TRPC7 is a receptor-operated DAG-activated channel in human keratinocytes

Muscarinic and purinergic receptors expressed in keratinocytes are an important part of a functional system for cell growth. While several aspects of this process are clearly dependent on Ca(2+) homeostasis, less is known about the mechanisms controlling Ca(2+) entry during epidermal receptor stimulation. We used patch-clamp technique to study responses to carbachol (CCh) and adenosine triphosphate (ATP) in HaCaT human keratinocytes. Both agonists induced large currents mediated by cation-selective channels about three times more permeable to Ca(2+) than Na(+), suggesting that they play an important role in receptor-operated Ca(2+) entry. CCh- and ATP-induced currents were inhibited by 1-[6-([(17beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino)hexyl]-1H-pyrrole-2,5-dione, a phospholipase C (PLC) blocker. Investigation of the pathways downstream of PLC activation revealed that InsP(3) did not affect the agonist responses. In contrast, 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeable analog of 1,2-diacylglycerol (DAG), evoked a similar cation current. This action appears to be direct, since the effects of activators or inhibitors of protein kinase C were comparatively small. Finally, transient receptor potential canonical 7 (TRPC7) specific knockdown by antisense oligonucleotides led to a decrease in ATP- and CCh-induced calcium entry, as well as OAG-evoked current. We concluded that activation of both muscarinic and purinergic receptors via a common DAG-dependent link opens Ca(2+)-permeable TRPC7 channels.     

Chronic interleukin-1 exposure triggers selection for Cebpa-knockout multipotent hematopoietic progenitors

The early events that drive myeloid oncogenesis are not well understood. Most studies focus on the cell-intrinsic genetic changes and how they impact cell fate decisions. We consider how chronic exposure to the proinflammatory cytokine, interleukin-1β (IL-1β), impacts Cebpa-knockout hematopoietic stem and progenitor cells (HSPCs) in competitive settings. Surprisingly, we found that Cebpa loss did not confer a hematopoietic cell–intrinsic competitive advantage; rather chronic IL-1β exposure engendered potent selection for Cebpa loss. Chronic IL-1β augments myeloid lineage output by activating differentiation and repressing stem cell gene expression programs in a Cebpa-dependent manner. As a result, Cebpa-knockout HSPCs are resistant to the prodifferentiative effects of chronic IL-1β, and competitively expand. We further show that ectopic CEBPA expression reduces the fitness of established human acute myeloid leukemias, coinciding with increased differentiation. These findings have important implications for the earliest events that drive hematologic disorders, suggesting that chronic inflammation could be an important driver of leukemogenesis and a potential target for intervention.     

Optical Isolator Utilizing Surface Plasmons

Feasibility of usage of surface plasmons in a new design of an integrated optical isolator has been studied. In the case of surface plasmons propagating at a boundary between a transition metal and a double-layer dielectric, there is a significant difference of optical loss for surface plasmons propagating in opposite directions. Utilizing this structure, it is feasible to fabricate a competitive plasmonic isolator, which benefits from a broad wavelength operational bandwidth and a good technological compatibility for integration into the Photonic Integrated Circuits (PIC). The linear dispersion relation was derived for plasmons propagating in a multilayer magneto-optical slab.     

Identification of reproducible ictal patterns based on quantified frequency analysis of intracranial EEG signals

