Action goals and the praxis network: an fMRI study.

The praxis representation network (PRN) of the left cerebral hemisphere is typically linked to the control of functional interactions with familiar tools. Surprisingly, little is known about the PRN engagement in planning and execution of tool-directed actions motivated by non-functional but purposeful action goals. Here we used functional neuroimaging to perform both univariate and multi-voxel pattern analyses (MVPA) in 20 right-handed participants who planned and later executed, with their dominant and non-dominant hands, disparate grasps of tools for different goals, including: (1) planning simple vs. demanding functional grasps of conveniently vs. inconveniently oriented tools with an intention to immediately use them, (2) planning simple-but non-functional-grasps of inconveniently oriented tools with a goal to pass them to a different person, (3) planning reaching movements directed at such tools with an intention to move/push them with the back of the hand, and (4) pantomimed execution of the earlier planned tasks. While PRN contributed to the studied interactions with tools, the engagement of its critical nodes, and/or complementary right hemisphere processing, was differently modulated by task type. E.g., planning non-functional/structural grasp-to-pass movements of inconveniently oriented tools, regardless of the hand, invoked the left parietal and prefrontal nodes significantly more than simple, non-demanding functional grasps. MVPA corroborated decoding capabilities of critical PRN areas and some of their right hemisphere counterparts. Our findings shed new lights on how performance of disparate action goals influences the extraction of object affordances, and how or to what extent it modulates the neural activity within the parieto-frontal brain networks.

Oxidation of ethidium-based probes by biological radicals: mechanism, kinetics and implications for the detection of superoxide.

Hydroethidine (HE) and hydropropidine ([Formula: see text]) are fluorogenic probes used for the detection of the intra- and extracellular superoxide radical anion ([Formula: see text]). In this study, we provide evidence that HE and [Formula: see text] react rapidly with the biologically relevant radicals, including the hydroxyl radical, peroxyl radicals, the trioxidocarbonate radical anion, nitrogen dioxide, and the glutathionyl radical, via one-electron oxidation, forming the corresponding radical cations. At physiological pH, the radical cations of the probes react rapidly with [Formula: see text], leading to the specific 2-hydroxylated cationic products. We determined the rate constants of the reaction between [Formula: see text] and the radical cations of the probes. We also synthesized N-methylated analogs of [Formula: see text] and HE which were used in mechanistic studies. Methylation of the amine groups was not found to prevent the reaction between the radical cation of the probe and the superoxide, but it significantly increased the lifetime of the radical cation and had a substantial effect on the profiles of the oxidation products by inhibiting the formation of dimeric products. We conclude that the N-methylated analogs of HE and [Formula: see text] may be used as a scaffold for the design of a new generation of probes for intra- and extracellular superoxide.

Characterization of Fluorescein-Based Monoboronate Probe and Its Application to the Detection of Peroxynitrite in Endothelial Cells Treated with Doxorubicin.

Boronate probes have emerged recently as a versatile tool for the detection of reactive oxygen and nitrogen species. Here, we present the characterization of a fluorescein-based monoboronate probe, a 4-(pinacol boronate)benzyl derivative of fluorescein methyl ester (FBBE), that proved to be useful to detect peroxynitrite in cell culture experiments. The reactivity of FBBE toward peroxynitrite as well hypochlorite, hydrogen peroxide, and tyrosyl hydroperoxide was determined. Second-order rate constants of the reactions of FBBE with peroxynitrite, HOCl, and H2O2 at pH 7.4 were equal to (2.8 ± 0.2) × 10(5) M(-1) s(-1), (8.6 ± 0.5) × 10(3) M(-1) s(-1), and (0.96 ± 0.03) M(-1) s(-1), respectively. The presence of glutathione completely blocked the oxidation of the probe by HOCl and significantly inhibited its oxidation by H2O2 and tyrosyl hydroperoxide but not by peroxynitrite. The oxidative conversion of the probe was also studied in the systems generating singlet oxygen, superoxide radical anion, and nitric oxide in the presence and absence of glutathione. Spectroscopic characterization of FBBE and its oxidation product has been also performed. The differences in the reactivity pattern were supported by DFT quantum mechanical calculations. Finally, the FBBE probe was used to study the oxidative stress in endothelial cells (Ea.hy926) incubated with doxorubicin, a quinone anthracycline antibiotic. In endothelial cells pretreated with doxorubicin, FBBE was oxidized, and this effect was reversed by PEG-SOD and L-NAME but not by catalase.

