Caloric Restriction Suppresses Microglial Activation and Prevents Neuroapoptosis Following Cortical Injury in Rats

Traumatic brain injury (TBI) is a widespread cause of death and a major source of adult disability. Subsequent pathological events occurring in the brain after TBI, referred to as secondary injury, continue to damage surrounding tissue resulting in substantial neuronal loss. One of the hallmarks of the secondary injury process is microglial activation resulting in increased cytokine production. Notwithstanding that recent studies demonstrated that caloric restriction (CR) lasting several months prior to an acute TBI exhibits neuroprotective properties, understanding how exactly CR influences secondary injury is still unclear. The goal of the present study was to examine whether CR (50% of daily food intake for 3 months) alleviates the effects of secondary injury on neuronal loss following cortical stab injury (CSI). To this end, we examined the effects of CR on the microglial activation, tumor necrosis factor-α (TNF-α) and caspase-3 expression in the ipsilateral (injured) cortex of the adult rats during the recovery period (from 2 to 28 days) after injury. Our results demonstrate that CR prior to CSI suppresses microglial activation, induction of TNF-α and caspase-3, as well as neurodegeneration following injury. These results indicate that CR strongly attenuates the effects of secondary injury, thus suggesting that CR may increase the successful outcome following TBI

Propofol-Induced Changes in Neurotrophic Signaling in the Developing Nervous System In Vivo

Several studies have revealed a role for neurotrophins in anesthesia-induced neurotoxicity in the developing brain. In this study we monitored the spatial and temporal expression of neurotrophic signaling molecules in the brain of 14-day-old (PND14) Wistar rats after the application of a single propofol dose (25 mg/kg i.p). The structures of interest were the cortex and thalamus as the primary areas of anesthetic actions. Changes of the protein levels of the brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), their activated receptors tropomyosin-related kinase (TrkA and TrkB) and downstream kinases Akt and the extracellular signal regulated kinase (ERK) were assessed by Western immunoblot analysis at different time points during the first 24 h after the treatment, as well as the expression of cleaved caspase-3 fragment. Fluoro-Jade B staining was used to follow the appearance of degenerating neurons. The obtained results show that the treatment caused marked alterations in levels of the examined neurotrophins, their receptors and downstream effector kinases. However, these changes were not associated with increased neurodegeneration in either the cortex or the thalamus. These results indicate that in the brain of PND14 rats, the interaction between Akt/ERK signaling might be one of important part of endogenous defense mechanisms, which the developing brain utilizes to protect itself from potential anesthesia-induced damage. Elucidation of the underlying molecular mechanisms will improve our understanding of the age-dependent component of anesthesia-induced neurotoxicity

Analysis of nuclear glucocorticoid receptor-DNA interaction in aged rat liver

Abstract: In order to contribute to the understanding of mechanisms by which regulatory proteins recognize genetic information stored in DNA, analyses of their interaction with specific nucleotides are usually performed. In this study, the electrophoretic mobility shift assay (EMSA) was applied to analyze the interaction of nuclear proteins from the liver of rats of different age i.e., young (3-month-old), middle- aged (12-month-old) and aged (24-month-old), with radioactively labelled synthetic oligonucleotide analogues, corresponding to GRE. The levels of GRE binding activity were assessed by quantitative densitometric scanning of the autoradiograms. The results showed statistically significant decreasing values of up to 78% and 49% in middle aged and old animals, respectively, compared to young animals (p LT 0.05). The specificity of the nuclear proteins-GRE interaction was demonstrated by competition experiments with unlabelled GRE. In a supershift assay, using the antibody BuGR2, it was shown that the GR proteins present in nuclear extracts have a high affinity for the GRE probe. The stabilities of the protein-DNA complexes were analysed and it was concluded that they changed during ageing.U cilju doprinosa razumevanju mehanizama pomoću kojih regulatorni proteini prepoznaju genetičku informaciju koju nosi DNK, analiziraju se njihove interakcije sa specifičnim nukleotidima. U ovom radu je metodom EMSA analizirana interakcija jedarnih proteina iz jetri pacova iz tri starosne grupe (mladi - 3 meseca, srednje doba – 12 meseci i stari – 24 meseca) sa sintetičkim, radioaktivno obeleženim, oligonukleotidnim analogom GRE. Nivo vezujuće aktivnosti GRE je određivan kvantitativno denzitometrijskom autoradiografijom. Rezultati su pokazali da postoji statistički značajan pad vrednosti GRE-vezujuće aktivnosti do 78% kod životinja srednjeg starosnog doba i do 49 % kod starih životinja, u poređenju sa vrednostima dobijenim za mlade životinje (p LT 0.05). Specifičnost interakcije jedarnih proteina i GRE je određena eksperimentima kompeticije sa neobeleženim GRE. Korišćenjem antitela BuGR2 pokazano je da je glukokortikoidni receptor protein koji u jedarnom ekstraktu ima najveći afinitet za GRE probu. Analizirana je stabilnost kompleksa protein-DNK i zaključeno je da se menja tokom starenja

