Demonstration of pyruvate recycling in primary cultures of neocortical astrocytes but not in neurons

Pyruvate recycling was studied in primary cultures of mouse cerebrocortical astrocytes, GABAergic cerebrocortical interneurons, and co-cultures consisting of both cell types by measuring production of [4-¹³C]glutamate from [3-¹³C]glutamate by aid of nuclear magnetic resonance spectroscopy. This change in the position of the label can only occur by entry of [3-¹³C]glutamate into the tricarboxylic acid (TCA) cycle, conversion of labeled -ketoglutarate to malate or oxaloacetate, malic enzyme-mediated decarboxylation of malate to pyruvate or phosphoenolpyruvate carboxykinase-mediated conversion of oxaloacetate to phosphoenolpyruvate and subsequent hydrolysis of the latter to pyruvate, and introduction of the labeled pyruvate into the TCA cycle, i.e., after exit of the carbon skeleton of pyruvate from the TCA cycle followed by re-entry of the same pyruvate molecules via acetyl CoA. In agreement with earlier observations, pyruvate recycling was demonstrated in astrocytes, indicating the ability of these cells to undertake complete oxidative degradation of glutamate. The recycled [4-¹³C]glutamate was not further converted to glutamine, showing compartmentation of astrocytic metabolism. Thus, absence of recycling into glutamine in the brain in vivo cannot be taken as indication that pyruvate recycling is absent in astrocytes. No recycling could be demonstrated in the cerebrocortical neurons. This is consistent with a previously demonstrated lack of incorporation of label from glutamate into lactate, and it also indicates that mitochondrial malic enzyme is not operational. Nor was there any indication of pyruvate recycling in the co-cultures. Although this may partly be due to more rapid depletion of glutamate in the co-cultures, this observation at the very least indicates that pyruvate recycling is not up-regulated in the neuronal-astrocytic co-cultures.     

Citrate,Beneficial or Deleterious in the CNS?

Cerebellar granule neurons were incubated with or without glucose (3 mM) in the presence or absence of citrate (20 mM) using normoxic and/or hypoxic incubation conditions. During 4 h of hypoglycemia and also during hypoxia plus hypoglycemia, citrate increased lactate dehydrogenase (LDH) leakage from the cells and decreased mitochondrial activity, the latter was also the case in the presence of glucose. After 24 h of hypoglycemia, however, citrate decreased LDH leakage slightly, possibly due to its metabolism in the tricarboxylic acid cycle under these conditions. It should be noted that during mild hypoxia plus hypoglycemia a reduced LDH leakage was observed when compared to hypoglycemia alone. The 4 h low oxygen period did protect the neurons also during the 20 h re-oxygenation period. The present study might indicate that incubation of brain cell cultures in an atmosphere of air (30% oxygen) and 5% CO₂, which is used in most laboratories, can be toxic and that oxygen concentration should be lowered considerably to mimic conditions in the brain.     

Metabolic Compartmentation in Cortical Synaptosomes: Influence of Glucose and Preferential Incorporation of Endogenous Glutamate into GABA

Metabolism of glutamine was determined under a variety of conditions to study compartmentation in cortical synaptosomes. The combined intracellular and extracellular amounts of [U-¹³C]GABA, [U-¹³C]glutamate and [U-¹³C]glutamine were the same in synaptosomes incubated with [U-¹³C]glutamine in the presence and absence of glucose. However, the concentration of these amino acids was decreased in the latter group, demonstrating the requirement for glucose to maintain the size of neurotransmitter pools. In hypoglycemic synaptosomes more [U-¹³C]glutamine was converted to [U-¹³C]aspartate, and less glutamate was re-synthesized from the tricarboxylic acid (TCA) cycle, suggesting use of the partial TCA cycle from -ketoglutarate to oxaloacetate for energy. Compartmentation was studied in synaptosomes incubated with glucose plus labeled and unlabeled glutamine and glutamate. Incubation with [U-¹³C]glutamine plus unlabeled glutamate gave rise to [U-¹³C]GABA but not labeled aspartate; however, incubation with [U-¹³C]glutamate plus unlabeled glutamine gave rise to [U-¹³C]aspartate, but not labeled GABA. Thus the endogenous glutamate formed via glutaminase in synaptic terminals is preferentially used for GABA synthesis, and is metabolized differently than glutamate taken up from the extracellular milieu.     

