Dopamine protects neurons against glutamate-induced excitotoxicity

Glutamate excitotoxicity is responsible for neuronal death in acute neurological disorders including stroke, trauma and neurodegenerative disease. Loss of calcium homeostasis is a key mediator of glutamate-induced cell death. The neurotransmitter dopamine (DA) is known to modulate calcium signalling, and here we show that it can do so in response to physiological concentrations of glutamate. Furthermore, DA is able to protect neurons from glutamate-induced cell death at pathological concentrations of glutamate. We demonstrate that DA has a novel role in preventing delayed calcium deregulation in cortical, hippocampal and midbrain neurons. The effect of DA in abolishing glutamate excitotoxicity can be induced by DA receptor agonists, and is abolished by DA receptor antagonists. Our data indicate that the modulation of glutamate excitotoxicity by DA is receptor-mediated. We postulate that DA has a major physiological function as a safety catch to restrict the glutamate-induced calcium signal, and thereby prevent glutamate-induced cell death in the brain.     

Seizure activity results in calcium- and mitochondria-independent ROS production via NADPH and xanthine oxidase activation

Seizure activity has been proposed to result in the generation of reactive oxygen species (ROS), which then contribute to seizure-induced neuronal damage and eventually cell death. Although the mechanisms of seizure-induced ROS generation are unclear, mitochondria and cellular calcium overload have been proposed to have a crucial role. We aim to determine the sources of seizure-induced ROS and their contribution to seizure-induced cell death. Using live cell imaging techniques in glioneuronal cultures, we show that prolonged seizure-like activity increases ROS production in an NMDA receptor-dependent manner. Unexpectedly, however, mitochondria did not contribute to ROS production during seizure-like activity. ROS were generated primarily by NADPH oxidase and later by xanthine oxidase (XO) activity in a calcium-independent manner. This calcium-independent neuronal ROS production was accompanied by an increase in intracellular [Na⁺] through NMDA receptor activation. Inhibition of NADPH or XO markedly reduced seizure-like activity-induced neuronal apoptosis. These findings demonstrate a critical role for ROS in seizure-induced neuronal cell death and identify novel therapeutic targets.Reactive oxygen species (ROS) contribute to neuronal damage and have been linked to excitotoxicity.^{1, 2, 3, 4} An increase in ROS generation has also been identified in acute neurologic disease such as stroke,^5,6 and recent evidence indicates that this may contribute to neuronal damage in seizures and epilepsy.^{7, 8, 9, 10} However, ROS measurements during seizure-like activity were predominantly performed in homogenates, extracellular fluids or brain regions with no clear demonstration of whether the ROS were of neuronal origin.^9,11,12 Moreover, these studies lacked the necessary temporal resolution to determine accurately the evolution of ROS generation during and after prolonged seizure activity. Such obstacles can be overcome by live cell imaging of ROS, which has emerged as a powerful tool to study disease mechanisms.¹³If seizure activity induces ROS production in neurons, then a critical question is which sources of ROS production are triggered by such activity. Previous studies have suggested that mitochondria are the primary source of ROS generation in seizure models.^8,14 However, there are alternative sources of ROS, in particular the enzymes NADPH oxidase and xanthine oxidase (XO). How these contribute to excitotoxicity during seizure activity is uncertain. That these enzymes may have an important role in seizure-induced ROS generation is suggested by two observations: (1) NMDA receptors have a pivotal role in seizure-induced neuronal damage¹⁵ and (2) direct pharmacologic activation of NMDA receptors can activate NADPH oxidase, increasing free radical production and consequently neuronal death.^5,16,17 There is also burgeoning evidence of a role for NADPH oxidase activation in chronic brain pathology secondary to psychosocial stress, which leads to the development of neuropathologic alterations, and also in neurodegenerative disease.^18,19Acute activation of NADPH oxidase in neurons has mainly been shown after direct pharmacologic activation of NMDA receptors via exposure to high levels of NMDA and this activation is calcium-dependent.^16,17 More recently, activation of NADPH oxidase has been shown during seizure activity.^9,20 These pathways also involved NMDA receptor activation and upregulation of NMDA receptor subunits NR1 and NR2B. Nonetheless, these studies used chemoconvulsant epilepsy models, which, in themselves, may have an impact on ROS generation. The mechanisms and relevance of activation of NADPH oxidase during seizure activity independent of chemoconvulsants is unclear, especially given the presence of alternative sources of ROS production. Moreover, XO may also represent a major potential source of ROS during periods of increased metabolism, such as that occuring during seizures. We have therefore asked whether NMDA receptor activation has a role in seizure-induced ROS production and which sources and mechanisms of ROS production are involved in its time course during seizure-like activity.Here, we demonstrate increased ROS generation during seizure-like activity. This is activity-dependent, but it is maintained by a Ca²⁺-independent pathway involving the activation of NMDA receptors, NADPH oxidase and XO at a later phase. Blocking NADPH oxidase and XO prevented seizure-induced neuronal cell death in vitro. We thus provide compelling evidence that these ROS-generating pathways are appropriate targets for preventing neuronal death in seizures.     

