The proteome of human brain after ischemic stroke.

Although stroke is among the most common causes of death and chronic disability worldwide, the proteome of the ischemic human brain remains unknown. Only a few studies have investigated the ischemic brain proteome in rodent stroke models. We performed a proteomic study of the human brain after ischemic stroke using a 2-dimensional differential gel electrophoresis-based proteomic approach.

New hierarchical phosphorylation pathway of the translational repressor eIF4E-binding protein 1 (4E-BP1) in ischemia-reperfusion stress.

Eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1) is a translational repressor that is characterized by its capacity to bind specifically to eIF4E and inhibit its interaction with eIF4G. Phosphorylation of 4E-BP1 regulates eIF4E availability, and therefore, cap-dependent translation, in cell stress. This study reports a physiological study of 4E-BP1 regulation by phosphorylation using control conditions and a stress-induced translational repression condition, ischemia-reperfusion (IR) stress, in brain tissue.

Analysis of the protein expression changes during taxol-induced apoptosis under translation inhibition conditions.

Taxol is currently used in chemotherapeutic treatments of different types of cancers. In this article, we demonstrate that taxol induces apoptosis and translation down-regulation in human embryonic kidney (HEK293T) cells. Antibody arrays are a promising new tool for the analysis of protein levels changes in cells responding to different stimuli.

Assessment of protein expression levels after transient global cerebral ischemia using an antibody microarray analysis.

An antibody microarray was used to analyze potential modifications in brain protein levels induced by ischemic reperfusion. Total brain extracts from rats subjected to 15 min of transient global ischemia followed by 3 days of reperfusion and sham control animals were compared within the same array. Separate arrays were run to analyze resistant (cortex) and vulnerable (CA1) regions to ischemia.

Eukaryotic initiation factors (eIF) 2alpha and 4E expression, localization, and phosphorylation in brain tumors.

Increased protein synthesis is regulated, in part, by two eukaryotic translation initiation factors (eIFs): eIF4E and eIF2alpha. One or both of these factors are often overexpressed in several types of cancer cells; however, no data are available at present regarding eIF4E and eIF2alpha levels in brain tumors. In this study, we analyzed the expression, subcellular localization and phosphorylation states of eIF4E and eIF2alpha in 64 brain tumors (26 meningiomas, 16 oligodendroglial tumors, and 22 astrocytomas) and investigated the correlation with the expression of MIB-1, p53, and cyclin D1 proteins as well.

Nitric oxide mediates NMDA-induced persistent inhibition of protein synthesis through dephosphorylation of eukaryotic initiation factor 4E-binding protein 1 and eukaryotic initiation factor 4G proteolysis.

Cerebral ischaemia causes long-lasting protein synthesis inhibition that is believed to contribute to brain damage. Energy depletion promotes translation inhibition during ischaemia, and the phosphorylation of eIF (eukaryotic initiation factor) 2alpha is involved in the translation inhibition induced by early ischaemia/reperfusion. However, the molecular mechanisms underlying prolonged translation down-regulation remain elusive.

Translation regulation after taxol treatment in NIH3T3 cells involves the elongation factor (eEF)2.

Changes to the translational machinery that occur during apoptosis have been described in the last few years. The two principal ways in which translational factors are modified during apoptosis are: (i) changes in protein phosphorylation and (ii) specific proteolytic cleavages. Taxol, a member of a new class of anti-tubulin drugs, is currently used in chemotherapeutic treatments of different types of cancers.

Regulatory proteins of eukaryotic initiation factor 2-alpha subunit (eIF2 alpha) phosphatase, under ischemic reperfusion and tolerance.

Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha), which is one of the substrates of protein phosphatase 1 (PP1), occurs rapidly during the first minutes of post-ischemic reperfusion after an episode of cerebral ischemia. In the present work, two experimental models of transient global ischemia and ischemic tolerance (IT) were used to study PP1 interacting/regulatory proteins following ischemic reperfusion. For that purpose we utilized PP1 purified by microcystin chromatography, as well as 2D DIGE of PP1alpha and PP1gamma immunoprecipitates.

