Silencing microRNA-134 produces neuroprotective and prolonged seizure-suppressive effects

Temporal lobe epilepsy is a common, chronic neurological disorder characterized by recurrent spontaneous seizures. MicroRNAs (miRNAs) are small, noncoding RNAs that regulate post-transcriptional expression of protein-coding mRNAs, which may have key roles in the pathogenesis of neurological disorders. In experimental models of prolonged, injurious seizures (status epilepticus) and in human epilepsy, we found upregulation of miR-134, a brain-specific, activity-regulated miRNA that has been implicated in the control of dendritic spine morphology. Silencing of miR-134 expression in vivo using antagomirs reduced hippocampal CA3 pyramidal neuron dendrite spine density by 21% and rendered mice refractory to seizures and hippocampal injury caused by status epilepticus. Depletion of miR-134 after status epilepticus in mice reduced the later occurrence of spontaneous seizures by over 90% and mitigated the attendant pathological features of temporal lobe epilepsy. Thus, silencing miR-134 exerts prolonged seizure-suppressant and neuroprotective actions; determining whether these are anticonvulsant effects or are truly antiepileptogenic effects requires additional experimentation.

Design, synthesis, and biological characterization of a peptide-mimetic antagonist for a tethered-ligand receptor

Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G protein-coupled receptors, which are enzymatically cleaved to expose a truncated extracellular N terminus that acts as a tethered activating ligand. PAR-1 is cleaved and activated by the serine protease α-thrombin, is expressed in various tissues (e.g., platelets and vascular cells), and is involved in cellular responses associated with hemostasis, proliferation, and tissue injury. We have discovered a series of potent peptide-mimetic antagonists of PAR-1, exemplified by RWJ-56110. Spatial relationships between important functional groups of the PAR-1 agonist peptide epitope SFLLRN were employed to design and synthesize candidate ligands with appropriate groups attached to a rigid molecular scaffold. Prototype RWJ-53052 was identified and optimized via solid-phase parallel synthesis of chemical libraries. RWJ-56110 emerged as a potent, selective PAR-1 antagonist, devoid of PAR-1 agonist and thrombin inhibitory activity. It binds to PAR-1, interferes with PAR-1 calcium mobilization and cellular function (platelet aggregation; cell proliferation), and has no effect on PAR-2, PAR-3, or PAR-4. By flow cytometry, RWJ-56110 was confirmed as a direct inhibitor of PAR-1 activation and internalization, without affecting N-terminal cleavage. At high concentrations of α-thrombin, RWJ-56110 fully blocked activation responses in human vascular cells, albeit not in human platelets; whereas, at high concentrations of SFLLRN-NH2, RWJ-56110 blocked activation responses in both cell types. Thus, thrombin activates human platelets independently of PAR-1, i.e., through PAR-4, which we confirmed by PCR analysis. Selective PAR-1 antagonists, such as RWJ-56110, should serve as useful tools to study PARs and may have therapeutic potential for treating thrombosis and restenosis.

Cardioprotection from ischemia by a brief exposure to physiological levels of ethanol: Role of epsilon protein kinase C

Recent epidemiological studies indicate beneficial effects of moderate ethanol consumption in ischemic heart disease. Most studies, however, focus on the effect of long-term consumption of ethanol. In this study, we determined whether brief exposure to ethanol immediately before ischemia also produces cardioprotection. In addition, because protein kinase C (PKC) has been shown to mediate protection of the heart from ischemia, we determined the role of specific PKC isozymes in ethanol-induced protection. We demonstrated that (i) brief exposure of isolated adult rat cardiac myocytes to 10–50 mM ethanol protected against damage induced by prolonged ischemia; (ii) an isozyme-selective ɛPKC inhibitor developed in our laboratory inhibited the cardioprotective effect of acute ethanol exposure; (iii) protection of isolated intact adult rat heart also occurred after incubation with 10 mM ethanol 20 min before global ischemia; and (iv) ethanol-induced cardioprotection depended on PKC activation because it was blocked by chelerythrine and GF109203X, two PKC inhibitors. Consumption of 1–2 alcoholic beverages in humans leads to blood alcohol levels of ≈10 mM. Therefore, our work demonstrates that exposure to physiologically attainable ethanol levels minutes before ischemia provides cardioprotection that is mediated by direct activation of ɛPKC in the cardiac myocytes. The potential clinical implications of our findings are discussed.

