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ContextHydrogen cyanide and methanethiol are two toxic gases that inhibit mitochondrial cytochrome c oxidase. Cyanide is generated in structural fires and methanethiol is released by decaying organic matter. Current treatments for cyanide exposure do not lend themselves to treatment in the field and no treatment exists for methanethiol poisoning. Sodium tetrathionate (tetrathionate), a product of thiosulfate oxidation, could potentially serve as a cyanide antidote, and, based on its chemical structure, we hypothesized it could react with methanethiol.ResultsWe show that tetrathionate, unlike thiosulfate, reacts directly with cyanide invitro under physiological conditions, and based on rabbit studies where we monitor cyanide poisoning in real-time, tetrathionate likely reacts directly with cyanide invivo. We found that tetrathionate administered by intramuscular injection rescues >80% of juvenile, young adult, and old adult mice from exposure to inhaled hydrogen cyanide gas that is >80% lethal. Tetrathionate also rescued young adult rabbits from intravenously administered sodium cyanide. Tetrathionate was reasonably well-tolerated by mice and rats, yielding a therapeutic index of ∼5 in juvenile and young adult mice, and ∼3.3 in old adult mice; it was non-mutagenic in Chinese Hamster ovary cells and by the Ames bacterial test. We found by gas chromatography-mass spectrometry that both tetrathionate and thiosulfate react with methanethiol to generate dimethyldisulfide, but that tetrathionate was much more effective than thiosulfate at recovering intracellular ATP in COS-7 cells and rescuing mice from a lethal exposure to methanethiol gas.ConclusionWe conclude that tetrathionate has the potential to be an effective antidote against cyanide and methanethiol poisoning.

A new sequential, tandem synthesis of functionalized α-arylnitriles via the bromination/cyanation/deformylation of substituted deoxybenzoin has developed. CuBr2-promoted bromination of substituted deoxybenzoins gives 2-bromo-2-arylacetophenne 3. The cyanation of 3 with sodium cyanide (NaCN) generates epoxynitrile. Then, a treatment of epoxynitrile with BF3·OEt2 results in the formation of functionalized α-arylnitriles 4 via a 1,2-aryl shift.

Double-13C-labeled imidazole-4-carboxamide (ICA: 13C2-1) and 5-aminoimidazole-4-carboxamide-1-β-riboside (AICAr: 13C2-5) were synthesized from stable isotope-labeled sodium cyanide and triethyl orthoformate. The key intermediate, 4-aminoimidazole-5-carboxamide (AICA: 13C2-4), should also provide access to double-13C-labeled histidine and adenosine derivatives via reported methods.

Okadaic acid (OA) and other toxins of the diarrheic shellfish poisoning (DSP) group are accumulated and transformed mainly in many bivalves, inside the digestive gland cells. In this work the absorption of okadaic acid by those cells has been studied by supplying the toxin dissolved in water and including it in oil droplets given to primary cell cultures, and by checking if the uptake is saturable and/or energy-dependent. Okadaic acid was found to be absorbed preferentially from the dissolved phase, and the uptake from oil droplets was substantially lower. The process did not require energy and was non-saturable, indicating that it involved a simple diffusion across the cellular membrane. Some apparent saturation was found due to the quick biotransformation of OA to 7-O-acyl esters.

A simple and efficient method has been developed for the synthesis of some dialkylaminonitrile derivatives of 2-formylbenzoic acid by simply mixing 2-formylbenzoic acids, secondary amines, and sodium cyanide via the Strecker reaction in water at room temperature without any activation or catalyst.

The 2-step synthesis of [14 C]sodium ferrocyanide decahydrate is described. Ferrous hydroxide was prepared from iron powder and then reacted with [14 C]sodium cyanide to afford the product. The [14 C]sodium ferrocyanide decahydrate was characterized by HPLC and 13 C NMR.

The title compound contains a guanidinium cation that was unexpectedly found during X-ray single crystal analysis of a copper(I) cyanide network expected to contain protonated N,N’-dimethyl-1,3-diaminopropane. The cation was presumably formed by reaction of the amine with cyanide ions in the aqueous sodium cyanide/copper cyanide mixtures used in the synthesis. The structure of the network solid features the guanidinium cation as a guest in an anionic two-dimensional polymeric framework with stoichiometry Cu2(CN)3−. Confirmation of the structure was provided by analytical, thermal gravimetric and infrared data.

