Interrelationships of Leucine and Glutamate Metabolism in Cultured Astrocytes

Abstract: The aim was to study the extent to which leu-cine furnishes α-NH₂ groups for glutamate synthesis via branched-chain amino acid aminotransferase. The transfer of N from leucine to glutamate was determined by incubating astrocytes in a medium containing [¹⁵N]leucine and 15 unlabeled amino acids; isotopic abundance was measured with gas chromatography-mass spectrometry. The ratio of labeling in both [¹⁵N]glutamate/[¹⁵N]leucine and [2-¹⁵N]glutamine/[¹⁵N]leucine suggested that at least one-fifth of all glutamate N had been derived from leucine nitrogen. At the same time, enrichment in [¹⁵N]leucine declined, reflecting dilution of the ¹⁶N label by the unlabeled amino acids that were in the medium. Isotopic abundance in [¹⁶N]-isoleucine increased very quickly, suggesting the rapidity of transamination between these amino acids. The appearance of ¹⁵N in valine was more gradual. Measurement of branched-chain amino acid transaminase showed that the reaction from leucine to glutamate was approximately six times more active than from glutamate to leucine (8.72 vs. 1.46 nmol/min/mg of protein). However, when the medium was supplemented with α-ketoisocaproate (1 mM), the ketoacid of leucine, the reaction readily ran in the “reverse” direction and intraastrocytic [glutamate] was reduced by ～50% in only 5 min. Extracellular concentrations of α-ketoisocaproate as low as 0.05 mM significantly lowered intracellular [glutamate]. The relative efficiency of branched-chain amino acid transamination was studied by incubating astrocytes with 15 unlabeled amino acids (0.1 mM each) and [¹⁵N]glutamate. After 45 min, the most highly labeled amino acid was [¹⁵N]alanine, which was closely followed by [¹⁵N]leucine and [¹⁵N]isoleucine. Relatively little ¹⁵N was detected in any other amino acids, except for [¹⁵N]serine. The transamination of leucine was ～17 times greater than the rate of [1-¹⁴C]leucine oxidation. These data indicate that leucine is a major source of glutamate nitrogen. Conversely, reamination of a-ketoisocaproate, the ketoacid of leucine, affords a mechanism for the temporary “buffering” of intracellular glutamate.     

Accumulation of cesium and radiocesium in forest litter in selected regions of poland and its influence on litter-to-mushroom transfer factor

The aim of this work was to check whether the stable cesium content in forest litter affects the value of radiocesium from litter-to-mushroom transfer factorTf or not. Total cesium in litter, measured by AAS, varied from 0.1–2.7 μg/g. These data, combined with earlier results for mushrooms, showed no simple correlation forTf. More complex relationships provided very high correlation coefficients, but their validity needs further investigation.     

Variations in stream water uptake by Eucalyptus camaldulensis with differing access to stream water

The stable isotopes ²H and ¹⁸O were used to determine the water sources of Eucalyptus camaldulensis at three sites with varying exposure to stream water, all underlain by moderately saline groundwater. Water uptake patterns were a function of the long-term availability of surface water. Trees with permanent access to a stream used some stream water at all times. However, water from soils or the water table commonly made up 50% of these trees' water. Trees beside an ephemeral stream had access to the stream 40–50% of the time (depending on the level of the stream). No more than 30% of the water they used was stream water when it was available. However, stream water use did not vary greatly whether the trees had access to the stream for 2 weeks or 10 months prior to sampling. Trees at the third site only had access to surface water during a flood. These trees did not change their uptake patterns during 2 months inundation compared with dry times, so were not utilising the low-salinity flood water. Pre-dawn leaf water potentials and leaf ¹³C measurements showed that the trees with permanent access to the stream experienced lower water stress and had lower water use efficiencies than trees at the least frequently flooded site. The trees beside the ephemeral stream appeared to change their water use efficiency in response to the availability of surface water; it was similar to the perennial-stream trees when stream water was available and higher at other times. Despite causing water stress, uptake of soil water and groundwater would be advantageous to E. camaldulensis in this semi-arid area, as it would provide the trees with a supply of nutrients and a reliable source of water. E. camaldulensis at the study site may not be as vulnerable to changes in stream flow and water quality as previously thought.     