MEKK1 suppresses oxidative stress-induced apoptosis of embryonic stem cell-derived cardiac myocytes

A combination of in vitro embryonic stem (ES) cell differentiation and targeted gene disruption has defined complex regulatory events underlying oxidative stress-induced cardiac apoptosis, a model of postischemic reperfusion injury of myocardium. ES cell-derived cardiac myocytes (ESCM) having targeted disruption of the MEKK1 gene were extremely sensitive, relative to wild-type ESCM, to hydrogen peroxide-induced apoptosis. In response to oxidative stress, MEKK1−/− ESCM failed to activate c-Jun kinase (JNK) but did activate p38 kinase similar to that observed in wild-type ESCM. The increased apoptosis was mediated through enhanced tumor necrosis factor α production, a response that was positively and negatively regulated by p38 and the MEKK1-JNK pathway, respectively. Thus, MEKK1 functions in the survival of cardiac myocytes by inhibiting the production of a proapoptotic cytokine. MEKK1 regulation of the JNK pathway is a critical response for the protection against oxidative stress-induced apoptosis in cardiac myocytes.

Remodeling of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor subunit composition in hippocampal neurons after global ischemia

Transient global ischemia induces selective delayed cell death, primarily of principal neurons in the hippocampal CA1. However, the molecular mechanisms underlying ischemia-induced cell death are as yet unclear. The present study shows that global ischemia triggers a pronounced and cell-specific reduction in GluR2 [the subunit that limits Ca2+ permeability of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors] in vulnerable CA1 neurons, as evidenced by immunofluorescence of brain sections and Western blot analysis of microdissected hippocampal subfields. At 72 h after ischemia (a time before cell death), virtually all CA1 pyramidal neurons exhibited greatly reduced GluR2 immunolabeling throughout their somata and dendritic processes. GluR2 immunolabeling was unchanged in pyramidal cells of the CA3 and granule cells of the dentate gyrus, regions resistant to ischemia-induced damage. Immunolabeling of the AMPA receptor subunit GluR1 was unchanged in CA1, CA3, and dentate gyrus. Western analysis indicated that GluR2 subunit abundance was markedly reduced in CA1 at 60 and 72 h after the ischemic insult; GluR1 abundance was unchanged in all subfields at all times examined. These findings, together with the previous observation of enhanced AMPA-elicited Ca2+ influx in postischemic CA1 neurons, show that functional GluR2-lacking, Ca2+-permeable AMPA receptors are expressed in vulnerable neurons before cell death. Thus, the present study provides an important link in the postulated causal chain between global ischemia and delayed death of CA1 pyramidal neurons.

Inhibition of poly(ADP-ribose) polymerase activity is insufficient to induce tetraploidy

