Uptake, Release, and Metabolism of Citrate in Neurons and Astrocytes in Primary Cultures

Abstract: Synthesis, uptake, release, and oxidative metabolism of citrate were investigated in neurons and astrocytes cultured from cerebral cortex or cerebellum. In addition, the possible role of citrate as a donor of the carbon skeleton for biosynthesis of neurotransmitter glutamate was studied. All cell types expressed the enzyme citrate synthase at a high activity, the cerebellar granule neurons containing the enzyme at a higher activity than that found in the astrocytes from the two brain regions or the cortical neurons. Saturable citrate uptake could not be detected in any of the cell types, but the astrocytes, and, in particular, those of cerebellar origin, had a very active de novo synthesis and release of citrate (～70 nmol × h^?1× mg of protein^?1). The rate of release of citrate from neurons was <5% of this value. Using [¹⁴C]citrate it could be shown that citrate was oxidatively metabolized to ¹⁴CO₂ at a modest rate (～1 nmol × n^?1× mg^?1 of protein) with slightly higher rates in astrocytes compared with neurons. Experiments designed to investigate the ability of exogenously supplied citrate to serve as a precursor for synthesis of transmitter glutamate in cerebellar granule neurons failed to demonstrate this. Rather than citrate serving this purpose it may be suggested that astrocytically released citrate may regulate the extracellular concentration of Ca²⁺ and Mg²⁺ by chelation, thereby modulating neuronal excitability.     

Diversity and infectivity of arbuscular mycorrhizal fungi in agricultural soils of the Sichuan Province of mainland China

Knowledge about the presence and diversity of arbuscular mycorrhizal fungi (AMF) in a specific area is an essential first step for utilizing these fungi in any application. The community composition of AMF in intensively managed agricultural soil in the Sichuan Province of southwest China currently is unknown. In one set of samples, AMF were trapped in pot cultures from 40 fields growing legumes in the Panxi region, southeast Sichuan. In a second set of samples, the MPN method with four-fold dilutions and maize as host was used to estimate infective propagules in soil from another 50 agricultural sites throughout the province. Soil types were heterogeneous and were classified as purple, yellow, paddy and red. Crops at each site were either maize, wheat or sweet orange. From this set of soil, AMF spores were also extracted and identified. Including all ninety soils, thirty glomeromycotan species in Glomus (20 species), Acaulospora (four species), Scutellospora (three species), Ambispora (one species), Archaeospora (one species) and Paraglomus (one species) were identified. Yellow, red and purple soils yielded similar numbers of AMF species, while AMF species diversity was clearly lower in paddy soil. In trap culture soils, the most frequent species were Glomus aggregatum or Glomus intraradices, Glomus claroideum and Glomus etunicatum. The species Acaulospora capsicula, Acaulospora delicata, G. aggregatum (or intraradices), G. claroideum, Glomus epigaeum, G. etunicatum, Glomus luteum, Glomus monosporum, Glomus mosseae and Glomus proliferum were successfully cultured as single-species pot cultures in Plantago lanceolata. The three most frequent species in field soils were G. mosseae, Glomus caledonium and Glomus constrictum. MPN values varied between 17 and 3334 propagules 100 g soil⁻¹ among the fifty field sites sampled. Regression analysis, including host&soil, log(P) and pH as explanatory variables explained 59% of the variation in log(MPN). The highest MPN estimates were found in purple soil cropped with maize and citrus, 324 and 278 propagules 100 g soil⁻¹, respectively. The lowest MPN value, 54 propagules 100 g soil⁻¹, was measured in wheat in purple and yellow soil. Despite intensive agricultural management that can include often repeated tillage, our examination of 90 agricultural sites revealed that soils of the Sichuan region have moderate to high numbers of infective AMF propagules as well as a high AMF species diversity. This opens possibilities for further studies and utilization of AMF in agriculture and horticulture in the Sichuan province, People’s Republic of China.     

