Alexander V. Glushakov

Enzyme-based lactic acid detection using AlGaN∕GaN high electron mobility transistors with ZnO nanorods grown on the gate region

The detection of lactic acid with ZnO nanorod-gated AlGaN∕GaN high electron mobility transistors (HEMTs) was demonstrated. The array of ZnO nanorods provided a large effective surface area with a high surface-to-volume ratio and a favorable environment for the immobilization of lactate oxidase. The HEMT drain-source current showed a rapid response when various concentrations of lactic acid solutions were introduced to the gate area of the HEMT sensor. The HEMT could detect lactic acid concentrations from 167nM to 139μM. Our results show that portable, fast response, and wireless-based lactic acid detectors can be realized with AlGaN∕GaN HEMT based sensors.

L-phenylalanine selectively depresses currents at glutamatergic excitatory synapses

To explore the hypothesis that L‐phenylalanine (L‐Phe) depresses glutamatergic synaptic transmission and thus contributes to brain dysfunction in phenylketonuria (PKU), the effects of L‐Phe on spontaneous and miniature excitatory postsynaptic currents (s/mEPSCs) in rat and mouse hippocampal and cerebrocortical cultured neurons were studied using the patch‐clamp technique. L‐Phe depressed the amplitude and frequency of both N‐methyl‐D‐aspartate (NMDA) and non‐NMDA components of glutamate receptor (GluR) s/mEPSCs. The IC50 of L‐Phe to inhibit non‐NMDAR mEPSC frequency was 0.98 ± 0.13 mM, a brain concentration seen in classical PKU. In contrast, D‐Phe had a significantly smaller effect, whereas L‐leucine, an amino acid that competes with L‐Phe for brain transporter, had no effect on mEPSCs. Unlike GluR s/mEPSCs, GABA receptor mIPSCs were not attenuated by L‐Phe. A high extracellular concentration of glycine prevented the attenuation by L‐Phe of NMDAR current, activated by exogenous agonist, and of NMDAR s/mEPSC amplitude, but not of NMDAR s/mEPSC frequency. On the other hand, L‐Phe significantly depressed non‐NMDAR current activated by low but not high concentrations of exogenous agonists. Glycine‐independent attenuation of NMDAR s/mEPSC frequency suggests decreased presynaptic glutamate release caused by L‐Phe, whereas decreased amplitudes of NMDAR and non‐NMDAR s/mEPSCs are consistent with competition of L‐Phe for the glycine‐ and glutamate‐binding sites of NMDARs and non‐NMDARs, respectively. The finding that GluR activity is significantly depressed at conditions characteristic of classical PKU indicates a potentially important contribution of impaired GluR function to PKU‐related mental retardation and provides important insights into the potential physiological consequences of impaired GluR function.

Putative Role of Prostaglandin Receptor in Intracerebral Hemorrhage

Each year, approximately 795,000 people experience a new or recurrent stroke. Of all strokes, 84% are ischemic, 13% are intracerebral hemorrhage (ICH) strokes, and 3% are subarachnoid hemorrhage strokes. Despite the decreased incidence of ischemic stroke, there has been no change in the incidence of hemorrhagic stroke in the last decade. ICH is a devastating disease 37–38% of patients between the ages of 45 and 64 die within 30 days. In an effort to prevent ischemic and hemorrhagic strokes we and others have been studying the role of prostaglandins and their receptors. Prostaglandins are bioactive lipids derived from the metabolism of arachidonic acid. They sustain homeostatic functions and mediate pathogenic mechanisms, including the inflammatory response. Most prostaglandins are produced from specific enzymes and act upon cells via distinct G-protein coupled receptors. The presence of multiple prostaglandin receptors cross-reactivity and coupling to different signal transduction pathways allow differentiated cells to respond to prostaglandins in a unique manner. Due to the number of prostaglandin receptors, prostaglandin-dependent signaling can function either to promote neuronal survival or injury following acute excitotoxicity, hypoxia, and stress induced by ICH. To better understand the mechanisms of neuronal survival and neurotoxicity mediated by prostaglandin receptors, it is essential to understand downstream signaling. Several groups including ours have discovered unique roles for prostaglandin receptors in rodent models of ischemic stroke, excitotoxicity, and Alzheimer disease, highlighting the emerging role of prostaglandin receptor signaling in hemorrhagic stroke with a focus on cyclic-adenosine monophosphate and calcium (Ca2+) signaling. We review current ICH data and discuss future directions notably on prostaglandin receptors, which may lead to the development of unique therapeutic targets against hemorrhagic stroke and brain injuries alike.