Poly(ADP-ribose) polymerase (PARP) knockout mice are resistant to murine models of human diseases such as cerebral and myocardial ischemia, traumatic brain injury, diabetes, Parkinsonism, endotoxic shock and arthritis, implicating PARP in the pathogenesis of these diseases. Potent selective PARP inhibitors are therefore being evaluated as novel therapeutic agents in the treatment of these diseases. Inhibition or depletion of PARP, however, increases genomic instability in cells exposed to genotoxic agents. We recently demonstrated the presence of a genomically unstable tetraploid population in PARP–/– fibroblasts and its loss after stable transfection with PARP cDNA. To elucidate whether the genomic instability is attributable to PARP deficiency or lack of PARP activity, we investigated the effects of PARP inhibition on development of tetraploidy. Immortalized wild-type and PARP–/– fibroblasts were exposed for 3 weeks to 20 µM GPI 6150 (1,11b-dihydro-[2H]benzopyrano[4,3,2-de]isoquinolin-3-one), a novel small molecule specific competitive inhibitor of PARP (Ki = 60 nM) and one of the most potent PARP inhibitors to date (IC50 = 0.15 µM). Although GPI 6150 initially decreased cell growth in wild-type cells, there was no effect on cell growth or viability after 24 h. GPI 6150 inhibited endogenous PARP activity in wild-type cells by ∼91%, to about the residual levels in PARP–/– cells. Flow cytometric analysis of unsynchronized wild-type cells exposed for 3 weeks to GPI 6150 did not induce the development of tetraploidy, suggesting that, aside from its catalytic function, PARP may play other essential roles in the maintenance of genomic stability.

Distinct neurochemical features of acute and persistent pain

To address the neurochemistry of the mechanisms that underlie the development of acute and persistent pain, our laboratory has been studying mice with deletions of gene products that have been implicated in nociceptive processing. We have recently raised mice with a deletion of the preprotachykinin-A gene, which encodes the peptides substance P (SP) and neurokinin A (NKA). These studies have identified a specific behavioral phenotype in which the animals do not detect a window of “pain” intensities; this window cuts across thermal, mechanical, and chemical modalities. The lowered thermal and mechanical withdrawal thresholds that are produced by tissue or nerve injury, however, were still present in the mutant mice. Thus, the behavioral manifestations of threshold changes in nociceptive processing in the setting of injury do not appear to require SP or NKA. To identify relevant neurochemical factors downstream of the primary afferent, we are also studying the dorsal horn second messenger systems that underlie the development of tissue and nerve injury-induced persistent pain states. We have recently implicated the γ isoform of protein kinase C (PKCγ) in the development of nerve injury-induced neuropathic pain. Acute pain processing, by contrast, is intact in the PKCγ-null mice. Taken together, these studies emphasize that there is a distinct neurochemistry of acute and persistent pain. Persistent pain should be considered a disease state of the nervous system, not merely a prolonged acute pain symptom of some other disease conditions.

Bcl-2 potentiates the maximal calcium uptake capacity of neural cell mitochondria.

Expression of the human protooncogene bcl-2 protects neural cells from death induced by many forms of stress, including conditions that greatly elevate intracellular Ca2+. Considering that Bcl-2 is partially localized to mitochondrial membranes and that excessive mitochondrial Ca2+ uptake can impair electron transport and oxidative phosphorylation, the present study tested the hypothesis that mitochondria from Bcl-2-expressing cells have a higher capacity for energy-dependent Ca2+ uptake and a greater resistance to Ca(2+)-induced respiratory injury than mitochondria from cells that do not express this protein. The overexpression of bcl-2 enhanced the mitochondrial Ca2+ uptake capacity using either digitonin-permeabilized GT1-7 neural cells or isolated GT1-7 mitochondria by 1.7 and 3.9 fold, respectively, when glutamate and malate were used as respiratory substrates. This difference was less apparent when respiration was driven by the oxidation of succinate in the presence of the respiratory complex I inhibitor rotenone. Mitochondria from Bcl-2 expressors were also much more resistant to inhibition of NADH-dependent respiration caused by sequestration of large Ca2+ loads. The enhanced ability of mitochondria within Bcl-2-expressing cells to sequester large quantities of Ca2+ without undergoing profound respiratory impairment provides a plausible mechanism by which Bcl-2 inhibits certain forms of delayed cell death, including neuronal death associated with ischemia and excitotoxicity.

Pharmacological modulation of heat shock factor 1 by antiinflammatory drugs results in protection against stress-induced cellular damage.