Yolk sphere formation is initiated in oocytes before development of patency in follicles of the moth,Plodia interpunctella

We describe a provitellogenic stage, a previously unrecognized stage of follicle development in moths, and show that oocytes begin yolk sphere formation prior to the development of patency by the follicular epithelium. The vitellogenic activities of follicles from pharate adult femalePlodia interpunctella (Hübner) were determined by visualizing the subunits of vitellin (YP1 and YP3) and the follicular epithelium yolk protein (YP2 and YP4) using monospecific antisera to each subunit to immunolabel whole-mounted ovaries or ultrathin sections. At 92 h after pupation, yolk spheres that contained only YP2 began to proliferate in the oocytes. The inter-follicular epithelial cell spaces were closed at 92 h making vitellogenin inaccessible to the oocyte, and consequently, the vitellin subunits were not observed in the yolk spheres. YP2 uptake most likely occurred across the brush border from the follicular epithelial cells to the oocyte at this time. At 105 h, the inter-follicular epithelial cell spaces appeared closed yet trace amounts of labeling for vitellin were observed in the spaces and also in the yolk spheres along with YP2. Equivalent labeling for all four YPs in yolk spheres was finally observed at 112 h after pupation when the follicular epithelium had become patent. These data indicate that the provitellogenic stage is an extended transition period between the previtellogenic and vitellogenic stages that lasts for approximately 13 h, and it is marked at the beginning by YP2 yolk sphere formation in the oocyte and at the end by patency in the follicular epithelium.     

A novel isotope labeling protocol for bacterially expressed proteins

Summary A novel protocol for isotopically labeling bacterially expressed proteins is presented. This method circumvents problems related to poor cell growth, commonly associated with the use of minimal labeled media, and problems with protein induction encountered, less commonly, when using enriched labeled media. The method involves initially growing the bacterial cells to high optical density in a commercially available enriched labeled medium. Following a suitable growth period, the cells are transferred to a different (minimal) labeled medium, appropriate for induction. The method is demonstrated using the protein melanoma growth stimulating activity (MGSA).     

Effect of endotoxin on lipid order and motion in erythrocyte membranes

Electron paramagnetic resonance employing a lipid-specific spin label has been used to investigate the molecular effects of endotoxin on the physical state of bilayer lipids in rat erythrocyte membranes. When added at a concentration as low as 40 μg/ml to whole blood (plasma plus leukocytes present), decreased membrane lipid motion was found in subsequently washed and spin-labeled intact erythrocytes (P < 0.02). However, if endotoxin were added to washed, plasma plus leukocyte-free intact erythrocytes, no change in the motion of the spin label was found, suggesting that plasma-soluble substances and/or leukocytes are required to produce the change in the physical state of lipids. The decreased lipid motion found in these studies is discussed with reference to the known decreased deformability of endotoxin-treated red cells and to the pathogenesis of sepsis.     

Exogenous 13C glucose oxidation during exercise: North American vs Western European studies

The purpose of this study was to test the hypothesis that the well-documented changes in background ¹³C enrichment of expired CO₂ observed in response to exercise and carbohydrate ingestion, in subjects living on a North American diet, are not present in subjects living on a Western European diet. The experimental protocol used by Pirnay et al. in 1977 and by Krzentowski et al. in 1984 in subjects living on a Western European diet (4 h of exercise on a treadmill at 50% VO_2max with ingestion of 100 g of glucose in 400 ml of water) was duplicated as closely as possible in six subjects living on a North American diet. The actual amounts of exogenous glucose oxidized, computed with a high artificial ¹³C enrichment of glucose (+189.7 ¹³C PDB-1) which allows one to neglect the 1–2 changes in ¹³C background, were [mean (SEM)] 54.7 (5.4) and 84.2 (3.4) g over 2 h and 4 h of exercise, respectively. These values compare well with data computed by Pirnay et al. [56.6 (13.1) and 94.9 (4.2) g] and by Krzentowski et al. [55.0 (6.2) and 88.0 (4.5) g] using a natural enrichment of glucose (–11.21 and –10.63 ¹³C PDB-1, respectively) assuming no change in ¹³C background in their Western European subjects. Under the same assumption and using a natural enrichment of glucose (–11.30 ¹³C PDB-1) the oxidation of exogenous glucose was overestimated by 30–40% in our North American subjects. This result indicates that because of a lower input of ¹³C in their diet, the difference between the isotopic composition of carbohydrate and fat stores are smaller, and changes in ¹³C background are small or absent in response to moderate workload in Western European subjects, when compared to their North American counterparts.     