Reaction of N-(tosylmethyl)-substituted ureas, thioureas, and N′-cyanoguanidines, prepared by condensation of the corresponding amides with various aldehydes and p-toluenesulfinic acid, with NaCN has been studied. The outcome of the reaction is strongly dependent on the amide nature and reaction conditions. Generally, N-(tosylmethyl)ureas afford α-ureido nitriles, N-(tosylmethyl)-N′-cyanoguanidines transform into 4-amino-2-cyanimino-1,5-dihydro-2H-imidazoles, and N-(tosylmethyl)thioureas give complex mixtures of various imidazole derivatives. The prepared α-ureido nitriles are selectively converted into the corresponding α-ureido amides by treatment with conc. HCl at room temperature. Under basic conditions, α-ureido nitriles cyclize into 4-amino-1,5-dihydro-2H-imidazole-2-ones. Treatment of 4-amino-2-cyanimino-1,5-dihydro-2H-imidazoles with conc. HCl at room temperature lead to hydrolysis of both the amidine fragment and cyano group to give hydrochlorides of 2-(carbamoylimino)imidazolidin-4-ones which are easily decarbamoylated to form hydrochlorides of 2-iminoimidazolidin-4-ones. Structure of the prepared imidazoles and some mechanistic aspects of cyanide-anion amidoalkylation with N-(tosylmethyl)ureas are discussed based on DFT calculations.

Caterpillars of the greater wax moth, Galleria mellonella, are shown to be a useful invertebrate organism for examining mitochondrial toxicants (inhibitors of electron transport) and testing putative antidotes. Administration of sodium azide, sodium cyanide, or sodium (hydro)sulfide by intra-haemocoel injection (through a proleg) results in a dose-dependent paralysed state in the larvae lasting from1 to ~40 min. The duration of paralysis is easily monitored, because if turned onto their backs, the larvae right themselves onto their prolegs once they are able to move again. The efficacy of putative antidotes to the three toxicants can routinely be assessed by observing shortened periods of paralysis with larvae given toxicant and antidote compared to larvae administered only the same dose of toxicant. The validity of the approach is demonstrated with agents previously shown to be antidotal towards cyanide intoxication in mice; namely, sodium nitrite and CoN

The alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344 uses free cyanide and several metal−cyanide complexes as the sole nitrogen source and tolerates high concentrations of metals like copper, zinc and iron, which are present in the jewelry wastewaters. To understand deeply the regulatory mechanisms involved in the transcriptional regulation of cyanide-containing wastewaters detoxification by P. pseudoalcaligenes CECT5344, RNA-Seq has been performed from cells cultured with a cyanide-containing jewelry wastewater, sodium cyanide or ammonium chloride as the sole nitrogen source. Small RNAs (sRNAs) that may have potential regulatory functions under cyanotrophic conditions were identified. In total 20 sRNAs were identified to be differentially expressed when compared the jewelry residue versus ammonium as nitrogen source, 16 of which could be amplified successfully by RT-PCR. As predicted targets of these 16 sRNAs were several components of the nit1C gene cluster encoding the nitrilase NitC essential for cyanide assimilation, the cioAB gene cluster that codes for the cyanide-insensitive cytochrome bd-type terminal oxidase, the medium length-polyhydroxyalkanoates (ml-PHAs) gene cluster, and gene clusters related with a global nitrogen limitation response like those coding for glutamine synthase and urease. Other targets were non-clustered genes (or their products) involved in metal resistance and iron acquisition, such as metal extrusion systems and the ferric uptake regulatory (Fur) protein, and a GntR-like regulatory family member probably involved in the regulation of the cyanide assimilation process in the strain CECT5344. Induction of genes targeted by sRNAs in the jewelry residue was demonstrated by qRT-PCR.

