Glial Metabolism of Valine

The three essential amino acids, valine, leucine and isoleucine, constitute the group of branched-chain amino acids (BCAAs). BCAAs are rapidly taken up into the brain parenchyma, where they serve several distinct functions including that as fuel material in brain energy metabolism. As one function of astrocytes is considered the production of fuel molecules that support the energy metabolism of adjacent neural cells in brain. Astroglia-rich primary cultures (APC) were shown to rapidly dispose of the BCAAs, including valine, contained in the culture medium. While the metabolisms of leucine and isoleucine by APC have already been studied in detail, some aspects of valine metabolism remained to be determined. Therefore, in the present study an NMR analysis was performed to identify the ¹³C-labelled metabolites that are generated by APC during catabolism of [U-¹³C]valine and that are subsequently released into the incubation medium. The results presented show that APC (1) are potently disposing of the valine contained in the incubation medium; (2) are capable of degrading valine to the tricarboxylic acid (TCA) cycle member succinyl-CoA; and (3) release into the extracellular milieu valine catabolites and compounds generated from them such as [U-¹³C]2-oxoisovalerate, [U-¹³C]3-hydroxyisobutyrate, [U-¹³C]2-methylmalonate, [U-¹³C]isobutyrate, and [U-¹³C]propionate as well as several TCA cycle-dependent metabolites including lactate. This article is dedicated to Dr. George DeVries.     

Elevated CO2 concentration and temperature effects on the partitioning of chemical components along juvenile Scots pine stems (Pinus sylvestris L.)

The effects of doubled ambient [CO₂] and different temperature levels on young Pinus sylvestris growing in phytotron chambers were studied. Five chambers were supplied with ~380 (‘ambient air’) and five with ~700 μmol mol⁻¹ CO₂ (‘elevated [CO₂]’). Temperature levels in the chambers ranged in increment steps of 2°C from −4°C to +4°C relative to the long-term monthly (day and night) average air temperature levels in Berlin–Dahlem. Substrate was medium fertile; soil moisture and air humidity were kept constant. After three vegetation periods twigs and stems were harvested, weighed, homogenized, and analyzed chemically. There was no significant temperature effect on wood mass accumulation, clearest positive [CO₂] effect occurred in the youngest twigs. In total, wood mass increased by 28.5% at doubled ambient [CO₂]. N-contents (percentage) decreased at elevated [CO₂] in the uppermost stem sections and not in twig wood causing wider C/N ratios in total. In response to elevated temperature, N-contents decreased slightly in twigs (~0.3%). Traces of free glucose, fructose and sucrose, which decreased from the top to the bottom, were found in stem wood, in contrast to traces of starch that increased from the top to the bottom. In response to elevated [CO₂] only a little more (0.05%) was accumulated in the top shoot and in tendency; glucose, fructose, and sucrose contents were lower at the bottom of stems as compared to the control. There was no obvious response of these non-structural carbohydrates to elevated temperature except for starch that decreased to half of the content from the lowest to the highest temperature level. Among the hemicellulose compounds, rhamnose and arabinose declined from the top shoot to the bottom of stem, whereas 4-O-methyl-d-glucuronic-acid, mannose, and xylose increased. Contents (percentage) of galactose remained approximately stable along the stem. The clearest positive effect of elevated [CO₂] along the whole stem was found for mannose with differences of 0.6–0.3%. In contrast to rhamnose and arabinose that showed a negative response to elevated [CO₂], mannose was reduced towards the uppermost stem sections. The 4-O-methyl-d-glucuronic-acid was slightly lowered at the bottom, and galactose and xylose showed no [CO₂] response. The only hemicellulose compound which reacted to temperature elevation was galactose. It increased slightly (~0.1% per 1°C). Cellulose and lignin (Klason) behaved oppositely: cellulose increased and lignin decreased from the top to the bottom. These structural components behaved reversely also in response to elevated [CO₂]. In stem parts above the bottom section, cellulose content was slightly higher at elevated [CO₂], and lignin content was slightly lower at the bottom. Lignin reacted to temperature elevation by a very slight increase on the average (~0.1% per one 1°C). Cellulose, however, decreased by ~0.2% per 1°C temperature elevation. The importance of persistent sinks of carbon in woody plant parts is discussed in respect to the greenhouse effect.     