Synthesis of the dodecasaccharide fragment representing the O-polysaccharide of Vibrio cholerae O:1, serotype Ogawa, bearing an aglycon offering flexibility for chemical linking to proteins

Two azidohexasaccharide building blocks, of which the glycosyl acceptor was the 5-(methoxycarbonyl)pentyl glycoside, were coupled using the trichloroacetimidate technology. The 12 azido functions present in the dodecasaccharide thus formed were then converted to amino groups using hydrogen sulfide as a reducing reagent. Subsequent N-acylation with 4-O-benzyl-L-glycero-tetronic acid, followed by catalytic debenzylation yielded the desired spacer-equipped, title dodecasaccharide. This revised version was published online in November 2006 with corrections to the Cover Date.     

Laccase-induced grafting on plasma-pretreated polypropylene

A new environmentally friendly strategy for the sustainable functionalization of inert man-made polymer surfaces is mapped out for the first time using a combination of plasma pretreatment and enzymatic postgrafting. The efficiency of enzymatic covalent binding is investigated by grafting methacrylate monomers possessing different amino groups, primary, tertiary, and quaternary, onto a polypropylene surface using plasma pretreatment. Subsequent enzymatic grafting, using laccase and guaiacol sulfonic acid (GSA), is determined by surface analytical techniques, such as attenuated total reflectance Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. The grafting of GSA in the presence of a laccase is proven by a 10-fold increase in sulfur compared to the control. The covalent coupling between GSA and primary amine groups is determined by HPLC-MS using hexylamine as a model substrate. The advantage of technology is in the strong covalent binding of functional groups onto the synthetic polymer's surface, which could then be suitably tailored by enzymes possessing substrate specificity and regional selectivity.     

Thoracic intramedullary sarcoidosis mimicking an intramedullary tumor

Sarcoidosis is a chronic, systemic granulomatous reticulosis of unknown origin, characterized by formation of hard tubercles and noncaseatinggranulomas. Since other infectious diseases such as berylliosis, mycobacterium and fungal infections may present with a noncaseating granulomas, histological diagnosis of sarcoidosis is made using the elimination method. Central nervous system manifestations of sarcoidosis may be present in 5-10% of the cases involving cranial nerves, leptomeninges and third ventricle respectively. Any part of the central nervous system can be affected. Involvement of spinal cord in sarcoidosis is extremely rare and presents with only 0.3-0.4% in patients with systemic sarcoidosis. Intramedullary sarcoidosis is a rare first manifestation of the disease and it can mimic an intramedullary tumor, which is often manifested with symptoms that initiate from spinal cord compression, resulting in paraparesis, sensory disorders and sphincter dysfunction. We present a case of intramedullary sarcoidosis that mimics a tumor of the thoracic spinal cord. Clinical features, neuroradiological, pathohistological findings, laboratory analysis and surgical treatment of such a rare entity are being discussed.     