Purpose: The identification of the epileptogenic zone (EZ) is crucial for planning epilepsy surgery in patients with drug‐resistant partial epilepsy. This task may require intracerebral encephalography (EEG) monitoring, the results of which are usually interpreted by visual presurgical inspection. A computer‐assisted method for rapidly identifying reproducible ictal patterns based on the analysis of time, frequency, and spatial domains of stereo‐EEG (SEEG) signals is described here. Methods: A new method for EZ detection was tested on SEEG recordings performed by intracerebral electrodes in eight patients with pharmacoresistant partial epilepsy. SEEG data were exported to a program developed in LabView. Key Findings: Prevalent frequencies during seizure events were evaluated by Fourier transform and further integral algorithms. Different frequencies and the relative powers were simultaneously evaluated in all recording leads. Patterns characterized by specific and prevalent frequencies were identified in a subset of recording sites during both seizure onset and seizure development. Three‐dimensional (3D) maps of the measurements obtained from each recording channel were reconstructed on magnetic resonance coordinates to visualize the spatial distribution of the EZ. With this method, the reproducibility of ictal patterns in the same patient was characterized. The boundaries of the EZ identified with this algorithm correlated well with the EZ recognized with the traditional approach (n = 8). The spatial distribution of specific SEEG signals associated with different types of seizures was also analyzed in two patients. Significance: We describe a computer‐assisted method to acquire information on EZ boundaries and to verify reproducibility of seizure patterns from intracerebral recordings performed in patients with pharmacoresistant partial epilepsies.     

A guinea pig model of mesial temporal lobe epilepsy following nonconvulsive status epilepticus induced by unilateral intrahippocampal injection of kainic acid

Purpose: Models of temporal lobe epilepsy are commonly utilized to study focal epileptogenesis and ictogenesis. The criteria that define animal models representative of human mesial temporal lobe may vary in different laboratories. We describe herein a focal epilepsy model of mesial temporal (hippocampal) origin that relies on the analysis of interictal and ictal electroencephalography (EEG) patterns and on their correlation with seizure symptoms and neuropathologic findings. The study is based on guinea pigs, a species seldom utilized to develop chronic epilepsy models. Methods: Young adult guinea pigs were bilaterally implanted under isoflurane anesthesia with epidural electrodes over somatosensory cortex and depth electrodes in CA1 hippocampal region. A stainless steel guide cannula was positioned unilaterally in the right dorsal hippocampus to inject 1 μl of 0.9% NaCl solution containing 1 μg kainic acid (KA). One week after surgery, continuous 24 h/day video‐EEG monitoring was performed 48 h before and every other week after KA injection, for no <1 month. EEG data were recorded wide‐band at 2 kHz. After video‐EEG monitoring, brains were analyzed for thionine and Timm staining and glial fibrillary acid protein (GFAP) immunostaining. Key Findings: Unilateral injection of KA in dorsal hippocampus of guinea pigs induces an acute nonconvulsive status epilepticus (SE) that terminates within 24 h (n = 22). Chronic seizures with very mild motor signs (undetectable without EEG monitoring) and highly variable recurrence patterns appear in 45.5% (10 of 22) KA‐treated animals, with variable delays from the initial SE. In these animals interictal events, CA1 cell loss, gliosis, and altered Timm staining pattern were observed. The induction of a chronic condition did not correlate with the duration of the nonconvulsive acute SE, but correlated with the extension and quality of neuropathologic damage. Significance: We demonstrate that a model of hippocampal (mesial temporal lobe) epilepsy can be developed in the guinea pig by intrahippocampal injection of KA. Seizure events in this model show little behavioral signs and may be overlooked without extensive video‐EEG monitoring. The establishment of a chronic epileptic condition correlates with the extension of the hippocampal damage (mainly cell loss and gliosis) and not with the intensity of the initial SE.     

Changes in action potential features during focal seizure discharges in the entorhinal cortex of the in vitro isolated guinea pig brain