Mechanism of oxidative conversion of Amplex® Red to resorufin: Pulse radiolysis and enzymatic studies.

Amplex® Red (10-acetyl-3,7-dihydroxyphenoxazine) is a fluorogenic probe widely used to detect and quantify hydrogen peroxide in biological systems. Detection of hydrogen peroxide is based on peroxidase-catalyzed oxidation of Amplex® Red to resorufin. In this study we investigated the mechanism of one-electron oxidation of Amplex® Red and we present the spectroscopic characterization of transient species formed upon the oxidation. Oxidation process has been studied by a pulse radiolysis technique with one-electron oxidants (N3(•), CO3(•-),(•)NO2 and GS(•)). The rate constants for the Amplex® Red oxidation by N3(•) ((2)k=2.1·10(9)M(-1)s(-1), at pH=7.2) and CO3(•-) ((2)k=7.6·10(8)M(-1)s(-1), at pH=10.3) were determined. Two intermediates formed during the conversion of Amplex® Red into resorufin have been characterized. Based on the results obtained, the mechanism of transformation of Amplex® Red into resorufin, involving disproportionation of the Amplex® Red-derived radical species, has been proposed. The results indicate that peroxynitrite-derived radicals, but not peroxynitrite itself, are capable to oxidize Amplex® Red to resorufin. We also demonstrate that horseradish peroxidase can catalyze oxidation of Amplex® Red not only by hydrogen peroxide, but also by peroxynitrite, which needs to be considered when employing the probe for hydrogen peroxide detection.

Toward selective detection of reactive oxygen and nitrogen species with the use of fluorogenic probes--Limitations, progress, and perspectives.

Over the last 40 years, there has been tremendous progress in understanding the biological reactions of reactive oxygen species (ROS) and reactive nitrogen species (RNS). It is widely accepted that the generation of ROS and RNS is involved in physiological and pathophysiological processes. To understand the role of ROS and RNS in a variety of pathologies, the specific detection of ROS and RNS is fundamental. Unfortunately, the intracellular detection and quantitation of ROS and RNS remains a challenge. In this short review, we have focused on the mechanistic and quantitative aspects of their detection with the use of selected fluorogenic probes. The challenges, limitations and perspectives of these methods are discussed.

Co-lateralized bilingual mechanisms for reading in single and dual language contexts: evidence from visual half-field processing of action words in proficient bilinguals.

When reading, proficient bilinguals seem to engage the same cognitive circuits regardless of the language in use. Yet, whether or not such "bilingual" mechanisms would be lateralized in the same way in distinct-single or dual-language contexts is a question for debate. To fill this gap, we tested 18 highly proficient Polish (L1) -English (L2) childhood bilinguals whose task was to read aloud one of the two laterally presented action verbs, one stimulus per visual half field. While in the single-language blocks only L1 or L2 words were shown, in the subsequent mixed-language blocks words from both languages were concurrently displayed. All stimuli were presented for 217 ms followed by masks in which letters were replaced with hash marks. Since in non-simultaneous bilinguals the control of language, skilled actions (including reading), and representations of action concepts are typically left lateralized, the vast majority of our participants showed the expected, significant right visual field advantage for L1 and L2, both for accuracy and response times. The observed effects were nevertheless associated with substantial variability in the strength of the lateralization of the mechanisms involved. Moreover, although it could be predicted that participants' performance should be better in a single-language context, accuracy was significantly higher and response times were significantly shorter in a dual-language context, irrespective of the language tested. Finally, for both accuracy and response times, there were significant positive correlations between the laterality indices (LIs) of both languages independent of the context, with a significantly greater left-sided advantage for L1 vs. L2 in the mixed-language blocks, based on LIs calculated for response times. Thus, despite similar representations of the two languages in the bilingual brain, these results also point to the functional separation of L1 and L2 in the dual-language context.

