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.     

Apolipoprotein A-II regulates HDL stability and affects hepatic lipase association and activity

The effect of apolipoprotein A-II (apoA-II) on the structure and stability of HDL has been investigated in reconstituted HDL particles. Purified human apoA-II was incorporated into sonicated, spherical LpA-I particles containing apoA-I, phospholipids, and various amounts of triacylglycerol (TG), diacylglycerol (DG), and/or free cholesterol. Although the addition of PC to apoA-I reduces the thermodynamic stability (free energy of denaturation) of its alpha-helices, PC has the opposite effect on apoA-II and significantly increases its helical stability. Similarly, substitution of apoA-I with various amounts of apoA-II significantly increases the thermodynamic stability of the particle alpha-helical structure. ApoA-II also increases the size and net negative charge of the lipoprotein particles. ApoA-II directly affects apoA-I conformation and increases the immunoreactivity of epitopes in the N and C termini of apoA-I but decreases the exposure of central domains in the molecule (residues 98-186). ApoA-II appears to increase HL association with HDL and inhibits lipid hydrolysis. ApoA-II mildly inhibits PC hydrolysis in TG-enriched particles but significantly inhibits DG hydrolysis in DG-rich LpA-I. In addition, apoA-II enhances the ability of reconstituted LpA-I particles to inhibit VLDL-TG hydrolysis by HL. Therefore, apoA-II affects both the structure and the dynamic behavior of HDL particles and selectively modifies lipid metabolism.     

Stimulating effect of growth hormone on type IV collagen production by endothelial cells cultured in normal and high glucose

Collagen IV accumulation is characteristic of diabetic angiopathy. To test the possible contribution of GH, we studied its effects on collagen IV production by human umbilical vein endothelial cells at 5.5 and 16.7 mmol/l glucose. GH (100 ng/ml) markedly increased collagen IV level in the culture supernatant and in the insoluble extracellular matrix and cell fraction at both glucose concentrations. This stimulating effect of GH was additional to that of high glucose. It was more pronounced on collagen IV than on total protein synthesis. GH increased free latent gelatinase activity slightly at normal and markedly at high glucose. Using GF109203X, a PKC inhibitor, we observed that high glucose, but not GH, activated PKC. These two factors stimulating collagen IV production appear to work through different pathways, favoring an additivity of their effects. This supports the contribution of high plasma GH in diabetic vascular basement membrane thickening.     

Preference for sucralose predicts behavioral responses to sweet and bittersweet tastants

Rats can be classified as either sucralose avoiders (SA) or sucralose preferrers (SP) based on their behavioral responses in 2-bottle preference, 1-bottle intake, and brief-access licking tests. The present study demonstrates that this robust phenotypic variation in the preference for sucralose predicts acceptance of saccharin, an artificial sweetener with a purported concentration-dependent "bitter" side taste and a 0.25 M sucrose solution adulterated with increasing concentrations of quinine hydrochloride (QHCl). Specifically, SA displayed decreased preference for and intakes of saccharin (≥41.5 mM) and sucrose-QHCl (>0.5 mM QHCl) solutions, relative to SP. In a second experiment involving brief-access (30-s) tests, SP and SA did not differ in their unconditioned licking responses across a range of sodium chloride or QHCl solutions (0.03-1 mM). However, the acceptability threshold for sucrose was lower in SA, relative to SP (0.06 and 0.13 M, respectively). Our findings suggest that phenotypic differences in sucralose preference are indicative of a more general difference in the hedonic processing of stimuli containing "bittersweet" or "sweet" taste qualities.     