Volume-regulated anion channel (VRAC) is a glutamate-permeable channel that is activated by physiological and pathological cell swelling and promotes ischemic brain damage. However, because VRAC opening requires cytosolic ATP, it is not clear if and how its activity is sustained in the metabolically compromised CNS. In the present study, we used cultured astrocytes - the cell type which shows prominent swelling in stroke - to model how metabolic stress and changes in gene expression may impact VRAC function in the ischemic and post-ischemic brain. The metabolic state of primary rat astrocytes was modified with chemical inhibitors and examined using luciferin-luciferase ATP assays and a Seahorse analyzer. Swelling-activated glutamate release was quantified with the radiotracer D-[3 H]aspartate. The specific contribution of VRAC to swelling-activated glutamate efflux was validated by RNAi knockdown of the essential subunit, leucine-rich repeat-containing 8A (LRRC8A); expression levels of VRAC components were measured with qRT-PCR. Using this methodology, we found that complete metabolic inhibition with the glycolysis blocker 2-deoxy-D-glucose and the mitochondrial poison sodium cyanide reduced astrocytic ATP levels by > 90% and abolished glutamate release from swollen cells (via VRAC). When only mitochondrial respiration was inhibited by cyanide or rotenone, the intracellular ATP levels and VRAC activity were largely preserved. Bypassing glycolysis by providing the mitochondrial substrates pyruvate and/or glutamine led to partial recovery of ATP levels and VRAC activity. Unexpectedly, the metabolic block of VRAC was overridden when ATP-depleted cells were exposed to extreme cell swelling (≥ 50% reduction in medium osmolarity). Twenty-four hour anoxic adaptation caused a moderate reduction in the expression levels of the VRAC component LRRC8A, but no significant changes in VRAC activity. Overall, our findings suggest that (i) astrocytic VRAC activity and metabolism can be sustained by low levels of glucose and (ii) the inhibitory influence of diminishing ATP levels and the stimulatory effect of cellular swelling are the two major factors that govern VRAC activity in the ischemic brain.

Soil and water samples were collected from various regions of SIPCOT and nearby Vanappadi Lake, Ranipet, Tamilnadu, India. Based on their colony morphology and their stability during subculturing, 72 bacteria were isolated, of which 14 isolates were actinomycetes. Preliminary selection was carried out to exploit the ability of the microorganisms to utilize sodium cyanate as nitrogen source. Those organisms that were able to utilize cyanate were subjected to secondary screening viz., utilization of sodium cyanide as the nitrogen source. The oxygenolytic cleavage of cyanide is dependent on cyanide monooxygenase which obligately requires pterin cofactor for its activity. Based on this, the organisms capable of utilizing sodium cyanide were tested for the presence of pterin. Thin layer chromatography (TLC) of the cell extracts using n-butanol: 5 N glacial acetic acid (4:1) revealed that 10 out of 12 organisms that were able to utilize cyanide had the pterin-related blue fluorescent compound in the cell extract. The cell extracts of these 10 organisms were subjected to high performance thin layer chromatography (HPTLC) for further confirmation using a pterin standard. Based on the incubation period, cell biomass yield, peak height and area, strain VPW3 was selected and was identified as Bacillus subtilis. The Rf value of the cell extract was 0.73 which was consistent with the 0.74 Rf value of the pterin standard when scanned at 254 nm. The compound was extracted and purified by preparative High Performance Liquid Chromatography (HPLC). Characterization of the compound was performed by ultraviolet spectrum, fluorescence spectrum, Electrospray Ionization-Mass Spectrometry (ESI-MS), and Nuclear Magnetic Resonance spectroscopy (NMR). The compound is proposed to be 6-propionyl pterin (2-amino-6-propionyl-3H-pteridin-4-one).

A general approach to 2-substituted ethyl 5-aminothiazole-4-carboxylates is reported herein. Both aliphatic and aromatic thioamides undergo 1,2-addition to ethyl glyoxylate to give hemiaminals which, when treated with acetyl chloride, undergo elimination to the corresponding imines. Upon exposure to aqueous sodium cyanide the imines undergo a Strecker addition–cyclization reaction to provide the target heterocycles in modest to good yields over the 3-step process.

