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.     

De novo-synthesized ceramide signals apoptosis in astrocytes via extracellular signal-regulated kinase.

Recent observations support the importance of ceramide synthesis de novo in the induction of apoptosis. However, the downstream targets of de novo-synthesized ceramide are unknown. Here we show that palmitate incorporated into ceramide and induced apoptotic DNA fragmentation in astrocytes. These effects of palmitate were exacerbated when fatty acid breakdown was uncoupled and were not evident in neurons, which show a very low capacity to take up and metabolize palmitate. Palmitate-induced apoptosis of astrocytes was prevented by L-cycloserine and fumonisin B1, two inhibitors of ceramide synthesis de novo, and by PD098059, an inhibitor of the extracellular signal-regulated kinase (ERK) cascade. Accordingly, palmitate activated ERK by a process that was dependent on ceramide synthesis de novo and Raf-1, but independent of kinase suppressor of Ras. Other potential targets of ceramide in the control of cell fate, namely, c-Jun amino-terminal kinase, p38 mitogen-activated protein kinase, and protein kinase B, were not significantly affected in astrocytes exposed to palmitate. Results show that the Raf-1/ERK cascade is the selective downstream target of de novo-synthesized ceramide in the induction of apoptosis in astrocytes and also highlight the importance of ceramide synthesis de novo in apoptosis of astrocytes, which might have pathophysiological relevance.     

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.     

Effect of thiostrepton and 3′-terminal fragments of aminoacyl-tRNA on EF-Tu and ribosome-dependent GTP hydrolysis

The effect of the antibiotics thiostrepton and micrococcin on EF-T_u-catalyzed (ribosome-dependent) GTP hydrolysis in the presence of A-Phe, C-A-Phe, or C-C-A-Phe (related to the sequence of the 3′-terminus of aminoacyl-tRNA)(System I) or by methanol (‘uncoupled GTPase’, System II) was investigated. In System I, thiostrepton increases the binding affinities of the effectors to the EF-T_u·GTP·70 S ribosome complex, as well as the extent of the GTP hydrolysis, while the K^GTP_m is virtually unchanged. Similarly, in the uncoupled system (System II) and in the absence of effectors, thiostrepton significantly increases V^GTP_max, whereas K^GTP_m remains unaffected. Micrococcin is without any effect in both systems. The ‘uncoupled GTPase’ (in System II) is also strongly inhibited by C-A-Phe. The results indicate the crucial role of the EF-T_u site which binds the aminoacylated C-C-A terminus of aminoacyl-tRNA in promoting GTP hydrolysis. It follows that the binding of the model effectors (such as C-C-A-Phe) to that site is favorably influenced by the interaction of thiostrepton with the 50 S ribosomal subunit, whereas thiostrepton, per se, does not influence the affinity of EF-T_u for GTP.     

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.