Sinistrals are rarely "right": evidence from tool-affordance processing in visual half-field paradigms.

Although current neuroscience and behavioral studies provide substantial understanding of tool representations (e.g., the processing of tool-related affordances) in the human brain, most of this knowledge is limited to right-handed individuals with typical organization of cognitive and manual skills. Therefore, any insights from these lines of research may be of little value in rehabilitation of patients with atypical laterality of praxis and/or hand dominance. To fill this gap, we tested perceptual processing of man-made objects in 18 healthy left-handers who were likely to show greater incidence of right-sided or bilateral (atypical) lateralization of functions. In the two experiments reported here, participants performed a tool vs. non-tool categorization task. In Experiment 1, target and distracter objects were presented for 200 ms in the left (LVF) or right (RVF) visual field, followed by 200 ms masks. In Experiment 2, the centrally presented targets were preceded by masked primes of 35 ms duration, again presented in the LVF or RVF. Based on results from both studies, i.e., response times (RTs) to correctly discriminated stimuli irrespective of their category, participants were divided into two groups showing privileged processing in either left (N = 9) or right (N = 9) visual field. In Experiment 1, only individuals with RVF advantage showed significantly faster categorization of tools in their dominant visual field, whereas those with LVF advantage revealed merely a trend toward such an effect. In Experiment 2, when targets were preceded by identical primes, the "atypical" group showed significantly facilitated categorization of non-tools, whereas the "typical" group demonstrated a trend toward faster categorization of tools. These results indicate that in subjects with atypically organized cognitive skills, tool-related processes are not just mirror reversed. Thus, our outcomes call for particular caution in neurorehabilitation directed at left-handed individuals.

Nitroxyl (HNO) reacts with molecular oxygen and forms peroxynitrite at physiological pH. Biological Implications.

Nitroxyl (HNO), the protonated one-electron reduction product of NO, remains an enigmatic reactive nitrogen species. Its chemical reactivity and biological activity are still not completely understood. HNO donors show biological effects different from NO donors. Although HNO reactivity with molecular oxygen is described in the literature, the product of this reaction has not yet been unambiguously identified. Here we report that the decomposition of HNO donors under aerobic conditions in aqueous solutions at physiological pH leads to the formation of peroxynitrite (ONOO(-)) as a major intermediate. We have specifically detected and quantified ONOO(-) with the aid of boronate probes, e.g. coumarin-7-boronic acid or 4-boronobenzyl derivative of fluorescein methyl ester. In addition to the major phenolic products, peroxynitrite-specific minor products of oxidation of boronate probes were detected under these conditions. Using the competition kinetics method and a set of HNO scavengers, the value of the second order rate constant of the HNO reaction with oxygen (k = 1.8 × 10(4) m(-1) s(-1)) was determined. The rate constant (k = 2 × 10(4) m(-1) s(-1)) was also determined using kinetic simulations. The kinetic parameters of the reactions of HNO with selected thiols, including cysteine, dithiothreitol, N-acetylcysteine, captopril, bovine and human serum albumins, and hydrogen sulfide, are reported. Biological and cardiovascular implications of nitroxyl reactions are discussed.

Antithrombotic effects of pyridinium compounds formed from trigonelline upon coffee roasting.

