Inhibition of sterol synthesis by delta 5-sterols in a sterol auxotroph of yeast defective in oxidosqualene cyclase and cytochrome P-450

Synthesis of ergosterol is demonstrated in the GL7 mutant of Saccharomyces cerevisiae. This sterol auxotroph has been thought to lack the ability to synthesize sterols due both to the absence of 2,3-oxidosqualene cyclase and to a heme deficiency eliminating cytochrome P-450 which is required in demethylation at C-14. However, when the medium sterol was 5 alpha-cholestan-3 beta-ol, 5 alpha-cholest-8(14)-en-3 beta-ol, or 24 beta-methyl-5 alpha-cholest-8(14)-en-3 beta-ol, sterol synthesis was found to proceed yielding 1-3 fg/cell of ergosterol (24 beta-methylcholesta-5,7,22E-trien-3 beta-ol). Ergosterol was identified by mass spectroscopy, gas and high performance liquid chromatography, ultraviolet spectroscopy, and radioactive labeling from [3H]acetate. Except for some cholest-5-en-3 beta-ol (cholesterol) which was derived from the 5 alpha-cholestan-3 beta-ol, the stanol and the two 8(14)-stenols were not significantly metabolized confirming the absence of an isomerase for migration of the double bond from C-8(14) to C-7. Drastic reduction of ergosterol synthesis to not more than 0.06 fg/cell was observed when the medium sterol either had a double bond at C-5, as in the case of cholesterol, or could be metabolized to a sterol with such a bond. Thus, both 5 alpha-cholest-8(9)-en-3 beta-ol and 5 alpha-cholest-7-en-3 beta-ol (lathosterol) were converted to cholesta-5,7-dien-3 beta-ol (7-dehydrocholesterol), and the presence of the latter dienol depressed the level of ergosterol. The most attractive of the possible explanations for our observations is the assumption of two genetic compartments for synthesis of sterols, one of which has and one of which has not been affected by the two mutations. The ability, despite the mutations, to synthesize small amounts of ergosterol which could act to regulate the cell cycle may also explain why this mutant can grow aerobically with cholesterol (acting in the bulk membrane role) as the sole exogenous sterol.     

Control of fungal sterol C-24 transalkylation: importance to developmental regulation

When cultures of Gibberella fujikuroi are incubated with 24-epiiminolanosterol the introduction of a methyl group into sterol side chains at C-24 is blocked inducing a mycelial accumulation of lanosterol and 24-desalkylsterols, i.e., having the cholestane side chain. The altered sterol composition lead to aberrant mycelial membranes resulting in growth inhibition. A compensatory physiological response to the ensuing hyphal death was induction of asexual sporulation. The results are interpreted to imply that regulation of sterol C-24 transalkylation may be a mechanism to mediate life cycle events of fungi.     

Mutagen formation in fried meat emulsion containing various amounts of creatine

The formation of mutagens in fried, minced meat emulsion was evaluated by the Ames Salmonella test system. Exogenous addition of creatine to the emulsion prior to frying greatly enhanced the mutagenicity of the emulsion. Addition of 5% creatine resulted in a 40-fold increase in the mutagenicity of the fried meat emulsion in the frameshift test strain, TA98, and in a 8-fold increase in the base substitution test strain, TA100. The present results suggest that creatine is an important factor in the mutagen formation in fried meat products.     

Studies on the C-24 configurations of Δ7-sterols in theseeds of Cucurbita maxima

Uncertainties surrounding the structures of the Δ⁷-sterols in the seeds of Cucurbita maxima have been resolved. Seven components were found by TLC, GLC, HPLC, mass spectrometry and ¹H NMR. They were 24β-ethyl-5α-cholesta-7,22,25(27)-trien-3β-ol, 24β-ethyl-5α-cholesta-7,25(27)-dien-3gb-ol, avenasterol, spinasterol, 24-dihydrospinasterol, 24ζ-methyllathosterol and 25(27)-dehydrofungisterol. The ¹H NMR spectra indicated that the sterols with an ethyl substituent at C-24 occurred in the absence of their C-24 epimers. This seems to be the first instance of the detection of 25(27)-dehydrofungisterol in a higher plant.     