Temporal lobe seizures in humans correlate with stereotyped electrophysiological patterns that can be reproduced in animal models to study the cellular and network changes responsible for ictogenesis. Seizure-like discharges that mimic seizure patterns in humans were induced in the entorhinal cortex of the in vitro isolated guinea pig brain by 3-min arterial applications of the GABA(A) receptor antagonist bicuculline. The onset of seizure is characterized by a paradoxical interruption of firing for several seconds in principal neurons coupled with both enhanced interneuronal firing and increased extracellular potassium (Gnatkovsky et al. 2008). The evolution of action potential features from firing break to excessive and synchronous activity associated with the progression of seizure itself is analyzed here. We utilized phase plot analysis to characterize action potential features of entorhinal cortex neurons in different phases of a seizure. Compared with preictal action potentials, resumed spikes in layer II-III neurons (n = 17) during the early phase of the seizure-like discharge displayed 1) depolarized threshold, 2) lower peak amplitude, 3) depolarized voltage of repolarization and 4) decelerated depolarizing phase, and 5) spike doublettes. Action potentials in deep-layer principal cells (n = 8) during seizure did not show the marked feature changes observed in superficial layer neurons. Action potential reappearance correlated with an increase in extracellular potassium. High-threshold, slow-action potentials similar to those observed in the irregular firing phase of a seizure were reproduced in layer II-III neurons by direct cortical application of a highly concentrated potassium solution (12-24 mM). We propose that the generation of possibly nonsomatic action potentials by increased extracellular potassium represents a crucial step toward reestablish firing after an initial depression in an acute model of temporal lobe seizures. Resumed firing reengages principal neurons into seizure discharge and promotes the transition toward the synchronized burst firing that characterizes the late phase of a seizure.     

ECT2 and RASAL2 mediate mesenchymal-amoeboid transition in human astrocytoma cells

Malignant astrocytomas are highly invasive brain tumors. The Rho family of cytoskeletal GTPases are key regulators of astrocytoma migration and invasion; expression of the guanine nucleotide exchange factor ECT2 is elevated in primary astrocytomas and predicts both survival and malignancy. Mice bearing orthotopically implanted astrocytoma cells with diminished ECT2 levels following ECT2 knockdown exhibit longer survival. Although ECT2 is normally expressed in the nucleus, we show that ECT2 is aberrantly localized to the cytoplasm in both astrocytoma cell lines and primary human astrocytomas, and colocalizes with RAC1 and CDC42 at the leading edge of migrating astrocytoma cells. Inhibition of ECT2 expression by RNA interference resulted in decreased RAC1 and CDC42 activity, but no change in RHO activity, suggesting that ECT2 is capable of activating these pro-migratory Rho family members. ECT2 overexpression in astrocytoma cells resulted in a transition to an amoeboid phenotype that was abolished with the ROCK inhibitor, Y-27632. Cytoplasmic fractionation of astrocytoma cells followed by ECT2 immunoprecipitation and mass spectrometry were used to identify protein-binding partners that modulate the activity of ECT2 toward RAC1 and RHO/ROCK. We identified RASAL2 as an ECT2-interacting protein that regulates RHO activity in astrocytoma cells. RASAL2 knockdown leads to a conversion to an amoeboid phenotype. Our studies reveal that ECT2 has a novel role in mesenchymal-amoeboid transition in human astrocytoma cells.     

The psycho- and neurotropic profiling of novel 3-(N-R,R′-aminomethyl)-2-methyl-1H-quinolin-4-ones in vivo

The article presents the study of psycho- and neurotropic properties of novel 3-(N-R,R′-aminomethyl)-2-methyl-1H-quinolin-4-ones in vivo. The research was carried out using the open field test, elevated plus maze, rotarod test, tail suspension test, passive avoidance test after scopolamine-induced amnesia and acute normobaric hypoxia with hypercapnia. As a result, two promising substances have been found. According to our results 3-[[(4-methoxyphenyl)amino]methyl]-2-methyl-1H-quinolin-4-one in the dose of 10?mg/kg shows a specific sedative effect and a considerable anti-amnesic activity. The most interesting N-[(2-methyl-4-oxo-1H-quinolin-3-yl)methyl]-N-phenylbenzamide (100?mg/kg) combines a potent anti-anxiety action, the anti-amnesic activity and a considerable antihypoxic effect. They are of interest for further profound studies as promising psychoactive compounds.