Coffee may exert a preventive effect on arterial thrombosis. Trigonelline is one of the most abundant compounds in coffee that undergoes pyrolysis upon roasting of coffee beans. The aim of the present study was to identify pyridinium compounds formed upon trigonelline pyrolysis and coffee roasting and to investigate the effect of three of them, i.e., 1-methylpyridine and 1,3- and 1,4-dimethylpyridine, on experimentally induced arterial thrombosis in rats. 1,3- and 1,4-dimethylpyridine but not 1-methylpyridine inhibited arterial thrombus formation. 1,3-Dimethylpyridine inhibited platelet aggregation and reduced fibrin formation in platelet-rich plasma, whereas 1,4-dimethylpyridine increased the plasma level of 6-keto-PGF1α. 1,4-Dimethylpyridine slightly increased rat tissue plasminogen activator plasma activity. In summary, we demonstrated that pyridinium compounds display mild antithrombotic properties due to stimulation by prostacyclin release (1,4-dimethylpyridine) and inhibition of platelet aggregation (1,3-dimethylpyridine). Those pyridinium compounds may, to some extent, be responsible for the beneficial effects of coffee drinking.

On the use of fluorescence lifetime imaging and dihydroethidium to detect superoxide in intact animals and ex vivo tissues: a reassessment.

Recently, D.J. Hall et al. reported that ethidium (E(+)) is formed as a major product of hydroethidine (HE) or dihydroethidium reaction with superoxide (O2(-)) in intact animals with low tissue oxygen levels (J. Cereb. Blood Flow Metab. 32:23-32, 2012). The authors concluded that measurement of E(+) is an indicator of O2(-) formation in intact brains of animals. This finding is in stark contrast to previous reports using in vitro systems showing that 2-hydroxyethidium, not ethidium, is formed from the reaction between O2(-) and HE. Published in vivo results support the in vitro findings. In this study, we performed additional experiments in which HE oxidation products were monitored under different fluxes of O2(-). Results from these experiments further reaffirm our earlier findings (H. Zhao et al., Free Radic. Biol. Med. 34:1359, 2003). We conclude that whether in vitro or in vivo, E(+) measured by HPLC or by fluorescence lifetime imaging is not a diagnostic marker product for O2(-) reaction with HE.

Reaction between peroxynitrite and triphenylphosphonium-substituted arylboronic acid isomers: identification of diagnostic marker products and biological implications.

Aromatic boronic acids react rapidly with peroxynitrite (ONOO(-)) to yield phenols as major products. This reaction was used to monitor ONOO(-) formation in cellular systems. Previously, we proposed that the reaction between ONOO(-) and arylboronates (PhB(OH)2) yields a phenolic product (major pathway) and a radical pair PhB(OH)2O(•-)···(•)NO2 (minor pathway). [Sikora, A. et al. (2011) Chem. Res. Toxicol. 24, 687-697]. In this study, we investigated the influence of a bulky triphenylphosphonium (TPP) group on the reaction between ONOO(-) and mitochondria-targeted arylboronate isomers (o-, m-, and p-MitoPhB(OH)2). Results from the electron paramagnetic resonance (EPR) spin-trapping experiments unequivocally showed the presence of a phenyl radical intermediate from meta and para isomers, and not from the ortho isomer. The yield of o-MitoPhNO2 formed from the reaction between o-MitoPhB(OH)2 and ONOO(-) was not diminished by phenyl radical scavengers, suggesting a rapid fragmentation of the o-MitoPhB(OH)2O(•-) radical anion with subsequent reaction of the resulting phenyl radical with (•)NO2 in the solvent cage. The DFT quantum mechanical calculations showed that the energy barrier for the dissociation of the o-MitoPhB(OH)2O(•-) radical anion is significantly lower than that of m-MitoPhB(OH)2O(•-) and p-MitoPhB(OH)2O(•-) radical anions. The nitrated product, o-MitoPhNO2, is not formed by the nitrogen dioxide radical generated by myeloperoxidase in the presence of the nitrite anion and hydrogen peroxide, indicating that this specific nitrated product may be used as a diagnostic marker product for ONOO(-). Incubation of o-MitoPhB(OH)2 with RAW 264.7 macrophages activated to produce ONOO(-) yielded the corresponding phenol o-MitoPhOH as well as the diagnostic nitrated product, o-MitoPhNO2. We conclude that the ortho isomer probe reported here is most suitable for specific detection of ONOO(-) in biological systems.