Molecular cloning and phylogenetic analysis of cereal type II metacaspase cDNA from wheat

A new cereal type II metacaspase full-length cDNA from wheat (Triticum aestivum L.) leaves, TaeMCAII, was for the first time successfully amplified and sequenced. The full-length sequence of the TaeMCAII cDNA of 1 551 bp contains a 1 218 bp open reading frame. The deduced protein encoded by the TaeMCAII cDNA consists of 405 amino acids with a calculated molecular mass of 44 kDa and an isoelectric point of 5.29. In response to wounding or heat shock, a similar sequence of ultrastructural events including the tonoplast rupture, chromatin condensation, degradation of chloroplasts and disappearance of cytoplasm and organelles were observed using transmission electron microscopy. As the observed changes in TaeMCAII mRNA level did not occur to be statistically significant wounding-induced programmed cell death (PCD) seems to be metacaspase-independent pathway. Interestingly, in PCD caused by a heat-shock treatment, the level of TaeMCAII mRNA remained unaltered until 48 h after the stress what suggests that TaeMCAII participates in later stages of PCD triggered by heat-shock. Phylogenetic analysis enabled to classify TaeMCAII as a type II metacaspase. Finally, homology modelling of the putative three-dimensional structure of the TaeMCAII protein and a topology analysis of its probable active site were performed.     

OXIDATION AND PHOSPHORYLATION PROCESSES IN BRAIN MITOCHONDRIA OF RATS EXPOSED TO CARBON DISULPHIDE

Abstract— The effects of acute and long-term exposure to CS₂ on oxidation and phosphorylation processes in brain mitochondria of rats were studied. Although rats developed different symptoms of poisoning, depending on the type of exposure, the brain mitochondria of both groups of animals exhibited the same types of disturbances in oxidative phosphorylation. The main characteristic of these disturbances was the uncoupling of oxidative phosphorylation indicated by lower respiratory control indices due to stimulation of oxidation of respiratory substrates by mitochondria in the metabolic state 4. This effect was accompanied by a decreased P:O ratio and a lower ATP-Pi exchange rate. An inhibitory effect of CS₂ on the energy transfer processes is also suggested.
The observed changes in oxidative phosphorylation were more distinct in the case of acute poisoning, with a longer period of an uninterrupted exposure enabling a more complete tissue saturation with CS₂, than in the case of long-term exposure with shorter periods of intoxication within the day.     

Stimulating effect of physical effort on the excretion of 5-hydroxyindoleacetic acid.

The excretion of 5-hydroxyindoleacetic acid, after work load, was studied. The physical effort evoked an increase in the excretion of 5-hydroxyindoleacetic acid. Under the control conditions no changes in the excretion of the investigated metabolite were found.     

Comparison of biochemical properties of DNA-topoisomerase I from normal and regenerating liver.

Biochemical properties of topoisomerase I from normal and regenerating rat liver were analysed using crude or fractionated nuclear extracts. We could not detect significative change in topoisomerase I content or activity (magnesium stimulation and inhibition by ATP) during the course of liver regeneration. Topoisomerase I can be resolved into two species of 97 kDa and 100 kDa, with the same pI of 8.2-8.6 as shown by two dimensional gel electrophoresis. The two polypeptides contained a non-phosphorylated precursor and others forms with variable degrees of phosphorylation. In-vitro dephosphorylation with alkaline phosphatase leads to the disappearance of the phosphorylated forms and inactivation of the enzyme. The affinity of topoisomerase I for chromatin (measured by salt elution) differs markedly between normal and regenerating liver: nearly 50% of topoisomerase I remained bound to the chromatin from normal liver at 250 mM NaCl whereas it was completely eluted from 24-h-regenerating-liver nuclei. The biological significance of these results is discussed.     

Downregulation of microsomal glutathione-S-transferase 1 modulates protective mechanisms in differentiated PC12 cells

Microsomal glutathione-S-transferase 1 (Mgst1) plays a specific role in protection of cells against oxidative stress. In this study, we assayed the effect of Mgst1 downregulation on cells behavior using differentiated PC12 line, a widely accepted neuronal model system. We have developed stable transfected cells with downregulated Mgst1 (PC12_M), which were differentiated with 1 mM dibutyryl-cAMP (db-cAMP). Mgst1 reduction induced necrosis, decreased ATP amount, and increased thiobarbituric acid reacting substances (TBARS) content. However, in PC12_M cell population, we detected more intensive neuritogenesis than that in mock-transfected cells. Interestingly, total glutathione as well as GSH level were significantly higher than those in control PC12 line. Real-time PCR and Western blot analyses showed elevated expression of enzymes involved in glutathione metabolism—a rate-limiting γ-glutamylcysteine ligase and glutathione reductase. The present study shows for the first time that under stress conditions induced by Mgst1 downregulation, a rescue pathway can be activated and thereby enables differentiated PC12 cells to survive. Since Mgst1expression was reported to decline with age, our results could represent a putative adaptive process during aging. It could also be an early mechanism protecting neuronal cells against some neurodegenerative insults.     

Study of permeation and blocker binding in TMEM16A calcium-activated chloride channels

We studied the effects of mutations of positively charged amino acid residues in the pore of X. tropicalis TMEM16A calcium-activated chloride channels: K613E, K628E, K630E; R646E and R761E. The activation and deactivation kinetics were not affected, and only K613E showed a lower current density. K628E and R761E affect anion selectivity without affecting Na⁺ permeation, whereas K613E, R646E and the double mutant K613E + R646E affect anion selectivity and permeability to Na⁺. Furthermore, altered blockade by the chloride channel blockers anthracene-9-carboxylic acid (A-9-C), 4, 4''-Diisothiocyano-2,2''-stilbenedisulfonic acid (DIDS) and T16inh-A01 was observed. These results suggest the existence of 2 binding sites for anions within the pore at electrical distances of 0.3 and 0.5. These sites are also relevant for anion permeation and blockade.