Recurrent activity in higher order, modality non-specific brain regions: a Granger causality analysis of autobiographic memory retrieval

It has been proposed that the workings of the brain are mainly intrinsically generated recurrent neuronal activity, with sensory inputs as modifiers of such activity in both sensory and higher order modality non-specific regions. This is supported by the demonstration of recurrent neuronal activity in the visual system as a response to visual stimulation. In contrast recurrent activity has never been demonstrated before in higher order modality non-specific regions. Using magneto-encephalography and Granger causality analysis, we tested in a paralimbic network the hypothesis that stimulation may enhance causal recurrent interaction between higher-order, modality non-specific regions. The network includes anterior cingulate/medial prefrontal and posterior cingulate/medial parietal cortices together with pulvinar thalami, a network known to be effective in autobiographic memory retrieval and self-awareness. Autobiographic memory retrieval of previous personal judgments of visually presented words was used as stimuli. It is demonstrated that the prestimulus condition is characterized by causal, recurrent oscillations which are maximal in the lower gamma range. When retrieving previous judgments of visually presented adjectives, this activity is dramatically increased during the stimulus task as ascertained by Granger causality analysis. Our results confirm the hypothesis that stimulation may enhance causal interaction between higher order, modality non-specific brain regions, exemplified in a network of autobiographical memory retrieval.     

Female Remating Behavior in a Lekking Moth

In polyandrous species, male reproductive success will at least partly be determined by males' success in sperm competition. To understand the potential for post‐mating sexual selection, it is therefore important to assess the extent of female remating. In the lekking moth Achroia grisella, male mating success is strongly determined by female choice based on the attractiveness of male ultrasonic songs. Although observations have indicated that some females will remate, only little is known about the level of sperm competition. In many species, females are more likely to remate if their first mating involved an already mated male than if the first male was virgin. Potentially, this is because mated males are less well able to provide an adequate sperm supply, nutrients, or substances inhibiting female remating. This phenomenon will effectively reduce the strength of pre‐copulatory sexual selection because attractive males with high mating success will be more susceptible to sperm competition. We therefore performed an experiment designed both to provide a more precise estimate of female remating probability and simultaneously to test the hypothesis that female remating is influenced by male mating history. Overall, approximately one of five females remated with a second male. Yet, although females mated to non‐virgin males were somewhat more prone to remate, the effect of male mating history was not significant. The results revealed, however, that heavier females were more likely to remate. Furthermore, we found that females' second copulations were longer, suggesting that, in accordance with theory, males may invest more sperm in situations with an elevated risk of sperm competition.     

The Common Variant in the FTO Gene Did Not Modify the Effect of Lifestyle Changes on Body Weight: The Finnish Diabetes Prevention Study

The common single‐nucleotide polymorphism in the FTO (fat mass and obesity associated) gene is consistently associated with an increased risk of obesity. However, the knowledge of a potential modifying effect of the FTO gene on changes in body weight achieved by lifestyle intervention is limited. We examined whether the FTO gene variant (rs9939609, T/A) is associated with body weight and BMI and long‐term weight changes in the Finnish Diabetes Prevention Study (DPS). Altogether, 522 (aged 40–65 years; BMI ≥25 kg/m²) subjects with impaired glucose tolerance (IGT) were randomized to control and lifestyle intervention groups. SNP rs9939609 was genotyped from 502 subjects. At baseline, those with the AA genotype had higher BMI than subjects with other genotypes (P = 0.006). The association was observed in women (P = 0.016) but not in men. During the 4‐year follow‐up, the subjects with the AA genotype had consistently the highest BMI (P = 0.009) in the entire study population. The magnitude of weight reduction was greater in the intervention group, but the risk allele did not modify weight change in either of the groups. Our results confirm the association between the common FTO variant and BMI in a cross‐sectional setting and during the long‐term lifestyle intervention. We did not observe association between FTO variant and the magnitude of weight reduction achieved by long‐term lifestyle intervention. Based on the results from the DPS, it is unlikely that the common variant of the FTO gene affects the success of lifestyle modification on weight loss.     

Time-dependent radiative forcing effects of forest fertilization and biomass substitution

Here we analyse the radiative forcing implications of forest fertilization and biomass substitution, with explicit consideration of the temporal patterns of greenhouse gas (GHG) emissions to and removals from the atmosphere (net emissions). We model and compare the production and use of biomass from a hectare of fertilized and non-fertilized forest land in northern Sweden. We calculate the annual net emissions of CO₂, N₂O and CH₄ for each system, over a 225-year period with 1-year time steps. We calculate the annual atmospheric concentration decay of each of these emissions, and calculate the resulting annual changes in instantaneous and cumulative radiative forcing. We find that forest fertilization can significantly increase biomass production, which increases the potential for material and energy substitution. The average carbon stock in tree biomass, forest soils and wood products all increase when fertilization is used. The additional GHG emissions due to fertilizer production and application are small compared to increases in substitution benefits and carbon stock. The radiative forcing of the 2 stands is identical for the first 15?years, followed by 2?years during which the fertilized stand produces slightly more radiative forcing. After year 18 the instantaneous and cumulative radiative forcing are consistently lower for the fertilized forest system. Both stands result in long-term negative radiative forcing, or cooling of the earth system. By the end of the 225-year simulation period, the cumulative radiative forcing reduction of the fertilized stand is over twice that of the non-fertilized stand. This suggests that forest fertilization and biomass substitution are effective options for climate change mitigation, as climate change is a long term issue.     