The activation of heat shock genes by diverse forms of environmental and physiological stress has been implicated in a number of human diseases, including ischemic damage, reperfusion injury, infection, neurodegeneration, and inflammation. The enhanced levels of heat shock proteins and molecular chaperones have broad cytoprotective effects against acute lethal exposures to stress. Here, we show that the potent antiinflammatory drug indomethacin activates the DNA-binding activity of human heat shock transcription factor 1 (HSF1). Perhaps relevant to its pharmacological use, indomethacin pretreatment lowers the temperature threshold of HSF1 activation, such that a complete heat shock response can be attained at temperatures that are by themselves insufficient. The synergistic effect of indomethacin and elevated temperature is biologically relevant and results in the protection of cells against exposure to cytotoxic conditions.

N-tert-butyl-alpha-phenylnitrone improves recovery of brain energy state in rats following transient focal ischemia.

Recent results have demonstrated that the spin trapping agent N-tert-butyl-alpha-phenylnitrone (PBN) reduces infarct size due to middle cerebral artery occlusion (MCAO), even when given after ischemia. The objective of the present study was to explore whether PBN influences recovery of energy metabolism. MCAO of 2-hr duration was induced in rats by an intraluminal filament technique. Brains were frozen in situ at the end of ischemia and after 1, 2, and 4 hr of recirculation. PBN was given 1 hr after recirculation. Neocortical focal and perifocal ("penumbra") areas were sampled for analyses of phosphocreatine (PCr), creatine, ATP, ADP, AMP, glycogen, glucose, and lactate. The penumbra showed a moderate-to-marked decrease and the focus showed a marked decrease in PCr and ATP concentrations, a decline in the sum of adenine nucleotides, near-depletion of glycogen, and an increase in lactate concentration after 2 hr of ischemia. Recirculation for 1 hr led to only a partial recovery of energy state, with little further improvement after 2 hr and signs of secondary deterioration after 4 hr, particularly in the focus. After 4 hr of recirculation, PBN-treated animals showed pronounced recovery of energy state, with ATP and lactate contents in both focus and penumbra approaching normal values. Although an effect of PBN on mitochondria cannot be excluded, the results suggest that PBN acts by preventing a gradual compromise of microcirculation. The results justify a reevaluation of current views on the pathophysiology of focal ischemic damage and suggest that a therapeutic window of many hours exists in stroke.

Signals involved in wound-induced proteinase inhibitor II gene expression in tomato and potato plants.

Chemical and physical signals have been reported to mediate wound-induced proteinase inhibitor II (Pin2) gene expression in tomato and potato plants. Among the chemical signals, phytohormones such as abscisic acid (ABA) and jasmonic acid (JA) and the peptide systemin represent the best characterized systems. Furthermore, electrical and hydraulic mechanisms have also been postulated as putative Pin2-inducing systemic signals. Most of the chemical agents are able to induce Pin2 gene expression without any mechanical wounding. Thus, ABA, JA, and systemin initiate Pin2 mRNA accumulation in the directly treated leaves and in the nontreated leaves (systemic) that are located distal to the treated ones. ABA-deficient tomato and potato plants do not respond to wounding by accumulation of Pin2 mRNA, therefore providing a suitable model system for analysis of the signal transduction pathway involved in wound-induced gene activation. It was demonstrated that the site of action of JA is located downstream to the site of action of ABA. Moreover, systemin represents one of the initial steps in the signal transduction pathway regulating the wound response. Recently, it was reported that heat treatment and mechanical injury generate electrical signals, which propagate throughout the plant. These signals are capable of inducing Pin2 gene expression in the nontreated leaves of wounded plants. Furthermore, electrical current application to tomato leaves leads to an accumulation of Pin2 mRNA in local and systemic tissues. Examination of photosynthetic parameters (assimilation and transpiration rate) on several types of stimuli suggests that heat-induced Pin2 gene expression is regulated by an alternative pathway from that mediating the electrical current and mechanical wound response.