CYP121 is a cytochrome P450 enzyme from Mycobacterium tuberculosis that catalyzes the formation of a C–C bond between the aromatic groups of its cyclodityrosine substrate (cYY). The crystal structure of CYP121 in complex with cYY reveals that the solvent-derived ligand remains bound to the ferric ion in the enzyme–substrate complex. Whereas in the generally accepted P450 mechanism, binding of the primary substrate in the active-site triggers the release of the solvent-derived ligand, priming the metal center for reduction and subsequent O2 binding. Here we employed sodium cyanide to probe the metal–ligand exchange of the enzyme and the enzyme–substrate complex. The cyano adducts were characterized by UV–vis, EPR, and ENDOR spectroscopies and X-ray crystallography. A 100-fold increase in the affinity of cyanide binding to the enzyme–substrate complex over the ligand-free enzyme was observed. The crystal structure of the [CYP121(cYY)CN] ternary complex showed a rearrangement of the substrate in the active-site, when compared to the structure of the binary [CYP121(cYY)] complex. Transient kinetic studies showed that cYY binding resulted in a lower second-order rate constant (kon (CN)) but a much more stable cyanide adduct with 3 orders of magnitude slower koff (CN) rate. A dynamic equilibrium between multiple high- and low-spin species for both the enzyme and enzyme–substrate complex was also observed, which is sensitive to changes in both pH and temperature. Our data reveal the chemical and physical properties of the solvent-derived ligand of the enzyme, which will help to understand the initial steps of the catalytic mechanism.

An efficient preparation of cyanohydrin ethyl carbonates via cyanocarbonation of aromatic and hetero-aromatic aldehydes with sodium cyanide and ethyl chlorocarbonate using commercial surfactants (5 mol %) in aqueous media at low temperature afforded almost quantitative isolated yields (≥94%) in 30 min. Aromatic aldehydes bearing electron-donating as well as electron-withdrawing groups have been successfully employed without any significant changes. Centrifugation of the reaction mixture allows accessible purification without the need of solvent extraction. Reaction conditions can be readily upscaled to multigrams.

An experimental study was conducted to evaluate the effect of 0.129 NaCN mg/l for 28 days on the behavior of tilapia and the activities of adenosine triphosphatases enzyme (Na+/K+, Ca2+ and Mg2+-ATPase) within gill filaments, liver and muscles. Also, to evaluate the stability of cyanide in stored, frozen fish tissues. The present results showed that the exposed fish had shown different modes of behaviors. During the first 14 days, the fish lost its equilibrium with excessive mucous secretion on its gill filaments and skin. On the other hand, tilapia showed a normal swimming behavior with no excessive mucus secretion at 21st and 28th days.In addition, the present result showed that the NaCN decreased the activities of ATPase within the investigated tissues during the exposure periods except for the 1st day. Furthermore, the present result showed that the mean concentration of cyanide gradually decreased within the frozen tissues and completely disappeared after 48 h within frozen liver and muscle. But, it disappeared after 72 h within frozen blood and gills. Keywords: Cyanide, Toxicity, ATPase activity, Behavior, Stability, Orechromis niloticus

Potassium ions cationized polyether cyanide [K+{PEG}CN−] was readily synthesized by mixing potassium cyanide and PEG400 at room temperature and used as a cost effective and comparatively safer alternative to the toxic sodium cyanide/acetic acid system for generating in situ HCN for the oxidative cyanation of tertiary amines with hydrogen peroxide using RuCl3 as catalyst. This method affords a facile approach to the synthesis of α-aminonitriles in high yields under solvent-free and acid-free reaction conditions.

A novel series of 2'-spiro pyrimidine pyranonucleosides has been designed and synthesized. Their precursors, 2'-C-cyano nucleosides 5a,b and 6a,b, were obtained by subjecting 1a,b to the sequence of selective protection of the primary hydroxyl group, acetalation, oxidation, and finally treatment with sodium cyanide. Deoxygenation at the 2'-position of cyanohydrins 5a,b or 6a,b led to the 2'-deoxy derivatives 9a,b. Fully deprotection of 5a,b, 6a,b, and 9a,b gave the desired 2'-C-cyano 7a,b, 8a,b, and 2'-C-cyano-2'-deoxy pyranonucleosides 10a,b, respectively. Mesylation of the corresponding cyanohydrins 5a,b and 6a,b afforded compounds 11a,b and 12a,b which after base treatment and subsequent deprotection furnished the spiro nucleosides 15a,b and 16a. The new analogues were evaluated for their potential cytostatic activities in cell culture.