Chemokines regulate small leucine-rich proteoglycans in the extracellular matrix of the pressure-overloaded right ventricle

Chemokines have been suggested to play a role during development of left ventricular failure, but little is known about their role during right ventricular (RV) remodeling and dysfunction. We have previously shown that the chemokine (C-X-C motif) ligand 13 (CXCL13) regulates small leucine-rich proteoglycans (SLRPs). We hypothesized that chemokines are upregulated in the pressure-overloaded RV, and that they regulate SLRPs. Mice with RV pressure overload following pulmonary banding (PB) had a significant increase in RV weight and an increase in liver weight after 1 wk. Microarray analysis (Affymetrix) of RV tissue from mice with PB revealed that CXCL10, CXCL6, chemokine (C-X3-C motif) ligand 1 (CX3CL1), chemokine (C-C motif) ligand 5 (CCL5), CXCL16, and CCL2 were the most upregulated chemokines. Stimulation of cardiac fibroblasts with these same chemokines showed that CXCL16 increased the expression of the four SLRPs: decorin, lumican, biglycan, and fibromodulin. CCL5 increased the same SLRPs, except decorin, whereas CX3CL1 increased the expression of decorin and lumican. CXCL16, CX3CL1, and CCL5 were also shown to increase the levels of glycosylated decorin and lumican in the medium after stimulation of fibroblasts. In the pressure-overloaded RV tissue, Western blotting revealed an increase in the total protein level of lumican and a glycosylated form of decorin with a higher molecular weight compared with control mice. Both mice with PB and patients with pulmonary stenosis had significantly increased circulating levels of CXCL16 compared with healthy controls measured by enzyme immunoassay. In conclusion, we have found that chemokines are upregulated in the pressure-overloaded RV and that CXCL16, CX3CL1, and CCL5 regulate expression and posttranslational modifications of SLRPs in cardiac fibroblasts. In the pressure-overloaded RV, protein levels of lumican were increased, and a glycosylated form of decorin with a high molecular weight appeared.     

Knockout of Mkp-1 exacerbates colitis in Il-10-deficient mice

Il-10-deficient mice develop colitis associated with exaggerated Th1/Th17 responses and are a valuable model of inflammatory bowel disease. Mkp-1 is a major negative regulator of MAPKs, and its expression is enhanced by IL-10. To understand the role of Mkp-1 in the regulation of intestinal mucosal immune responses, we studied the effect of Mkp-1 deletion on the pathogenesis of colitis in Il-10(-/-) mice. We found that knockout of Mkp-1 on an Il-10(-/-) background accelerated the development of colitis. Compared with Il-10(-/-) mice, colitis not only appeared earlier but also was more severe in Il-10(-/-)/Mkp-1(-/-) mice. Il-10(-/-) mice exhibited a mild intestinal inflammation in the specific pathogen-free environment, and rectal prolapse rarely appeared before 6 mo of age. In contrast, the majority of Il-10(-/-)/Mkp-1(-/-) mice developed severe colitis rapidly and presented with rectal prolapse after only 2-3 mo. The colon of Il-10(-/-)/Mkp-1(-/-) mice showed diffuse transmural chronic inflammation and mucosal hyperplasia, with significantly more proliferating crypt epithelial cells than those of Il-10(-/-) mice. In addition to the severe colitis, Il-10(-/-)/Mkp-1(-/-) mice also developed conjunctivitis and blepharitis. The colon of Il-10(-/-)/Mkp-1(-/-) mice contained significantly higher levels of proinflammatory cytokines and exhibited greater MAPK activities than did the colon of Il-10(-/-) mice. Splenocytes and lymphocytes from Il-10(-/-)/Mkp-1(-/-) mice produced higher levels of Th1 cytokines ex vivo upon activation than did cells from Il-10(-/-) mice. Our studies support a pivotal role of Mkp-1 as a negative regulator of mucosal immune responses and highlight its protective function against inflammatory bowel disease.