Portal cavernoma cholangiopathy : case report and systematic review

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Individual and Organizational Factors Influencing Technology Adoption for Construction Safety

Thesis (Master's)--University of Washington, 2016-06

Steatosis in chronic hepatitis C: Association with increased messenger RNA expression of collagen I, tumor necrosis factor-α and cytochrome P450 2E1

Background: Increased levels of tumor necrosis factor (TNF)-alpha and oxidative stress have been implicated as factors contributing to hepatic injury in fatty liver diseases. As steatosis is associated with an accelerated progression of fibrosis in chronic hepatitis C (HCV), we hypothesized that the messenger (m)RNA expression of genes involved with the production of reactive oxygen species, inflammation and cellular injury would be increased in liver tissue from subjects with steatosis and chronic HCV. Methods: Real-time polymerase chain reaction was performed to determine relative mRNA expression levels of collagen I, TNF-alpha, cytochrome P450 2E1 (CYP 2E1), transforming growth factor-beta1 and CD14 in liver biopsies from 38 patients with chronic HCV. The mRNA expression levels were compared between subjects with and without steatosis, fibrosis, and inflammation. Results: Multivariate analysis demonstrated that collagen I mRNA expression was increased by 199% in steatosis (P = 0.02), 85% in moderate to severe fibrosis (P = 0.02) and 157% in inflammation (P = 0.03). Livers of patients with steatosis also had an increase in TNF-alpha mRNA expression by 50% (P = 0.03) and CYP 2E1 expression by 37% (P = 0.04) compared with non-steatotic livers. Tumor necrosis factor-alpha protein was localized to Kupffer cells, bile ducts and portal inflammatory cells by immunohistochemistry. Conclusion: Increased expression of TNF-alpha may be involved in the pathogenesis of liver injury and progression of fibrosis in individuals who have steatosis in association with chronic HCV. (C) 2003 Blackwell Publishing Asia Pty Ltd.

Oxygen sensors and energy sensors act synergistically to achieve a graded alteration in gene expression: Consequences for assessing the level of neuroprotection in response to stressors

Changes in gene expression are associated with switching to an autoprotected phenotype in response to environmental and physiological stress. Ubiquitous molecular chaperones from the heat shock protein (HSP) superfamily confer neuronal protection that can be blocked by antibodies. Recent research has focused on the interactions between the molecular sensors that affect the increased expression of neuroprotective HSPs above constitutive levels. An examination of the conditions under which the expression of heat shock protein 70 (Hsp70) was up regulated in a hypoxia and anoxia tolerant tropical species, the epaulette shark (Hemiscyllium ocellatum), revealed that up-regulation was dependent on exceeding a stimulus threshold for an oxidative stressor. While hypoxic-preconditioning confers neuroprotective changes, there was no increase in the level of Hsp70 indicating that its increased expression was not associated with achieving a neuroprotected state in response to hypoxia in the epaulette shark. Conversely, there was a significant increase in Hsp70 in response to anoxic-preconditioning, highlighting the presence of a stimulus threshold barrier and raising the possibility that, in this species, Hsp70 contributes to the neuroprotective response to extreme crises, such as oxidative stress. Interestingly, there was a synergistic effect of coincident stressors on Hsp70 expression, which was revealed when metabolic stress was superimposed upon oxidative stress. Brain energy charge was significantly lower when adenosine receptor blockade, provided by treatment with aminophylline, was present prior to the final anoxic episode, under these circumstances, the level of Hsp70 induced was significantly higher than in the pair-matched saline treated controls. An understanding of the molecular and metabolic basis for neuroprotective switches, which result in an up-regulation of neuroprotective Hsp70 expression in the brain, is needed so that intervention strategies can be devised to manage CNS pathologies and minimise damage caused by ischemia and trauma. In addition, the current findings indicate that measurements of HSP expression per se may provide a useful correlate of the level of neuroprotection achieved in the switch to an autoprotected phenotype.