Protein dynamics studied by neutron scattering

This review of protein dynamics studied by neutron scattering focuses on data collected in the last 10 years. After an introduction to thermal neutron scattering and instrumental aspects, theoretical models that have been used to interpret the data are presented and discussed. Experiments are described according to sample type, protein powders, solutions and membranes. Neutron-scattering results are compared to those obtained from other techniques. The biological relevance of the experimental results is discussed. The major conclusion of the last decade concerns the strong dependence of internal dynamics on the macromolecular environment.     

A partial skeleton of Prodeinotherium bavaricum (Proboscidea, Mammalia) from the Middle Miocene of Unterzolling (Upper Freshwater Molasse, Germany)

Here, a partial skeleton of Prodeinotherium bavaricum from Unterzolling (Southern Germany) is documented. The following elements are preserved and described for the first time: cervical vertebrae 1-2 and 5-7, the first thoracic vertebra, one lumbar vertebra, trapezium, metacarpals 1-5, tibia, calcaneus, endo- and mesocuneiform, cuboid, the fourth metatarsal, and some phalanges. Comparisons with the skeletons of P. bavaricum from Franzensbad (Czech Republic) and Deinotherium giganteum from Eserovo (Bulgaria) show osteological differences that are described and discussed.     

Control of calcium homeostasis by angiotensin II in adrenal glomerulosa cells through activation of p38 MAPK

Angiotensin II-induced activation of aldosterone secretion in adrenal glomerulosa cells is mediated by an increase of intracellular calcium. We describe here a new Ca2+-regulatory pathway involving the inhibition by angiotensin II of calcium extrusion through the Na+/Ca2+ exchanger. Caffeine reduced both the angiotensin II-induced calcium signal and aldosterone production in bovine glomerulosa cells. These effects were independent of cAMP or calcium release from intracellular stores. The calcium response to angiotensin II was more sensitive to caffeine than the response to potassium, suggesting that the drug interacts with a pathway specifically elicited by the hormone. In calcium-free medium, calcium returned more rapidly to basal levels after angiotensin II stimulation in the presence of caffeine. Thapsigargin had no effect on these kinetics, but diltiazem, which inhibits the Na+/Ca2+ exchanger, markedly reduced the rate of calcium decrease and abolished caffeine action. The involvement of this exchanger was supported by the effect of cell depolarization and of a reduction of extracellular sodium on the rate of calcium extrusion. We also determined the mechanism of angiotensin II action on the exchanger. Phorbol esters reduced the rate of calcium extrusion, which was increased by baicalein, an inhibitor of lipoxygenases, and by SB 203580, an inhibitor of the p38 MAPK. Finally, we showed that angiotensin II acutely activates, in a caffeine-sensitive manner, p38 MAPK in glomerulosa cells. In conclusion, in bovine glomerulosa cells, the Na+/Ca2+ exchanger plays a crucial role in extruding calcium, and, by reducing its activity, angiotensin II influences the amplitude of the calcium signal. The hormone exerts its action on the exchanger through a caffeine-sensitive pathway involving the p38 MAPK and lipoxygenase products.     

The disulphide bonds in the catalytic domain of BACE are critical but not essential for amyloid precursor protein processing activity

beta-Site APP-cleaving enzyme (BACE) initiates the processing of the amyloid precursor protein (APP) leading to the generation of beta-amyloid, the main component of Alzheimer's disease senile plaques. BACE (Asp2, memapsin 2) is a type I transmembrane aspartic protease responsible for the beta-secretase cleavage of APP producing a soluble form of the ectodomain (sAPPbeta) and the membrane-bound, carboxy-terminal intermediates C99 and C89. BACE maturation involves cysteine bridge formation, N -glycosylation and propeptide removal. We investigated variants of BACE in which the disulphide bonds of the catalytic domain spanning between Cys216/Cys420, Cys278/Cys443 and Cys330/Cys380 were removed by mutagenesis. When transfected in cultured cells, these mutants showed impaired maturation. Nevertheless, a fraction of mutated protein retained both the competence to mature as well as the activity to process APP. For the generation of a functional enzyme the conserved Cys330/Cys380 bond was the most critical, whereas the two bonds between Cys216/Cys420 and Cys278/Cys443, which are typical for the membrane-bound BACE, appeared to be less important.     