Testing the diet-breadth trade-off hypothesis: differential regulation of novel plant secondary compounds by a specialist and a generalist herbivore

Specialist herbivores are predicted to have evolved biotransformation pathways that can process large doses of secondary compounds from the plant species on which they specialize. It is hypothesized that this physiological specialization results in a trade-off such that specialists may be limited in ability to ingest novel plant secondary compounds (PSCs). In contrast, the generalist foraging strategy requires that herbivores alternate consumption of plant species and PSC types to reduce the possibility of over-ingestion of any particular PSC. The ability to behaviorally regulate is a key component of this strategy. These ideas underpin the prediction that in the face of novel PSCs, generalists should be better able to maintain body mass and avoid toxic consequences compared to specialists. We explored these predictions by comparing the feeding behavior of two herbivorous rodents: a juniper specialist, Neotoma stephensi, and a generalist, Neotoma albigula, fed diets with increasing concentrations of phenolic resin extracted from the creosote bush (Larrea tridentata), which produces a suite of PSCs novel to both species. The specialist lost more mass than the generalist during the 15-day trial. In addition, although the specialist and generalist both regulated phenolic resin intake by reducing meal size while on the highest resin concentration (4%), the generalist began to regulate intake on the 2% diet. The ability of the generalist to regulate intake at a lower PSC concentration may be the source of the generalist’s performance advantage over the specialist. These data provide evidence for the hypothesis that the specialist’s foraging strategy may result in behavioral as well as physiological trade-offs in the ability to consume novel PSCs.     

Restoration of seminal plasma to stallion spermatozoa selected by colloid centrifugation increases sperm progressive motility but is detrimental to chromatin integrity

There is controversy about whether the presence of some seminal plasma (SP) in an equine insemination dose is necessary for promoting fertility. A new technique for improving stallion sperm quality, single layer centrifugation (SLC) using a species-specific colloid, Androcoll-E, selects a sperm subpopulation that is highly motile with normal morphology, intact membranes and good chromatin integrity from the rest of the ejaculate and removes SP. The present study was designed to investigate the effect of restoring homologous SP (5% and 10%) on the progressive motility, velocity, and chromatin integrity of SLC-selected stallion spermatozoa in 44 semen samples over time. Sperm progressive motility (P < 0.01) and the proportion with class A velocity (>50 μm/sec) were increased in samples where SP was restored, whereas the proportion with class B velocity (10 to 50 μm/sec) was decreased compared with SLC samples. However, after 24 h cold storage of treated samples, progressive motility was not different for the SP-treated groups compared with SLC, whereas chromatin damage DNA fragmentation index (%DFI) was higher. In contrast, adding SP to untreated 24 h-stored SLC samples did not affect progressive motility although it did increase the proportion of spermatozoa with class A velocity. There was individual variation between stallions whether 5% or 10% SP produced a greater increase in progressive motility. In conclusion, 5% to 10% SP can be added back to SLC-selected samples if considered necessary to optimize fertility. However, it should be added immediately before insemination rather than before storage of the sperm dose, to benefit from the transient increase in sperm progressive motility and avoid increased chromatin damage.     

A proteomic profile of washing fluid from the colorectal tract to search for potential biomarkers of colon cancer

Washing fluid (WF) from the colon rectal tract after surgical resection might represent a first step in obtaining a mixture of proteins derived from the secretion of tumoral epithelial cells potentially involved in the pathological progression of tissue. In this study, we performed a proteomic analysis of colorectal WF to search for potential biomarkers of colon cancer. The outcome of this approach might open the possibility of using WF to screen for the precancerous and early stages of colorectal cancer (CRC). Samples of WFs were obtained during surgery from 35 patients submitted to colon resection for suspicious adenocarcinoma or carcinoma, while the respective controls were obtained by washing the healthy sections. WFs were immediately centrifuged, concentrated and trichloroacetic acid (TCA) was added to obtain protein pellets. After two-dimensional gel electrophoresis (2DE), the protein patterns of malignant samples were compared with respective normal samples. Forty-one protein spots were found to be differentially expressed exhibiting ≥2 fold-change of mean value spot intensities. After mass spectrometry, these protein spots collapsed into 38 different proteins. Interestingly, 19 of the differentially expressed proteins identified in the study corresponded to those suggested as being potential biomarkers of CRC. In accordance with the literature, these proteins showed the same direction of change (up or down for all proteins). Our results suggest that WF has the potential of being a method for the exploration of clinical samples for biomarker and drug target discovery.     

