.e. pH less than 9). Methyl red was found to be a dark quencher species capable of functioning at this pH. Practical application of the chemiluminescence quenching assay system has been demonstrated using two model nucleic acid hybridisation assays based on intra- and intermolecular emitter/quencher pairs.

The type II fatty acid synthases (FASs) of higher plants (and Escherichia coli) contain three condensing enzymes called beta-ketoacyl-ACP synthases (KAS), where ACP is acyl-carrier-protein. We have used novel derivatives of the antibiotic thiolactomycin to inhibit these enzymes. Overall de novo fatty acid biosynthesis was measured using [1-(14)C]acetate substrate and chloroplast preparations from pea leaves, and [1-(14)C]laurate was used to distinguish between the effects of the inhibitors on KAS I from those on KAS II. In addition, the activities of these enzymes, together with the short-chain condensing enzyme, KAS III, were measured directly. Six analogues were tested and two, both with extended hydrocarbon side chains, were found to be more effective inhibitors than thiolactomycin. Incubations with chloroplasts and direct assay of the individual condensing enzymes showed that all three compounds inhibited the pea FAS condensing enzymes in the order KAS II > KAS I > KAS III. These results demonstrate the general activity of thiolactomycin and its derivatives against these FAS condensation reactions, and suggest that such compounds will be useful for further detailed studies of inhibition and for use as pharmaceuticals against Type II FASs of pathogens.

Induction of fatty acid desaturation is very important for the temperature adaptation of poikilotherms. However, in oxygen-limited late-exponential-phase Acanthamoebacastellanii cultures, oxygen alone was able to induce increased activity of a fatty acid desaturase that converts oleate into linoleate and which has been implicated in the temperature adaptation of this organism. Experiments with Δ10-nonadecenoate showed that the enzyme is an n-6 desaturase rather than a Δ12-desaturase. It also used preferentially 1-acyl-2-oleoyl-phosphatidylcholine as substrate and NAD(P)H as electron donor. The involvement of cytochrome b5 as an intermediate electron carrier was shown by difference spectra measurements and anti-(cytochrome b5) antibody experiments. Of the three protein components of the desaturase complex, oxygen only increased the activity of the terminal (cyanide-sensitive) protein during n-6 desaturase induction. The induction of this terminal protein paralleled well the increase in overall oleate n-6 desaturation. The ability of oxygen to induce oleate desaturase independently of temperature in this lower eukaryotic animal model is of novel intrinsic interest, as well as being important for the design of future experiments to determine the molecular mechanism of temperature adaptation in poikilotherms.

Induction of a microsomal oleate v v12 (n-6) desaturase which is mainly responsible for an increase in membrane lipid unsaturation at low temperature has been observed in the freeliving amoeba Acanthamoeba castellanii. In this study we show that the enzyme can also be regulated by oxygen independently of temperature in batch cultures grown to O P -limitation. Raising the oxygen concentration from below the lower limit of detection ( 6 0.1 W WM) to approximately air-saturation (230 W WM), whilst maintaining the growth temperature constant (30³C), increased lipid unsaturation and elevated n-6-desaturase activity 2.3-fold. Addition of the protein synthesis inhibitor, anisomycin, showed that increased desaturase activity was due to new protein synthesis rather than activation of pre-existing enzyme. These observations are important for future studies of the mechanism of temperature adaptation in poikilotherms.z 1998 Federation of European Biochemical Societies.

Aims: To develop rapid means of distinguishing between cysts and trophozoites of the opportunistic pathogen, Acanthamoeba castellanii, the causative agent of keratitis. Methods and Results: Fluorescence of Congo Red, Calco¯or White was speci®c for the endocyst wall; trophozoites did not become¯uorescent. The anionic oxonol dye, DiBAC 4 (3), did not penetrate the cytoplasmic membrane after short-term (<5 min) exposure, whereas cysts are permeable and become¯uorescent. Confocal scanning laser microscopy con®rmed these properties and large populations of organisms were analysed by¯ow cytometry. Conclusions: These data provide a rapid alternative to traditional haemocytometer or plate counts for discrimination of trophozoites from cysts. Signi®cance and Impact of the Study: Rapid and precise determination of the growth cycle of a dangerous ocular pathogen.