Enhancement of Antigenic Site Detection with Gold Labeled Secondary and Tertiary Antibodies Using the Immunogold-Silver Staining Method

We report a modification of the immunogold-silver staining method (IGSS) for localizing hepatic phosphoenolpyruvate carboxykinase (PEPCK) in tissue sections, and we compare the efficacy of localizing the primary antibody with either a 5 nm gold labeled secondary antibody or 5 nm gold labeled secondary and tertiary antibodies. Light microscope examination of 10 μm frozen sections demonstrated that the use of combined secondary and tertiary gold labeled antibodies was superior to using a secondary gold labeled antibody alone. The increased labeling density (number of colloidal gold particles/antigenic site/cell) achieved by combined gold labeled antibodies was confirmed by electron microscopy. The increased labeling density resulted in a two-thirds reduction in the time needed for the IGSS physical development of the silver shells and less background. We achieved intense specific staining of hepatocytes expressing PEPCK while minimizing background staining. The use of combined secondary and tertiary gold labeled antibodies enhances the signal-to-noise ratio, achieves high resolution and is a suitable method for use in both light and electron microscopy.     

PCR-mediated screening and labeling of DNA from clones

A simplified and economical protocol for DNA library screening and nonradioactive labeling is described. Bacterial clones are lysed in 1% of Triton X-100 and subjected to polymerase chain reaction in the presence of digoxigenin-11-dUTP to screen and simultaneously to label the DNA inserts. Bacteriallysates are stable in storage at −20°C and can be used repeatedly for PCR-mediated labeling. In this protocol, very low concentrations of dNTP, digoxigenin-dUTP, and primers are used in combination with a reduced reaction volume. This will considerably reduce the expense of screening and labeling bacterial clones and facilitate the exchange of DNA probes among laboratories.     

Trophic ecology of juvenile bull sharks (Carcharhinus leucas) in the Coyote estuary,Costa Rica

Bull shark (Carcharhinus leucas) is a near-threatened elasmobranch species capable of moving between the fresh and salty waters of tropical and subtropical coastal areas, for which we still lack important ecological information. During their first years of life, bull sharks use estuarine systems as nursery areas, making them highly susceptible to environmental and anthropogenic pressures. We studied the trophic ecology of juveniles found in the Coyote estuary, a potential nursery area in Costa Rica, to understand the potential impact of further bull shark declines and gain knowledge that could aid in their conservation. We analysed the trophic ecology of juvenile bull sharks [81–103 cm total length (TL)] in the Coyote estuary, Costa Rica, using stable isotopes of δ¹⁵N and δ¹³C. Since one problem using this technique in juveniles is the confounding effect of the maternal signature, we sampled different tissues (muscle and plasma), verified the status of the shark's umbilical scar and identified the size at which the isotope signature is a result of the animal's current diet. The isotopic values of the muscle tissue reflected the maternal isotopic signature. In contrast, plasma values reflected the diet of juvenile bull sharks >95 cm TL and with a closed umbilical scar. Juvenile bull sharks fed primarily on teleost fishes of the order Anguilliformes and Siluriformes, and have a high trophic position (≥4.0) in the Coyote estuary. Our findings suggest that this estuary is an important feeding site for juvenile bull sharks of the Pacific of Costa Rica. Thus, the protection of essential habitats such as the Coyote estuary will benefit not only bull shark conservation, but also the conservation of an array of fish species that also use this habitat as a rookery, many of which are of commercial interest.