Using yeast to study glucocorticoid receptor phosphorylation

The glucocorticoid receptor (GR) is a phosphoprotein and a member of the steroid/thyroid receptor superfamily of ligand dependent transcription factors. When the glucocorticoid receptor is expressed in yeast (Saccharomyces cerevisiae), it is competent for signal transduction and transcriptional regulation. We have studied the glucocorticoid receptor phosphorylation in yeast and demonstrated that the receptor is phosphorylated in both the absence and presence of hormone, on serine and threonine residues. This phosphorylation occurs within 15 min upon addition of radioactivity in both hormone treated and untreated cells. As reported for mammalian cells, additional phosphorylation occurs upon hormone binding and this phosphorylation is dependent on the type of the ligand. We have followed the hormone dependent receptor phosphorylation by electrophoretic mobility shift assay, and have shown that this mobility change is sensitive to phosphatase treatment. In addition, the appearance of hormone dependent phosphoisoforms of the receptor depends on the potency of the agonist used. Using this method we show that the residues contributing to the hormone dependent mobility shift are localized in one of the transcriptional activation domains, between amino acids 130-247. We altered the phosphorylation sites within this domain that correspond to the amino acids phosphorylated in mouse hormone treated cells. Using phosphopeptide maps we show that hormone changes the peptide pattern of metabolically labelled receptor, and we identify peptides which are phosphorylated in hormone dependent manner. Then we determine that phosphorylation of residues S224 and S232 is increased in the presence of hormone, whereas phosphorylation of residues T171 and S246 is constitutive. Finally, we show that in both yeast and mammalian cells the same residues on the glucocorticoid receptor are phosphorylated. Our results suggest that yeast cells would be a suitable system to study glucocorticoid receptor phosphorylation. The genetic manipulability of yeast cells, together with conservation of the phosphorylation of GR in yeast and mammalian cells and identification of hormone dependent phosphorylation, would facilitate the isolation of molecules involved in the glucocorticoid receptor phosphorylation pathway and further our understanding of this process. (C) 1998 Elsevier Science Ltd. All rights reserved

Regional and Temporal Profiles of Calpain and Caspase-3 Activities in Postnatal Rat Brain following Repeated Propofol Administration

Exposure of newborn rats to a variety of anesthetics has been shown to induce apoptotic neurodegeneration in the developing brain. We investigated the effect of the general anesthetic propofol on the brain of 7-day-old (P7) Wistar rats during the peak of synaptic growth. Caspase and calpain protease families most likely participate in neuronal cell death. Our objective was to examine regional and temporal patterns of caspase-3 and calpain activity following repeated propofol administration (20 mg/kg). P7 rats were exposed for 2, 4 or 6 h to propofol and killed 0, 4, 16 and 24 h after exposure. Relative caspase-3 and calpain activities were estimated by Western blot analysis of the proteolytic cleavage products of α-II-spectrin, protein kinase C and poly(ADP-ribose) polymerase 1. Caspase-3 activity and expression displayed a biphasic pattern of activation. Calpain activity changed in a region- and time-specific manner that was distinct from that observed for caspase-3. The time profile of calpain activity exhibited substrate specificity. Fluoro-Jade B staining revealed an immediate neurodegenerative response that was in direct relationship to the duration of anesthesia in the cortex and inversely related to the duration of anesthesia in the thalamus. At later post-treatment intervals, dead neurons were detected only in the thalamus 24 h following the 6-hour propofol exposure. Strong caspase-3 expression that was detected at 24 h was not followed by cell death after 2- and 4-hour exposures to propofol. These results revealed complex patterns of caspase-3 and calpain activities following prolonged propofol anesthesia and suggest that both are a manifestation of propofol neurotoxicity at a critical developmental stage

Effects of age and dexamethasone treatment on glucocorticoid response element and activating protein-1 binding activity in rat brain

The effect of dexamethasone (DEX) on glucocorticoid receptor (GR)-mediated gene expression was examined in the brain of young and aged rats. Electrophoretic mobility shift assays showed that DEX treatment led to an increase of glucocorticoid response element (GRE) binding activity in aged rats, whereas in young animals GRE binding activity was decreased. Western blot analysis and reverse transcriptase polymerase chain reaction confirmed that, in aged animals, the GR mRNA and the GR protein levels were increased on DEX treatment. The binding activity of GRE activating protein-1 (AP-1) site and cross-competition analysis demonstrated specific pattern of expression during the ageing and DEX treatment, suggesting that GR modulates the activity of transcription factors AP-1 (Fos/Jun proteins) through protein-protein interaction. On the basis of these results, it can be concluded that the composition of transcriptional complexes that bind to GRE and AP-I regulatory elements changes upon DEX treatment in an age-specific manner

"Elia in Wartime"

As I detailed in the last number of the Bulletin the Society recently acquired a cache of papers belonging to Herbert Grant Smith (1883-1974) stalwart member of the Charles Lamb Society and editor of the Charles Lamb Society Bulletin 1948-72. We were very pleased to be able to purchase this material, and, while currently housed in a banana box in the University of Leicester, it will find a home in the main CLS collection, now housed in the Guildhall Library in the City of London

Association between the frequency of altered genes, clinicopathological parameters and genomic instability.

<p>^a AA, anaplastic astrocytoma; GBM, glioblastoma multiforme;</p><p>^b Bold indicates statistically significant values;</p><p><i>ITGB1</i>- integrin, beta 1; <i>INPP5A</i>- inositol polyphosphate-5-phosphatase; <i>CPS1</i> - carbamoyl-phosphate synthetase 1, mitochondrial; <i>PROS1</i> - protein S (alpha).^c NP, number of patients per group; </p