Specific inhibition of N-methyl-D-aspartate receptor function in rat hippocampal neurons by L-phenylalanine at concentrations observed during phenylketonuria

Hippocampal N-methyl-D-aspartate receptors (NMDARs) are thought to be involved in the regulation of memory formation and learning. Investigation of NMDAR function during experimental conditions known to be associated with impaired cognition in vivo may provide new insights into the role of NMDARs in learning and memory. Specifically, the mechanism whereby high concentrations of L-phenylalanine (L-Phe) during phenylketonuria (>1.2 mM) cause mental retardation remains unknown. Therefore, the effects of L-Phe on NMDA-activated currents (INMDA) were studied in cultured hippocampal neurons from newborn rats using the patch-clamp technique. L-Phe specifically and reversibly attenuated INMDA in a concentration-dependent manner (IC50 = 1.71 ± 0.24 mM). In contrast, L-tyrosine (L-Tyr), an amino acid synthesized from L-Phe in normal subjects, did not significantly change INMDA. Although the L-Phe-INMDA concentration-response relationship was independent of the concentration of NMDA, it was shifted rightward by increasing the concentration of glycine. Consistent with an effect of L-Phe on the NMDAR glycine-binding site, L-Phe (1 mM) did not attenuate INMDA in the presence of D-alanine (10 μM). Furthermore, L-Phe significantly attenuated neither glutamate-activated current in the presence of MK-801, nor current activated by AMPA. The finding that L-Phe inhibits specifically NMDAR current in hippocampal neurons by competing for the glycine-binding site suggests a role for impaired NMDAR function in the development of mental retardation during phenylketonuria and accordingly an important role for NMDARs in memory formation and learning.

Neuroprotective Action of Halogenated Derivatives of L-Phenylalanine

Background and Purpose— The aromatic amino acid L-Phenylalanine (L-Phe) significantly and reversibly depresses excitatory glutamatergic synaptic transmission (GST) via a unique set of presynaptic and postsynaptic mechanisms. Therefore, we hypothesized that endogenous derivatives of L-Phe, which display potent antiglutamatergic activity, may safely and efficaciously protect the brain during conditions characterized by overactivation of glutamate receptors. Methods— We tested this hypothesis in vitro with a combination of patch-clamp and lactate dehydrogenase (LDH) analyses in rat cultured neurons exposed to simulated ischemia, and in vivo using a rat model of experimental stroke caused by transient middle cerebral artery occlusion (MCAO). Results— 3,5-diiodo-l-tyrosine (DIT) and 3,5-dibromo-l-tyrosine (DBrT), endogenous halogenated derivatives of L-Phe, attenuated GST by similar mechanisms as L-Phe, but with greater potency. For example, the IC50s for DIT and DBrT to depress the frequency of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptor-mediated mEPSCs were 104.6±14.1 μmol/L and 127.5±13.3 μmol/L, respectively. Depression of GST by DIT and DBrT persisted during energy deprivation. Furthermore, DBrT significantly reduced LDH release in neuronal cultures exposed to oxygen glucose deprivation. In rats subjected to transient MCAO, DBrT decreased the brain infarct volume and neurological deficit score to 52.7±14.1% and 57.1±12.0% of control values, respectively. DBrT neither altered atrioventricular nodal and intraventricular conduction in isolated heart, nor heart rate and blood pressure in vivo. Conclusion— DBrT, an endogenous halogenated derivative of L-Phe, shows promise as a representative of a novel class of neuroprotective agents by exerting significant neuroprotection in both in vitro and in vivo models of brain ischemia.

