INTERVENTION OF BRAIN CORTEX PHOSPHOLIPIDS IN PYRIDOXAL PHOSPHATE-DEPENDENT REACTIONS

We have examined the role of brain cortex phospholipids in regulating some enzymic reactions specific to the CNS. The study was carried out at the level of the reactions concerned with GABA formation, both in vivo and in vitro, and included investigation of the specificity of the effect. It was found that the brain cortex phospholipids enhance the transformation of exogenous vitamin B₆ into pyridoxal-5-phosphate by activating the pyridoxal 5-phosphate-kinase enzyme, in contrast to other phospholipids of different origins. The possible role of brain cortex phospholipids in regulating an enzyme contained in the soluble fraction is discussed. Moreover, it is suggested that the specific effect of the phospholipids from various sources is linked to their fatty acid composition and to be therefore dependent on the aliphatic chains.     

Effect of the cytoskeleton dynamic condition on the neuronal plasticity]

Infringement of the Lymnaea stagnalis cytoskeleton condition affected preservation and repeated development of plastic responses. Stabilising of the microtubules led to a dependence of the development and preservation dynamics of the plastic responses. Stabilising of the microfilaments transformed short-term plastic responses into long-term ones. The findings suggest a key role of reorganisation of the cytoskeleton in neuronal plasticity.     

Carbon flows in eutrophic Lake Rotsee: a <Superscript>13</Superscript>C-labelling experiment

The microbial segment of food webs plays a crucial role in lacustrine food-web functioning and carbon transfer, thereby influencing carbon storage and CO₂ emission and uptake in freshwater environments. Variability in microbial carbon processing (autotrophic and heterotrophic production and respiration based on glucose) with depth was investigated in eutrophic, methane-rich Lake Rotsee, Switzerland. In June 2011, ¹³C-labelling experiments were carried out at six depth intervals in the water column under ambient light as well as dark conditions to evaluate the relative importance of (chemo)autotrophic, mixotrophic and heterotrophic production. Label incorporation rates of phospholipid-derived fatty acid (PLFA) biomarkers allowed us to differentiate between microbial producers and calculate group-specific production. We conclude that at 6 m, net primary production (NPP) rates were highest, dominated by algal photoautotrophic production. At 10 m —the base of the oxycline— a distinct low-light community was able to fix inorganic carbon, while in the hypolimnion, heterotrophic production prevailed. At 2 m depth, high label incorporation into POC could only be traced to nonspecific PLFA, which prevented definite identification, but suggests cyanobacteria as dominating organisms. There was also depth zonation in extracellular carbon release and heterotrophic bacterial growth on recently fixed carbon. Large differences were observed between concentrations and label incorporation of POC and biomarkers, with large pools of inactive biomass settling in the hypolimnion, suggesting late-/post-bloom conditions. Net primary production (115 mmol C m^?2 d^?1) reached highest values in the epilimnion and was higher than glucose-based production (3.3 mmol C m^?2 d^?1, highest rates in the hypolimnion) and respiration (5.9 mmol C m^?2 d^?1, highest rates in the epilimnion). Hence, eutrophic Lake Rotsee was net autotrophic during our experiments, potentially storing large amounts of carbon.     

Hypermodified nucleoside carboxyl group as a target site for specific tRNA modification

The free carboxyl group of hypermodified nucleosides N6-methyl-N6-(threoninocarbonyl)adenosine (mt6A37) and 3-(3-amino-3-carboxypropyl)uridine (acp3U20:1) in tRNAmMet (yellow lupine), and N6-(threoninocarbonyl)adenosine (t6A37) in tRNAiMet (yellow lupine) can be converted quantitatively and under very mild conditions into the respective anilides in a reaction with aniline and a water-soluble carbodiimide. The tRNA reactions proceed with rates very similar to that reported previously for t6A nucleoside. Detailed analysis of the products of tRNA modification with [3H]aniline on tRNA (chromatography on BD-DEAE-cellulose), oligonucleotide (polyacrylamide gel electrophoresis) and nucleoside (HPLC on Aminex A6) levels clearly indicates that only the hypermodified nucleoside residues undergo the reaction. The site of modification is confirmed for mono-modified (at mt6A37) and bis-modified (at mt6A37 and acp3U20:1) tRNAmMet, and for mono-modified (at t6A37) tRNAiMet by sequence analysis using 5'end 32P-labeled tRNAs. The modification procedure seems to be universally applicable for all hypermodified nucleosides bearing a free carboxyl group and for different amine reagents designed for the studies on tRNA function.     

Imaging Ca2+ concentration changes at the secretory vesicle surface with a recombinant targeted cameleon.

Regulated exocytosis involves the Ca(2+)-triggered fusion of secretory vesicles with the plasma membrane, by activation of vesicle membrane Ca(2+)-binding proteins [1]. The Ca(2+)-binding sites of these proteins are likely to lie within 30 nm of the vesicle surface, a domain in which changes in Ca2+ concentration cannot be resolved by conventional fluorescence microscopy. A fluorescent indicator for Ca2+ called a yellow 'cameleon' (Ycam2) - comprising a fusion between a cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13 and an enhanced yellow-emitting GFP - which is targetable to specific intracellular locations, has been described [2]. Here, we generated a fusion between phogrin, a protein that is localised to secretory granule membranes [3], and Ycam2 (phogrin-Ycam2) to monitor changes in Ca2+ concentration ([Ca2+]) at the secretory vesicle surface ([Ca2+]gd) through alterations in fluorescence resonance energy transfer (FRET) between the linked cyan and yellow fluorescent proteins (CFP and YFP, respectively) in Ycam2. In both neuroendocrine PC12 and MIN6 pancreatic beta cells, apparent resting values of cytosolic [Ca2+] and [Ca2+](gd) were similar throughout the cell. In MIN6 cells following the activation of Ca2+ influx, the minority of vesicles that were within approximately 1 microm of the plasma membrane underwent increases in [Ca2+](gd) that were significantly greater than those experienced by deeper vesicles, and greater than the apparent cytosolic [Ca2+] change. The ability to image both global and compartmentalised [Ca2+] changes with recombinant targeted cameleons should extend the usefulness of these new Ca2+ probes.