Analytical techniques for assaying nitric oxide bioactivity

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood). NO may be considered one of the most important signaling molecules produced in our body, regulating essential functions including but not limited to regulation of blood pressure, immune response and neural communication. Therefore its accurate detection and quantification in biological matrices is critical to understanding the role of NO in health and disease. With such a short physiological half life of NO, alternative strategies for the detection of reaction products of NO biochemistry have been developed. The quantification of relevant NO metabolites in multiple biological compartments provides valuable information with regards to in vivo NO production, bioavailability and metabolism. Simply sampling a single compartment such as blood or plasma may not always provide an accurate assessment of whole body NO status, particularly in tissues. The ability to compare blood with select tissues in experimental animals will help bridge the gap between basic science and clinical medicine as far as diagnostic and prognostic utility of NO biomarkers in health and disease. Therefore, extrapolation of plasma or blood NO status to specific tissues of interest is no longer a valid approach. As a result, methods continue to be developed and validated which allow the detection and quantification of NO and NO-related products/metabolites in multiple compartments of experimental animals in vivo. The established paradigm of NO biochemistry from production by NO synthases to activation of soluble guanylyl cyclase (sGC) to eventual oxidation to nitrite (NO(2)(-)) and nitrate (NO(3)(-)) may only represent part of NO's effects in vivo. The interaction of NO and NO-derived metabolites with protein thiols, secondary amines, and metals to form S-nitrosothiols (RSNOs), N-nitrosamines (RNNOs), and nitrosyl-heme respectively represent cGMP-independent effects of NO and are likely just as important physiologically as activation of sGC by NO. A true understanding of NO in physiology is derived from in vivo experiments sampling multiple compartments simultaneously. Nitric oxide (NO) methodology is a complex and often confusing science and the focus of many debates and discussion concerning NO biochemistry. The elucidation of new mechanisms and signaling pathways involving NO hinges on our ability to specifically, selectively and sensitively detect and quantify NO and all relevant NO products and metabolites in complex biological matrices. Here, we present a method for the rapid and sensitive analysis of nitrite and nitrate by HPLC as well as detection of free NO in biological samples using in vitro ozone based chemiluminescence with chemical derivitazation to determine molecular source of NO as well as ex vivo with organ bath myography.     

Messenger RNA levels of estrogen receptors alpha and beta and progesterone receptors in the cyclic and inseminated/early pregnant sow uterus

The aim of the present study was to investigate differences in the expression of mRNAs for ERalpha, ERbeta and PR in the sow uterus at different stages of the estrous cycle as well as in inseminated sows at estrus and during early pregnancy by use of solution hybridization and in relation to plasma levels of estradiol and progesterone. Uterine samples were collected at different stages of the estrous cycle and after insemination/early pregnancy. In the endometrium, the expression of ERalpha mRNA and PR mRNA was similar for cyclic and early pregnant groups. Both were highest at early diestrus/70 h after ovulation and ERalpha mRNA was lowest at late diestrus/d 19 while PR mRNA was lowest at diestrus and late diestrus/d 11 and d 19. The expression of endometrial ERbeta was constantly low during the estrous cycle but higher expression was found in inseminated/early pregnant sows at estrus and 70 h after ovulation. In the myometrium, high expression of ERalpha mRNA and PR mRNA was observed at proestrus and estrus in cyclic sows and at estrus in newly inseminated sows. Higher expression of myometrial ERbeta mRNA was found in inseminated/early pregnant sows compared with cyclic sows, although significant only at estrus. In conclusion, the expression of mRNAs for ERalpha, ERbeta and PR in the sow uterus differed between endometrium and myometrium as well as with stages of the estrous cycle and early pregnancy. In addition to plasma steroid levels, the differences between cyclic and inseminated/early pregnant sows suggest that other factors, e.g. insemination and/or the presence of embryos, influence the expression of these steroid receptor mRNAs in the sow uterus.