Temporal and spatial dynamics underlying capacitative calcium entry in human colonic smooth muscle

Angiotensin converting enzyme (ACE) has been shown to be involved in regulation of apoptosis in nonintestinal tissues. This study examined the role of ACE in the modulation of intestinal adaptation utilizing ACE knockout mice (ACE-/-). A 60% small bowel resection (SBR) was used, since this model results in a significant increase in intestinal epithelial cell (EC) apoptosis as well as proliferation. Baseline villus height, crypt depth, and intestinal EC proliferation were higher, and EC apoptosis rates were lower in ACE-/- compared with ACE+/+ mice. After SBR, EC apoptosis rates remained significantly lower in ACE-/- compared with ACE+/+ mice. Furthermore, villus height and crypt depth after SBR continued to be higher in ACE-/- mice. The finding of a lower bax-to-bcl-2 protein ratio in ACE-/- mice may account for reduced EC apoptotic rates after SBR in ACE-/- compared with ACE+/+ mice. The baseline higher rate of EC proliferation in ACE-/- compared with ACE+/+ mice may be due to an increase in the expression of several EC growth factor receptors. In conclusion, ACE appears to have an important role in the modulation of intestinal EC apoptosis and proliferation and suggests that the presence of ACE in the intestinal epithelium has a critical role in guiding epithelial cell adaptive response.     

Stereoselectivity of Mucorales lipases toward triradylglycerols--a simple solution to a complex problem

The lipases from Rhizopus and Rhizomucor are members of the family of Mucorales lipases. Although they display high sequence homology, their stereoselectivity toward triradylglycerols (sn-2 substituted triacylglycerols) varies. Four different triradylglycerols were investigated, which were classified into two groups: flexible substrates with rotatable O'-C1' ether or ester bonds adjacent to C2 of glycerol and rigid substrates with a rigid N'-C1' amide bond or a phenyl ring in sn-2. Although Rhizopus lipase shows opposite stereopreference for flexible and rigid substrates (hydrolysis in sn-1 and sn-3, respectively), Rhizomucor lipase hydrolyzes both groups of triradylglycerols preferably in sn-1. To explain these experimental observations, computer-aided molecular modeling was applied to study the molecular basis of stereoselectivity. A generalized model for both lipases of the Mucorales family highlights the residues mediating stereoselectivity: (1) L258, the C-terminal neighbor of the catalytic histidine, and (2) G266, which is located in a loop contacting the glycerol backbone of a bound substrate. Interactions with triradylglycerol substrates are dominated by van der Waals contacts. Stereoselectivity can be predicted by analyzing the value of a single substrate torsion angle that discriminates between sn-1 and sn-3 stereopreference for all substrates and lipases investigated here. This simple model can be easily applied in enzyme and substrate engineering to predict Mucorales lipase variants and synthetic substrates with desired stereoselectivity.     

Electronic structure and biological activity of steroids

We present the analysis of the electronic structure for 31 steroids by using HeI UV photoelectron spectroscopy (UPS) and MO calculations. The electronic structure of molecules in the gas phase is related directly to steroid-receptor binding measurements. The results indicate that formally 'inert' sigma-skeleton plays a crucial role in diversifying the electronic structures of the title compounds ('ribbon-orbital effect'). This is an attempt to rationalize the biological activity of steroids (represented through steroid-receptor binding) by making direct correlation between spectroscopic and biological data.     

Angiopoietins have distinct modular domains essential for receptor binding,dimerization and superclustering

Angiopoietins are a recently discovered family of angiogenic factors that interact with the endothelial receptor tyrosine kinase Tie2, either as agonists (angiopoietin-1) or as context-dependent agonists/antagonists (angiopoietin-2). Here we show that angiopoietin-1 has a modular structure unlike any previously characterized growth factor. This modular structure consists of a receptor-binding domain, a dimerization motif and a superclustering motif that forms variable-sized multimers. Genetic engineering of precise multimers of the receptor-binding domain of angiopoietin-1, using surrogate multimerization motifs, reveals that tetramers are the minimal size required for activating endothelial Tie2 receptors. In contrast, engineered dimers can antagonize endothelial Tie2 receptors. Surprisingly, angiopoietin-2 has a modular structure and multimerization state similar to that of angiopoietin-1, and its antagonist activity seems to be a subtle property encoded in its receptor-binding domain.