Biomarkers for acute diagnosis and management of stroke in neurointensive care units

The effectiveness of current management of critically ill stroke patients depends on rapid assessment of the type of stroke, ischemic or hemorrhagic, and on a patient′s general clinical status. Thrombolytic therapy with recombinant tissue plasminogen activator (r-tPA) is the only effective treatment for ischemic stroke approved by the Food and Drug Administration (FDA), whereas no treatment has been shown to be effective for hemorrhagic stroke. Furthermore, a narrow therapeutic window and fear of precipitating intracranial hemorrhage by administering r-tPA cause many clinicians to avoid using this treatment. Thus, rapid and objective assessments of stroke type at admission would increase the number of patients with ischemic stroke receiving r-tPA treatment and thereby, improve outcome for many additional stroke patients. Considerable literature suggests that brain-specific protein biomarkers of glial [i.e. S100 calcium-binding protein B (S100B), glial fibrillary acidic protein (GFAP)] and neuronal cells [e.g., ubiquitin C-terminal hydrolase-L1 (UCH-L1), neuron-specific enolase (NSE), αII-spectrin breakdown products SBDP120, SBDP145, and SBDP150, myelin basic protein (MBP), neurofilament light chain (NF-L), tau protein, visinin-like protein-1 (VLP 1), NR2 peptide] injury that could be detected in the cerebrospinal fluid (CSF) and peripheral blood might provide valuable and timely diagnostic information for stroke necessary to make prompt management and decisions, especially when the time of stroke onset cannot be determined. This information could include injury severity, prognosis of short-term and long-term outcomes, and discrimination of ischemic or hemorrhagic stroke. This chapter reviews the current status of the development of biomarker-based diagnosis of stroke and its potential application to improve stroke care.

Modulation of nicotinic acetylcholine receptor activity in submucous neurons by intracellular messengers

The effects on acetylcholine-induced membrane currents (ACh currents), produced by agents known to modify the activity of intracellular messengers, were studied in the neurons of the guinea-pig ileum submucous plexus (SMP) using a whole-cell patch clamp recording method. The ACh currents were not affected by forskolin, the adenylate cyclase activator, regardless of whether or not ATP and GTP were present in the intracellular solution, and by phorbol 12-myristate 13-acetate, the protein kinase C activator. The ACh currents were strongly suppressed by thapsigargin, the microsomal calcium ATPase inhibitor, and genistein, the tyrosine protein kinase inhibitor. They were also suppressed by 3-isobutyl-1-methylxanthine, the cyclic-AMP phosphodiesterase inhibitor, regardless of the presence of forskolin in the extracellular solution and ATP and GTP in the intracellular solution. In addition, the currents were suppressed by activation of P2 purinoceptors with ATP, which could not be explained by a direct effect of ATP on nicotinic acetylcholine receptors (nAChRs). Reactive blue 2, the P2y purinoceptor antagonist, did not abolish inhibition of the ACh current by ATP. Alpha,beta-Imido-ATP and adenosine caused no membrane current responses and did not influence the ACh currents. These results suggest that the activity of the nAChRs in the SMP neurons is strongly suppressed by raised intracellular Ca2+ level, without involvement of protein kinases A and C, and may involve the participation of tyrosine kinase. The activity of nAChRs is also influenced by the activity of P2 purinoceptors; the mechanisms responsible for this influence are not yet clear. So, the activity of the SMP neuronal nAChRs is relatively independent on the intracellular signaling known to influence many other groups of transmitter-gated receptors of neuronal membrane.