Ubiquitination of both adeno-associated virus type 2 and 5 capsid proteins affects the transduction efficiency of recombinant vectors

In the presence of complementing adeno-associated virus type 2 (AAV-2) Rep proteins, AAV-2 genomes can be pseudotyped with the AAV-5 capsid to assemble infectious virions. Using this pseudotyping strategy, the involvement of the ubiquitin-proteasome system in AAV-5 and AAV-2 capsid-mediated infections was compared. A recombinant AAV-2 (rAAV-2) proviral luciferase construct was packaged into both AAV-2 and AAV-5 capsid particles, and transduction efficiencies in a number of cell lines were compared. Using luciferase expression as the end point, we demonstrated that coadministration of the viruses with proteasome inhibitors not only increased the transduction efficiency of rAAV-2, as previously reported, but also augmented rAAV-5-mediated gene transfer. Increased transgene expression was independent of viral genome stability, since there was no significant difference in the amounts of internalized viral DNA in the presence or absence of proteasome inhibitors. Western blot assays of immunoprecipitated viral capsid proteins from infected HeLa cell lysates and in vitro reconstitution experiments revealed evidence for ubiquitin conjugation of both AAV-2 and AAV-5 capsids. Interestingly, heat-denatured virus particles were preferential substrates for in vitro ubiquitination, suggesting that endosomal processing of the viral capsid proteins is a prelude to ubiquitination. Furthermore, ubiquitination may be a signal for processing of the capsid at the time of virion disassembly. These studies suggest that the previously reported influences of the ubiquitin-proteasome system on rAAV-2 transduction are also active for rAAV-5 and provide a clearer mechanistic framework for understanding the functional significance of ubiquitination.     

High-level production of the non-cariogenic sucrose isomer palatinose in transgenic tobacco plants strongly impairs development

Palatinose (isomaltulose, 6-O-alpha-D-glucopyranosyl-D-fructose) is a structural isomer of sucrose which is produced from sucrose by some bacterial strains as a reserve material during periods of low carbon availability. The ability to synthesise palatinose is not only advantageous for the bacteria but is also of industrial interest since palatinose is used as a sucrose substitute in food production. To explore the possibility of palatinose production in plants a recently isolated sucrose isomerase gene (palI; EC 5.4.99.11) from Erwinia rhapontici [F. B?rnke et al. (2001) J Bacteriol 183: 2425-2430] was cloned into a plant expression vector between the constitutive 35S CaMV promoter and the octopine synthase polyadenylation signal. To allow secretion of the protein into the apoplast the signal peptide of the potato proteinase inhibitor II was N-terminally fused to the pall coding region. Expression of the protein was verified by northern and western blot analyses. Efficient secretion of the protein was demonstrated by palI detection in intercellular fluids. Transgenic plants expressing palI accumulated high levels of palatinose. As a consequence, transgenic plants showed severe phenotypic alterations. Young leaves were curled and developed bleached areas during maturation. Flowers were misshapen and sterile. Based on nonaqueous fractionation experiments palatinose was found in several subcellular compartments, indicating limited membrane transport of the sugar. In contrast to results obtained with short-term feeding experiments, no evidence for palatinose-mediated regulation of photosynthetic or defence genes could be obtained in the transgenic palI-expressing tobacco plants. Based on our results we conclude that plants can efficiently be used as bioreactors for the production of palatinose. Furthermore, tissue-specific expression of palI should allow carbon allocation to specific tissues and/or cell-types to be modulated.     

Development of a test system for inhibitors of human aldosterone synthase (CYP11B2): screening in fission yeast and evaluation of selectivity in V79 cells

Aldosterone synthase (CYP11B2) is a mitochondrial cytochrome P450 enzyme catalyzing the last steps of aldosterone production in the adrenal cortex. A new pharmacological approach for the treatment of the aldosterone induced effects in congestive heart failure and all forms of hyperaldosteronism could be the use of CYP11B2 inhibitors. In search for such compounds, it was our goal to develop a cellular enzyme assay suitable for screening high numbers of compounds. An assay procedure for the evaluation of inhibitors using the human CYP11B2 expressed in fission yeast Schizosaccharomyces pombe was established and a series of 10 compounds was tested in this whole cellular system. Human 11beta-hydroxylase (CYP11B1), which catalyzes the production of glucocorticoids, shows more than 90% homology compared to human CYP11B2. As this enzyme should not be affected, strong inhibitors of CYP11B2 have to be tested for selectivity. For that purpose, an assay procedure with V79MZ cells that express human CYP11B1 and CYP11B2, respectively, was integrated into the evaluation process. Using these screening procedures a potent and rather selective non-steroidal inhibitor of human CYP11B2 was detected with an IC(50) value of 59nM. We also identified a very potent inhibitor of both enzymes showing a stronger inhibitory activity against the cortisol producing CYP11B1.