Proteomic characterization of protein phosphatase 1 complexes in ischemia-reperfusion and ischemic tolerance.

Serine/threonine protein phosphatase 1 (PP1) regulates multiple cellular processes. Protein phosphorylation-dephosphorylation is largely altered during ischemia and subsequent reperfusion. The brain is particularly vulnerable to stress resulting from ischemia-reperfusion (IR), however, the acquisition of ischemic tolerance (IT) protects against IR stress.

Characterization of the activity of human MAP kinase-interacting kinase Mnk1b.

Human mitogen-activated protein (MAP) kinase interacting kinase 1b (Mnk1b) is a splice variant of human Mnk1a, which has been identified in our laboratory [A. O'Loghlen, V.M.

A DNA aptamer population specifically detects Leishmania infantum H2A antigen.

Aptamers are short single-stranded DNA or RNA oligonucleotides that are selected in vitro by their affinity and specificity for the target. Binding is a consequence of the particular tertiary structure that they are able to acquire, depending on their sequence. Parasites of the genus Leishmania belongs to the lower eukaryote order Kinetoplastida that causes leishmaniosis in man and animals.

Calpain inhibition stimulates caspase-dependent apoptosis induced by taxol in NIH3T3 cells.

Taxol is an anticancer drug that triggers apoptosis in a wide spectrum of cancers such as ovarian, breast, lung, head and neck, and bladder carcinoma by both caspase-dependent and -independent apoptosis mechanisms. However, the exact signaling pathways involved in taxol-induced apoptosis strongly depend on the cellular background and they are not completely established yet. In this study we demonstrate that taxol induces caspase-3-independent apoptosis in NIH3T3 cells by a calpain-mediated mechanism.

Delayed postconditionig initiates additive mechanism necessary for survival of selectively vulnerable neurons after transient ischemia in rat brain.

1. The aim of this study was to validate the role of postconditioning, used 2 days after lethal ischemia, for protection of selectively vulnerable brain neurons against delayed neuronal death. 2.

Evidence for a role of second pathophysiological stress in prevention of delayed neuronal death in the hippocampal CA1 region.

In ischemic tolerance experiment, when we applied 5-min ischemia 2 days before 30-min ischemia, we achieved a remarkable (95.8%) survival of CA1 neurons. However, when we applied 5-min ischemia itself, without following lethal ischemia, we found out 45.

Anti-heat shock protein 90beta antibodies decrease pre-oligodendrocyte population in perinatal and adult cell cultures. Implications for remyelination in multiple sclerosis.

Lesions in the CNS of patients with multiple sclerosis (MS) often fail to remyelinate, resulting in neurological dysfunction. A key factor seems to be the inefficiency of oligodendrocyte precursor cells (OPCs). We recently reported antibodies against heat shock protein 90beta (Hsp90beta) in MS patients that recognized the antigen on the OPC surface.

Protective effect of L-trans-pyrrolidine-2,4-dicarboxilic acid preload against cell death induced by oxygen/glucose deprivation in differentiated PC12 cells.

It has been postulated that cellular glutamate is released into the extracellular fluid when the energy supply of the brain is compromised (i.e., anoxia or oxygen/glucose deprivation), and there the amino acid triggers the so-called excitotoxic cascade, causing neuronal death.

Suppression of human Mnk1 by small interfering RNA increases the eukaryotic initiation factor 4F activity in HEK293T cells.

Short-interfering RNAs (siRNAs) have proved to be a useful tool in studying gene function in plants, invertebrates and mammalian systems. Herein, we report the use of siRNAs for targeting the human MAP kinase-interacting kinase Mnk1 gene. This study demonstrates the efficacy of the designed siRNA in silencing Mnk1 in the human cell line HEK293T and shows that Mnk1 suppression decreases eukaryotic initiation factor 4E phosphorylation without causing any change in global protein synthesis rate and cell proliferation.

Identification and molecular characterization of Mnk1b, a splice variant of human MAP kinase-interacting kinase Mnk1.

