Glutamine and Aspartate Loading of Synaptosomes: A Reevaluation of Effects on Calcium-Dependent Excitatory Amino Acid Release

Guinea-pig cerebral cortical synaptosomes were preincubated for 60 min with 100 microM D-aspartate, L-aspartate, or L-glutamate. The total D- plus L-aspartate content of the synaptosomal fraction increased to 235%, 195%, or 164%, respectively, of the control. Despite this no increase was seen in the very low KCl evoked, Ca2+-dependent release of aspartate. Preincubation with the three amino acids changed the synaptosomal glutamate content to 78% (D-aspartate), 149% (L-aspartate), or 168% (L-glutamate) of control. However there was no statistically significant effect of these preincubations on the extent of Ca2+-dependent glutamate release. Thus the Ca2+-dependent release of aspartate and glutamate is not determined by the total synaptosomal content of these amino acids. The addition of 0.1-0.5 mM glutamine to the incubation caused a massive appearance of glutamate in the extrasynaptosomal medium. Analysis of specific activities showed that glutamine was hydrolysed directly by an extrasynaptosomal glutaminase, and that intrasynaptosomal glutamate was predominantly labelled by uptake of this glutaminase-derived glutamate. No increase was seen in the extent of Ca2+-dependent release of glutamate (by fluorimetry) either after preincubation with glutamine or in the continued presence of glutamine. Thus we are unable to confirm reports that glutamine expands the transmitter pool of glutamate. The extrasynaptosomal glutaminase activity in the synaptosomal preparation was inhibited by Ca2+ and activated by phosphate. Identical kinetics were obtained with "free" brain mitochondria, confirming the origin of the glutamine-derived glutamate.     

Validation of signature polarlipid fatty acid biomarkers for alkane-utilizing bacteria in soils and subsurface aquifer materials

Abstract Extractable cell membrane-derived polarlipid ester-linked fatty acids (PLFA) obtained from aerated soils gassed with methane or propane and from methane- and propane-oxidizing bacteria isolated from the soils were analyzed by capillary gas chromatography/mass spectrometry. Exposure of aerated soils to methane resulted in the formation of a high proportion of an unusual 18-carbon mono-unsaturated PLFA, 18:lw8c. High proportions of this fatty acid biomarker are found in monocultures from this soil grown in minimal media with methane. This PLFA has been previously established as associated with authentic type II methane-oxidizing bacteria. The microbiota in aerated soils exposed to hydrocarbons containing propane, formed a suite of PLFA characterized by high proportions of a 16-carbon mono-unsaturated acid, 16:lw6c, and an 18-carbon saturated fatty acid with an additional methyl branch at the 10 position, 10 Me 18:0. This PLFA pattern has been detected in several monocultures enriched from the soil with propane-amended minimal media. The correspondence of high proportions of these unusual mono-unsaturated PLFA in the isolated monocultures and in situ in the soils after stimulation with the appropriate hydrocarbon is a strong validation of the utility of these biomarkers in defining the community structure of the surface soil microbial community.     

The meaning of interaction parameters in two-state protein complexes

The theory of interaction parameters has thus far been based on the free-energy relationships in the formation of ternary complexes formed between a pair of ligands and a protein molecule. The concept has been formulted in terms of a thermodynamic square comprised of the free protein, the two binary complexes, and the ternary complex. However, an increasing number of proteins have been found to exist as equilibrium mixtures of two macrostates. The equilibrium constants for such two-state transitions vary quite considerably between the various binary and ternary complexes of a given protein. We show here that the interpretations of interaction parameters in such two-state systems, requiring the use of a thermodynamic cube, are much more complex than those based on the classic thermodynamic square commonly employed. We demonstrate the use of enthalpies of interaction and heat capacities of interaction to analyze the source of observed free enerigies of interaction in such systems. Specifically, we find that measured negative interaction parameters may arise simply from the inability of a system to achieve all of the positive component effects anticipated by the conventional formulation.     

Qualitative importance of the microbial loop and plankton community structure in a eutrophic lake during a bloom of cyanobacteria

Plankton community structure and major pools and fluxes of carbon were observed before and after culmination of a bloom of cyanobacteria in eutrophic Frederiksborg Slotssø, Denmark. Biomass changes of heterotrophic nanoflagellates, ciliates, microzooplankton (50 to 140 μm), and macrozooplankton (larger than 140 μm) were compared to phytoplankton and bacterial production as well as micro- and macrozooplankton ingestion rates of phytoplankton and bacteria. The carbon budget was used as a means to examine causal relationships in the plankton community. Phytoplankton biomass decreased and algae smaller than 20 μm replacedAphanizomenon after the culmination of cyanobacteria. Bacterial net production peaked shortly after the culmination of the bloom (510 μg C liter^?1 d^?1 and decreased thereafter to a level of approximately 124 μg C liter^?1 d^?1. Phytoplankton extracellular release of organic carbon accounted for only 4–9% of bacterial carbon demand. Cyclopoid copepods and small-sized cladocerans started to grow after the culmination, but food limitation probably controlled the biomass after the collapse of the bloom. Grazing of micro- and macrozooplankton were estimated from in situ experiments using labeled bacteria and algae. Macrozooplankton grazed 22% of bacterial net production during the bloom and 86% after the bloom, while microzooplankton (nauplii, rotifers and ciliates larger than 50 μm) ingested low amounts of bacteria and removed 10–16% of bacterial carbon. Both macro-and microzooplankton grazed algae smaller than 20 μm, although they did not control algal biomass. From calculated clearance rates it was found that heterotrophic nanoflagellates (40–440 ml^?1) grazed 3–4% of the bacterial production, while ciliates smaller than 50 μm removed 19–39% of bacterial production, supporting the idea that ciliates are an important link between bacteria and higher trophic levels. During and after the bloom ofAphanizomenon, major fluxes of carbon between bacteria, ciliates and crustaceans were observed, and heterotrophic nanoflagellates played a minor role in the pelagic food web.     