Role of the Prostaglandin E2 EP1 Receptor in Traumatic Brain Injury

Brain injuries promote upregulation of so-called proinflammatory prostaglandins, notably prostaglandin E2 (PGE2), leading to overactivation of a class of its cognate G-protein-coupled receptors, including EP1, which is considered a promising target for treatment of ischemic stroke. However, the role of the EP1 receptor is complex and depends on the type of brain injury. This study is focused on the investigation of the role of the EP1 receptor in a controlled cortical impact (CCI) model, a preclinical model of traumatic brain injury (TBI). The therapeutic effects of post-treatments with a widely studied EP1 receptor antagonist, SC-51089, were examined in wildtype and EP1 receptor knockout C57BL/6 mice. Neurological deficit scores (NDS) were assessed 24 and 48 h following CCI or sham surgery, and brain immunohistochemical pathology was assessed 48 h after surgery. In wildtype mice, CCI resulted in an obvious cortical lesion and localized hippocampal edema with an associated significant increase in NDS compared to sham-operated animals. Post-treatments with the selective EP1 receptor antagonist SC-51089 or genetic knockout of EP1 receptor had no significant effects on cortical lesions and hippocampal swelling or on the NDS 24 and 48 h after CCI. Immunohistochemistry studies revealed CCI-induced gliosis and microglial activation in selected ipsilateral brain regions that were not affected by SC-51089 or in the EP1 receptor-deleted mice. This study provides further clarification on the respective contribution of the EP1 receptor in TBI and suggests that, under this experimental paradigm, the EP1 receptor would have limited effects in modulating acute neurological and anatomical pathologies following contusive brain trauma. Findings from this protocol, in combination with previous studies demonstrating differential roles of EP1 receptor in ischemic, neurotoxic, and hemorrhagic conditions, provide scientific background and further clarification of potential therapeutic application of prospective prostaglandin G-protein-coupled receptor drugs in the clinic for treatment of TBI and other acute brain injuries.

Contribution of PGE2 EP1 receptor in hemin-induced neurotoxicity

Although hemin-mediated neurotoxicity has been linked to the production of free radicals and glutamate excitotoxicity, the role of the prostaglandin E2 (PGE2)-EP1 receptor remains unclear. Activation of the EP1 receptor in neurons results in increased intracellular calcium levels; therefore, we hypothesize that the blockade of the EP1 receptor reduces hemin neurotoxicity. Using postnatal primary cortical neurons cultured from wild-type (WT) and EP1−/− mice, we investigated the EP1 receptor role in hemin neurotoxicity measured by lactate dehydrogenase (LDH) cell survival assay. Hemin (75 μM) induced greater release of LDH in WT (34.7 ± 4.5%) than in EP1−/− (27.6 ± 3.3%) neurons. In the presence of the EP1 receptor antagonist SC-51089, the hemin-induced release of LDH decreased. To further investigate potential mechanisms of action, we measured changes in the intracellular calcium level [Ca2+]i following treatment with 17-phenyl trinor PGE2 (17-pt-PGE2) a selective EP1 agonist. In the WT neurons, 17-pt-PGE2 dose-dependently increased [Ca2+]i. However, in EP1−/− neurons, [Ca2+]i was significantly attenuated. We also revealed that hemin dose-dependently increased [Ca2+]i in WT neurons, with a significant decrease in EP1−/− neurons. Both 17-pt-PGE2 and hemin-induced [Ca2+]i were abolished by N-methyl-D-aspartic (NMDA) acid receptor and ryanodine receptor blockers. These results suggest that blockade of the EP1 receptor may be protective against hemin neurotoxicity in vitro. We speculate that the mechanism of hemin neuronal death involves [Ca2+]i mediated by NMDA acid receptor-mediated extracellular Ca2+ influx and EP1 receptor-mediated intracellular release from ryanodine receptor-operated Ca2+ stores. Therefore, blockade of the EP1 receptor could be used to minimize neuronal damage following exposure to supraphysiological levels of hemin.

Efficacy of 3,5-dibromo-L-phenylalanine in rat models of stroke, seizures and sensorimotor gating deficit.

Background and purpose:  Abnormal glutamatergic activity is implicated in neurologic and neuropsychiatric disorders. Selective glutamate receptor antagonists were highly effective in animal models of stroke and seizures but failed in further clinical development because of serious side effects, including an almost complete set of symptoms of schizophrenia. Therefore, the novel polyvalent glutamatergic agent 3,5‐dibromo‐L‐phenylalanine (3,5‐DBr‐L‐Phe) was studied in rat models of stroke, seizures and sensorimotor gating deficit.