In this paper, we report the identification and molecular characterization of a splice variant of human Mnk1 which has been named as Mnk1b. Human Mnk1b mRNA is homologous to human Mnk1 mRNA but lacking a region corresponding to exon 19, which causes a change in the reading frame generating a stop codon. The resulting protein lacks the last 89 amino acids at the C-terminal region that are replaced by 12 amino acids with an entirely new sequence.

Antibodies reactive to heat shock protein 90 induce oligodendrocyte precursor cell death in culture. Implications for demyelination in multiple sclerosis.

Oligodendrocyte precursor cells (OPCs) are extremely efficient at remyelination. These cells persist in the adult human central nervous system and can proliferate. However, the failure to remyelinate is a pathological characteristic of the human demyelinating disease multiple sclerosis (MS), which suggests that these cells are ineffective in this disorder.

Ischaemic preconditioning in the rat brain: effect on the activity of several initiation factors, Akt and extracellular signal-regulated protein kinase phosphorylation, and GRP78 and GADD34 expression.

Translational repression induced during reperfusion of the ischaemic brain is significantly attenuated by ischaemic preconditioning. The present work was undertaken to identify the components of the translational machinery involved and to determine whether translational attenuation selectively modifies protein expression patterns during reperfusion. Wistar rats were preconditioned by 5-min sublethal ischaemia and 2 days later, 30-min lethal ischaemia was induced.

Selection of aptamers against KMP-11 using colloidal gold during the SELEX process.

SELEX procedure is a methodology in which single stranded oligonucleotides are selected from a wide variety of sequences based on their interaction with a target molecule. We have designed a novel SELEX methodology using colloidal gold to select high affinity single stranded DNA aptamers against Leishmania infantum KMP-11. Kinetoplastid membrane protein-11 (KMP-11) is a major component of the cell membrane of kinetoplastid parasites.

Role of protein synthesis in the ischemic tolerance acquisition induced by transient forebrain ischemia in the rat.

Although ischemic preconditioning of the heart and brain is a well-documented neuroprotective phenomenon, the mechanism underlying the increased resistance to severe ischemia induced by a preceding mild ischemic exposure remains unclear. In this study we have determined the effect of ischemic preconditioning on ischemia/reperfusion-associated translation inhibition in the neocortex and hippocampus of the rat. We studied the effect of the duration on the sublethal ischemic episode (3, 4, 5 or 8 min), as well as the amount of time elapsed between sublethal and lethal ischemia on the cell death 7 days after the last ischemic episode.

Initiation factor 2B activity is regulated by protein phosphatase 1, which is activated by the mitogen-activated protein kinase-dependent pathway in insulin-like growth factor 1-stimulated neuronal cells.

We have previously demonstrated that insulin-like growth factor 1 (IGF1) induces eukaryotic initiation factor 2B (eIF2B) activation in neuronal cells through the phosphatidylinositol 3 kinase/glycogen synthase kinase 3 pathway as well as by activation of the mitogen-activated protein kinase (MAPK)-activating kinase (MEK)/MAPK signaling pathway (Quevedo, C., Alcázar, A., and Salinas, M.

Low concentrations of glutamate induce apoptosis in cultured neurons: implications for amyotrophic lateral sclerosis.

Evidence is accumulating that excessive glutamate concentration in the extracellular space is neurotoxic and plays a role in amyotrophic lateral sclerosis (ALS). However, the published results on glutamate levels in cerebrospinal fluid (CSF) and on glutamate-mediated toxicity of CSF in ALS disease remain controversial. In this report, we studied CSF from patients with sporadic ALS and controls to determine glutamate concentrations, and then analyzed the neurotoxic effect of glutamate at the concentrations present in CSF from ALS patients on cultured cortical neuronal cells.

Cerebral postischemic reperfusion-induced demethylation of the protein phosphatase 2A catalytic subunit.

Brain reperfusion after a period of global ischemia induces changes in the phosphorylation state of a great number of proteins. Neuronal responses to ischemia and reperfusion are quite different depending on the brain region, and phosphorylation changes may be implicated in this tissue-specific response. For this reason, we have used both biochemical and immunohistochemical methods to investigate the potential role of PP2A, the most abundant Ser/Thr phosphatase in the brain, in ischemic injury.