Rapid detection of chromosome aneuploidies in uncultured amniocytes by using fluorescence in situ hybridization (FISH).

Herein we report the results of the first major prospective study directly comparing aneuploidy detection by fluorescence in situ hybridization of interphase nuclei with the results obtained by cytogenetic analysis. We constructed probes derived from specific subregions of human chromosomes 21, 18, 13, X, and Y that give a single copy-like signal when used in conjunction with suppression hybridization. A total of 526 independent amniotic fluid samples were analyzed in a blind fashion. All five probes were analyzed on 117 samples, while subsets of these five probes were used on the remaining samples (because of insufficient sample size), for a total of over 900 autosomal hybridization reactions and over 400 sex chromosome hybridization reactions. In this blind series, 21 of 21 abnormal samples were correctly identified. The remaining samples were correctly classified as disomic for these five chromosomes. The combination of chromosome-specific probe sets composed primarily of cosmid contigs and optimized hybridization/detection allowed accurate chromosome enumeration in uncultured human amniotic fluid cells, consistent with the results obtained by traditional cytogenetic analysis.     

A serum-free system for culturing human placental trophoblasts

Summary We have compared hormone production by early gestation and term human placental trophoblasts cultured in Ham's F10 medium containing 10% fetal bovine serum with that by cells cultured in serum-free HB102 medium. Mean daily production of progesterone on Days 3 to 7 was approximately 25% less by both early gestation and term cells cultured in HB102 as compared to Ham's F10, but production was maintained at a stable level for at least 7 d longer than the cells in Ham's. Estradiol production from 10⁻⁶ M dehydroepiandrosterone by both early gestation and term cells was comparable in both media. Human placental lactogen production on Days 3 to 7 was 40% less by cells cultured in HB102. Human chorionic gonadotropin (hCG) output by early gestation cells was also 50% less in HB102 but term cells in HB102 produced twice as much hCG as those in Ham's F10. 3B-Hydroxysteroid dehydrogenase (3BHSD) activity in early gestation and term cells and 11B-hydroxysteroid dehydrogenase (11BHSD) activity of early gestation cultures was comparable in the two media. 11BHSD activity was decreased in the term cultures, and this decrease was more marked in Ham's than in HB102. Sulfatase and aromatase activities in the early gestation cultures were comparable in both media; sulfatase activity was comparable and aromatase activity only 20% less in the term cells cultured in HB102. These results indicate that serum-free HB102 supports differential function of human trophoblast cells and is useful for studies of placental activity for as long as 14 d in culture.     

Lateral diffusion in planar lipid bilayers: a fluorescence recovery after photobleaching investigation of its modulation by lipid composition, cholesterol, or alamethicin content and divalent cations.

In spite of the fact that planar lipid bilayers are still the best-suited artificial membrane system for the study of reconstituted ion channels and receptors, data dealing with their physical characterization, especially as regards dynamics, are scanty. A combined electrical and optical chamber was designed and allowed fluorescence recovery after photobleaching recovery curves to be recorded from stable virtually solvent-free bilayers. D, the lateral diffusion coefficient of N-(7-nitrobenzoyl-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn- glycero-3-phosphoethanolamine, was found to be relatively insensitive to the phospholipid composition (headgroup, chain unsaturation, etc.), whereas inclusion of 33-50% cholesterol in the membrane reduced D by a factor of 2. Divalent cations significantly reduced D of negatively charged bilayers. These results compare well with data gathered on other model and natural systems. In addition, the incorporation of the voltage-dependent pore-former alamethicin did slightly reduce lipid lateral mobility. This study demonstrates the feasibility of such experiments with planar bilayers, which are amenable to physical constraints, and thus offers new opportunities for systematic studies of structure-function relationships in membrane-associating molecules.     

Energy metabolism of Chironomus anthracinus (Diptera: Chironomidae) from the profundal zone of Lake Esrom,Denmark, as a function of body size,temperature and oxygen concentration

The profundal zone of Lake Esrom, Denmark has a dense population of Chironomus anthracinus, which survives 2–4 months of oxygen depletion each summer during stratification. The metabolism of 3rd and 4th instar larvae was examined in regard to variation in biomass and temperature. Respiration at air saturation was described by a curvilinear multiple regression relating oxygen consumption to individual AFDW and temperature. At 10 °C and varying oxygen regimes the O₂ consumption and CO₂ production of 4th instar larvae were almost unaltered from saturation to about 3 mg O₂ l^–1, but decreased steeply below this level. The respiratory quotient increased from 0.82 at saturation to about 3.4 at oxygen concentrations near 0.5 mg O₂ l^–1. This implied a shift from aerobic to partially anaerobic metabolism. At 0.5 mg O₂ l^–1 the total energy production equalled 20% of the rate at saturation of which more than one third was accounted for by anaerobic degradation of glycogen. This corresponded to a daily loss of 12 µg mg AFDW^–1 or approximately 5% of the body reserves. At unchanged metabolic rate the glycogen store would last three weeks, but long term oxygen deficiency causes a further suppression of the energy metabolism in C. anthracinus.