Centriole as microtubule-organizing centers for marginal bands of molluscan erythrocytes

The erythrocytes of blood clams (arcidae) are flattened, elliptical, and nucleated. They contain elliptical marginal bands (MBs) of microtubules, each physically associated with a pair of centrioles marginal bands (MBs) of microtubles, each physically associated with a pair of centrioles (Cohen, W., and I. Nemhauser, 1980, J. Cell Biol., 86:286-291). The MBs were found to be cold labile in living cells, disappearing within 1-2 h at 0 degrees C. After the cells had been rewarmed for 1-2 h, continuous MBs with associated centrioles were once again present. Time-course studies utilizing phase contrast, antitubulin immunofluorescence, and electron microscopy of cytoskeletons prepared during rewarming revealed structural evidence of centriole participation in MB reassembly. At the earliest stage of reassembly, a continuous MB was not present. Instead, relatively short and straight microtubules focused on a pointed centriolar “pole,” and none were present elsewhere in the cytoskeleton. Thin continuous MBs then formed, still pointed in the centriolar region. Subsequently, the MBs regained ellipticity, with their thickness gradually increasing but not reaching that of controls even after several hours of rewarming. At these later time points, microtubules still radiated from the centrioles and joined the MBs some distance away. In the presence of 0.1 mM colchicines, MB reassembly was arrested at the pointed stage. Electron microscopic observations indicate that pericentriolar material is involved in microtubule nucleation in this system, rather than the centriolar triplets directly. The results suggest a model in which the centrioles and associated material nucleate assembly and growth of microtubules in diverging directions around the cell periphery. Microtubules of opposite polarity would then pass each other at the end of the cell distal to the centrioles, with continued elongation eventually closing the MB ellipse behind the centriole pair.     

A reappraisal of sterol biosynthesis and metabolism in aphids

Aphids of Schizaphis graminum (Rondani) (biotype C) reared on its host-plant, Sorghum bicolor (L.) Moench, sequestered campesterol, stigmasterol and sitosterol. Aphids reared for 72 hr on holidic diets supplemented with [4-¹⁴C]-sitosterol contained both [¹⁴C]-sitosterol and [¹⁴C]-cholesterol, indicating that these aphids are capable of dealkylation at C-24. When aphids were reared on artificial diets containing [2-¹⁴C]-mevalonic acid, no detectable amounts of radioactively labelled desmethyl sterols, nor metabolic intermediates in sterol synthesis (i.e. squalene, 2,3-oxidosqualene, 4,4-dimethyl and 4-monomethyl sterols) were found to accumulate in their tissues. The relevance of these findings to previous research suggesting the ability of aphids, via their symbiotes, to synthesize sterols is discussed.     

New biologically active hybrid bacteriocins constructed by combining regions from various pediocin-like bacteriocins: the C-terminal region is important for determining specificity.

The pediocin-like bacteriocins, produced by lactic acid bacteria, are bactericidal polypeptides with very similar primary structures. Peptide synthesis followed by reverse-phase and ion-exchange chromatographies yielded biologically active pediocin-like bacteriocins in amounts and with a purity sufficient for characterizing their structure and mode of action. Despite similar primary structures, the pediocin-like bacteriocins, i.e., pediocin PA-1, sakacin P, curvacin A, and leucocin A, differed in their relative toxicities against various bacterial strains. On the basis of the primary structures, the polypeptides of these bacteriocins were divided into two modules: the relatively hydrophilic and well conserved N-terminal region, and the somewhat more diverse and hydrophobic C-terminal region. By peptide synthesis, four new biologically active hybrid bacteriocins were constructed by interchanging corresponding modules from various pediocin-like bacteriocins. All of the new hybrid bacteriocin constructs had bactericidal activity. The relative sensitivity of different bacterial strains to a hybrid bacteriocin was similar to that to the bacteriocin from which the C-terminal module was derived and quite different from that to the bacteriocin from which the N-terminal was derived. Thus, the C-terminal part of the pediocin-like bacteriocins is an important determinant of the target cell specificity. The synthetic bacteriocins were more stable than natural isolates, presumably as a result of the absence of contaminating proteases. However, some of the synthetic bacteriocins lost activity, but this was detectable only after months of storage. Mass spectrometry suggested that this instability was due to oxidation of methionine residues, resulting in a 10- to 100-fold reduction in activity.     

