David Bellido

Gilthead sea bream liver proteome altered at low temperatures by oxidative stress

Gilthead sea bream exposed to the cold show multiple physiological alterations, particularly in liver. A typical cold‐stress response was reproduced in gilthead sea bream acclimated to 20°C (Warm group) when the water temperature was lowered to 8°C (Cold group). After 10 days, thiobarbituric acid reactive substances in the liver had increased by 50%, and nitric oxide had increased twofold. This indicates that lipid peroxidation and oxidative stress had occurred. Protein profiles of liver from fish in warm and cold environments were obtained by 2‐DE. Quantification of differential expression by matching spots showed that a total of 57 proteins were altered significantly. Many proteins were downregulated following cold exposure, including actin, the most abundant protein in the proteome; enzymes of amino acid metabolism; and enzymes with antioxidant capacity, such as betaine‐homocysteine‐methyl transferase, glutathione‐S‐transferase and catalase. Some proteins associated with protective action were upregulated at low temperatures, including peroxiredoxin, thioredoxin and lysozyme; as well as enzymes such as aldehyde dehydrogenase and adenosin‐methionine synthetase. However, the upregulation of proteases, proteasome activator protein and trypsinogen‐like protein indicated an increase in proteolysis. Increases in elongation factor‐1α, the GAPDH oxidative form, tubulin and Raf‐kinase inhibitor protein indicated oxidative stress and the induction of apoptosis. These data indicate that cold exposure induced oxidative damage in hepatocytes.

Intracellular distribution of hepatic glucokinase and glucokinase regulatory protein during the fasted to refed transition in rats

We have studied the intracellular distribution in vivo of glucokinase (GK) and glucokinase regulatory protein (GKRP) in livers of fasted and refed rats, using specific antibodies against both proteins and laser confocal fluorescence microscopy. GK was found predominantly in the nucleus of hepatocytes from starved rats. GK was translocated to the cytoplasm in livers of 1‐ and 2‐h refed animals, but returned to the nucleus after 4 h. GKRP concentrated in the hepatocyte nuclei and its distribution did not change upon refeeding. These results show that, in physiological conditions, GKRP is present predominantly in the nuclei of hepatocytes and that the translocation of hepatic GK from and to the nucleus is operative in vivo.

Presence of Plagiotrochus Mayr, 1881 in the Himalayan area, with redescription of Plagiotrochus semicarpifoliae (Cameron, 1902) COMB. N. (Hymenoptera: Cynipidae)

Plagiotrochus semicarpifoliae comb n. is transferred to Plagiotrochus Mayr from Callirhytis Foerster, 1869, and adults of the agamic form are redescribed and compared with other species of the genus. The plesiomorphic traits of P. semicarpifoliae suggest a Southeast Asian origin for Plagiotrochus, which could have migrated with their hosts to the Mediterranean area, where the most derived forms are present.

Acetylation of GAGA factor modulates its interaction with DNA.

GAGA is a Drosophila transcription factor that shows a high degree of post-translational modification. Here, we show that GAGA factor is acetylated in vivo. Lysine residues K325 and K373 on basic regions BR1 and BR3 of the DNA binding domain, respectively, are shown to be acetylated by PCAF. While BR1 is strictly required to stabilize DNA binding, BR3 is dispensable. However, acetylation of both lysine residues, either alone or in combination, weakens the binding to DNA. Despite the high degree of conservation of K325 and K373 in flies, their mutation to glutamine does not affect DNA binding. Molecular dynamics simulations, using acetylated K325 and a K325Q mutant of GAGA DNA binding domain in complex with DNA, are fully consistent with these results and provide a thermodynamic explanation for this observation. We propose that while K325 and K373 are not essential for DNA binding they have been largely conserved for regulatory purposes, thus highlighting a key regulatory system for GAGA factor in flies.

Intracellular distribution of digoxigenin-labeled phosphorothioate oligonucleotides.

Publisher Summary The great potential of antisense oligonucleotides as selective inhibitors of gene expression has led to their development and use for therapeutic purposes. Antisense technology has had to satisfy several conditions, such as stability to nucleases, enhancement of cellular permeability, and selectivity for the target sequence. Some of these requirements have been addressed by the chemical development of nucleic acid analogs. Although modifications such as phosphorothioate substitutions have been used mainly for improved stability and C-5 propyne or morpholino modifications have been used for higher affinity, the major concern is, at present, inefficient cellular uptake. Carrier systems such as cationic liposomes, nanoparticles, or supramolecular biovectors have been proposed to enhance cellular uptake and nuclear accumulation. Nevertheless, the characterization of intracellular distribution is important in assessing an effective antisense effect. As an alternative approach to the study of the distribution of antisense oligonucleotides, this chapter describes the synthesis and purification of digoxigenin-labeled phosphorothioate oligonucleotides, and their use in the characterization of intracellular distribution in human Epstein-Barr virus-transformed B cells using immunocytochemistry with both confocal and electron microscopy.