Alcadein Cleavages by Amyloid ��-Precursor Protein (APP) ��- and ��-Secretases Generate Small Peptides, p3-Alcs, Indicating Alzheimer Disease-related ��-Secretase Dysfunction

Alcadeins (Alcs) constitute a family of neuronal type I membrane proteins, designated Alc_α, Alc_β, and Alc_γ. The Alcs express in neurons dominantly and largely colocalize with the Alzheimer amyloid precursor protein (APP) in the brain. Alcs and APP show an identical function as a cargo receptor of kinesin-1. Moreover, proteolytic processing of Alc proteins appears highly similar to that of APP. We found that APP α-secretases ADAM 10 and ADAM 17 primarily cleave Alc proteins and trigger the subsequent secondary intramembranous cleavage of Alc C-terminal fragments by a presenilin-dependent γ-secretase complex, thereby generating “APP p3-like” and non-aggregative Alc peptides (p3-Alcs). We determined the complete amino acid sequence of p3-Alc_α, p3-Alc_β, and p3-Alc_γ, whose major species comprise 35, 37, and 31 amino acids, respectively, in human cerebrospinal fluid. We demonstrate here that variant p3-Alc C termini are modulated by FAD-linked presenilin 1 mutations increasing minor β-amyloid species Aβ42, and these mutations alter the level of minor p3-Alc species. However, the magnitudes of C-terminal alteration of p3-Alc_α, p3-Alc_β, and p3-Alc_γ were not equivalent, suggesting that one type of γ-secretase dysfunction does not appear in the phenotype equivalently in the cleavage of type I membrane proteins. Because these C-terminal alterations are detectable in human cerebrospinal fluid, the use of a substrate panel, including Alcs and APP, may be effective to detect γ-secretase dysfunction in the prepathogenic state of Alzheimer disease subjects.     

Song dialects in the yellowhammer Emberiza citrinella : bioacoustic variation between and within dialects

The yellowhammer Emberiza citrinella is a common European bird that sings in dialects that for decades have been distinguished by the existence of one single element (called a “specific”). In this study we looked into other possibilities for dialect discrimination, measuring 24 different variables. For the first time, multivariate statistics were used to discriminate dialect in yellowhammer song. Two similar dialects (XlB and XsB) that are not clearly defined in the literature were studied. Statistics incorporated (1) all variables, (2) no variables of “specific” elements, and (3) no variables under the influence of these “specific” variables. Multivariate statistics support dialect discrimination by ear and confirmed that only one element in yellowhammer song characterises dialect. In addition, we looked for local differences within two dialects and found that one local observation area showed a higher separation than the other sites (Meck1). However, as yet there is insufficient evidence for the existence of a new subdialect. The experiments comply with the current laws of the study area.     

Insights into tRNA-Dependent Amidotransferase Evolution and Catalysis from the Structure of the Aquifex aeolicus Enzyme

Many bacteria form Gln-tRNA^Gln and Asn-tRNA^Asn by conversion of the misacylated Glu-tRNA^Gln and Asp-tRNA^Asn species catalyzed by the GatCAB amidotransferase in the presence of ATP and an amide donor (glutamine or asparagine). Here, we report the crystal structures of GatCAB from the hyperthermophilic bacterium Aquifex aeolicus, complexed with glutamine, asparagine, aspartate, ADP, or ATP. In contrast to the Staphylococcus aureus GatCAB, the A. aeolicus enzyme formed acyl-enzyme intermediates with either glutamine or asparagine, in line with the equally facile use by the amidotransferase of these amino acids as amide donors in the transamidation reaction.A water-filled ammonia channel is open throughout the length of the A. aeolicus GatCAB from the GatA active site to the synthetase catalytic pocket in the B-subunit. A non-catalytic Zn²⁺ site in the A. aeolicus GatB stabilizes subunit contacts and the ammonia channel. Judged from sequence conservation in the known GatCAB sequences, the Zn²⁺ binding motif was likely present in the primordial GatB/E, but became lost in certain lineages (e.g., S. aureus GatB). Two divalent metal binding sites, one permanent and the other transient, are present in the catalytic pocket of the A. aeolicus GatB. The two sites enable GatCAB to first phosphorylate the misacylated tRNA substrate and then amidate the activated intermediate to form the cognate products, Gln-tRNA^Gln or Asn-tRNA^Asn.     

Life‐style changes of a halophilic archaeon analyzed by quantitative proteomics

Quantitative proteomics based on isotopic labeling has become the method of choice to accurately determine changes in protein abundance in highly complex mixtures. Isotope‐coded protein labeling (ICPL), which is based on the nicotinoylation of proteins at lysine residues and free N‐termini was used as a simple, reliable and fast method for the comparative analysis of three different cellular states of the halophilic archaeon Halobacterium salinarum through pairwise comparison. The labeled proteins were subjected to SDS‐PAGE, in‐gel digested and the proteolytic peptides were separated by LC and analyzed by MALDI‐TOF/TOF MS. Automated quantitation was performed by comparing the MS peptide signals of ¹²C and ¹³C nicotinoylated isotopic peptide pairs. The transitions between (i) aerobic growth in complex versus synthetic medium and (ii) aerobic versus anaerobic/phototrophic growth, both in complex medium, provide a wide span in nutrient and energy supply for the cell and thus allowed optimal studies of proteome changes. In these two studies, 559 and 643 proteins, respectively, could be quantified allowing a detailed analysis of the adaptation of H. salinarum to changes of its living conditions. The subtle cellular response to a wide variation of nutrient and energy supply demonstrates a fine tuning of the cellular protein inventory.