Transposition in Lactobacillus sake and its abolition of lactocin S production by insertion of IS1163, a new member of the IS3 family.

This report presents the nucleotide sequence and insertional activity of IS1163, which is a new member of the IS3 family of transposable elements. Analysis of spontaneous mutants of the lactocin S-producing Lactobacillus sake strain L45 show that the bacteriocin-negative phenotype is due to either loss of the producer plasmid or the insertion of IS1163 into the lactocin S operon (las operon). The data further show that insertional inactivation of the lactocin S operon is the result of a transposition event involving a chromosomally located donor copy of IS1163. Although the insertions described are clustered within a 250-bp region of the las operon, there are no features of the insertion sites to suggest target-specific insertion of IS1163. The overlapping, frameshifted organization of the two major open reading frames found in IS1163 is typical for the IS3 family, but the structure of the putative frameshift region includes features which distinguish IS1163 from the other members of the group. The insertional activity of IS1163 in L. sake L45 has aided in identifying regions of pCIM1 essential for lactocin S production and may have further practical applications as a mutational tool in L. sake.     

The consequences of a drastic fish stock reduction in the large and shallow Lake Wolderwijd,The Netherlands. Can we understand what happened?

In 1990 an experiment started in the large and shallow lake Wolderwijd (2700 ha, mean depth 1.5 m) to improve the water quality. About 75% of the fish stock was removed (425 000 kg fish). The fish was mainly composed of bream and roach. In May 600000 young pikes (3–4 cm) were introduced.In May 1991 the water became very clear (Secchi depth 1.8 m) during a spring bloom of large Daphnia. Then the grazing by zooplankton was eight times higher than the primary production of algae and the total suspended matter concentration became very low. Compared to the situation before the fish reduction, the grazing had increased only slightly, while the primary production had decreased significantly in early spring. The fish stock reduction might have contributed to the reduction in primary production by a reduced internal nutrient load. The clear water period lasted six weeks. Daphnia disappeared in July due to food limitation, the algal biomass increased and the Secchi depth became 50 cm. Daphnia did not recover during summer, due to predation that was not caused by 0 + fish but by the mysid shrimp Neomysis integer. Neomysis could develop abundantly, because of the reduced biomass of the predator perch. The production of young fish had been low because of the cold spring weather. The cold weather was probably also responsible for the slow increase in density of macrophytes. After 1991, perch probably can control Neomysis. Due to lack of spawning places and shelter for 0 + pike, pike was probably not able to control the production of 0 + fish. In a lake of this scale, it will not be easy to get more than 50% coverage of macrophytes, which seems necessary to keep the algal biomass low by nutrient competition. Therefore, we expect also in the future a decrease in transparency in the summer. Locally, especially near Characeae, the water might stay clear.     

Five fundamental ways in which complex food webs may spiral out of control

Theory suggests that increasingly long, negative feedback loops of many interacting species may destabilize food webs as complexity increases. Less attention has, however, been paid to the specific ways in which these ‘delayed negative feedbacks’ may affect the response of complex ecosystems to global environmental change. Here, we describe five fundamental ways in which these feedbacks might pave the way for abrupt, large-scale transitions and species losses. By combining topological and bioenergetic models, we then proceed by showing that the likelihood of such transitions increases with the number of interacting species and/or when the combined effects of stabilizing network patterns approach the minimum required for stable coexistence. Our findings thus shift the question from the classical question of what makes complex, unaltered ecosystems stable to whether the effects of, known and unknown, stabilizing food-web patterns are sufficient to prevent abrupt, large-scale transitions under global environmental change.