Food (cassava) linamarin is metabolized into neurotoxicants cyanide and cyanate, metabolites of which we sought to elucidate the differential toxicity effects on memory. Young 6–8 weeks old male rats were treated intraperitoneally with either 2.5 mg/kg body weight (bw) cyanide (NaCN), or 50 mg/kg bw cyanate (NaOCN), or 1 μl/g bw saline, daily for 6 weeks. Short-term and long-term memories were assessed using a radial arm maze (RAM) testing paradigm. Toxic exposures had an influence on short-term working memory with fewer correct arm entries (F 2, 19 = 4.57 p

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:30:32Z No. of bitstreams: 0 Made available in DSpace on 2014-05-27T11:30:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-09-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) This study examined the distribution and orientation of gill O2 chemoreceptors in Oreochromis niloticus and their role in cardiorespiratory responses to graded hypoxia. Intact fish, and a group with the first gill arch excised (operated), were submitted to graded hypoxia and their cardiorespiratory responses (oxygen uptake - V̇O2, breathing frequency - fR, ventilatory stroke volume - VT, gill ventilation - V̇G, O2 extraction from the ventilatory current - EO2, and heart rate - fH) were compared. Their responses to bolus injections of NaCN into the bloodstream (internal) or ventilatory water stream (external) were also determined. The V̇O2 of operated fish was significantly lower at the deepest levels of hypoxia. Neither reflex bradycardia nor ventilatory responses were completely abolished by bilateral excision of the first gill arch. EO2 of the operated group was consistently lower than the intact group. The responses to internal and external NaCN included transient decreases in fH and increases in fR and Vamp (ventilation amplitude). These cardiorespiratory responses were attenuated but not abolished in the operated group, indicating that chemoreceptors are not restricted to the first gill arch, and are sensitive to oxygen levels in both blood and water. © 2013 Elsevier Inc. Department of Physiological Sciences Laboratory of Zoophysiology and Comparative Biochemistry Federal University of São Carlos (UFSCar), P.O. Box 676, Via Washington Luis, km 235, 13565-905 São Carlos, SP Department of Zoology and Botany São Paulo State University - UNESP/IBILCE, Rua Cristóvão Colombo, 2265, 15054-000 - São José do Rio Preto, SP National Institute of Science and Technology - Comparative Physiology (FAPESP/CNPq) Department of Zoology and Botany São Paulo State University - UNESP/IBILCE, Rua Cristóvão Colombo, 2265, 15054-000 - São José do Rio Preto, SP FAPESP: 10/03599-2

Neural stem/progenitor cells (NSPCs) are multipotent cells within the embryonic and adult brain that give rise to both neuronal and glial cell lineages. Maintenance of NSPC multi-potency is promoted by low oxygen tension, although the metabolic underpinnings of this trait have not been described. In this study, we investigated the metabolic state of undifferentiated NSPCs in culture, and tested their relative reliance on oxidative versus glycolytic metabolism for survival, as well as their dependence on hypoxia inducible factor-1alpha (HIF-1α) expression for maintenance of metabolic phenotype. Unlike primary neurons, NSPCs from embryonic and adult mice survived prolonged hypoxia in culture. In addition, NSPCs displayed greater susceptibility to glycolytic inhibition compared with primary neurons, even in the presence of alternative mitochondrial TCA substrates. NSPCs were also more resistant than neurons to mitochondrial cyanide toxicity, less capable of utilizing galactose as an alternative substrate to glucose, and more susceptible to pharmacological inhibition of the pentose phosphate pathway by 6-aminonicotinamide. Inducible deletion of exon 1 of the Hif1a gene improved the ability of NSPCs to utilize pyruvate during glycolytic inhibition, but did not alter other parameters of metabolism, including their ability to withstand prolonged hypoxia. Taken together, these data indicate that NSPCs have a relatively low requirement for oxidative metabolism for their survival and that hypoxic resistance is not dependent upon HIF-1α signaling.

We report a new method for stabilizing appreciable loadings (~1 wt %) of isolated gold atoms on titania and show that these catalyze the low-temperature water-gas shift reaction. The method combines a typical gold deposition/precipitation method with UV irradiation of the titania support suspended in ethanol. Dissociation of H2O on the thus-created Au-O-TiO(x) sites is facile. At higher gold loadings, nanoparticles are formed, but they were shown to add no further activity to the atomically bound gold on titania. Removal of this "excess" gold by sodium cyanide leaching leaves the activity intact and the atomically dispersed gold still bound on titania. The new materials may catalyze a number of other reactions that require oxidized active metal sites.

Background Sirtuins are NAD+ dependent deacetylases, which regulate mitochondrial energy metabolism as well as cellular response to stress. The NAD/NADH-system plays a crucial role in oxidative phosphorylation linking sirtuins and the mitochondrial respiratory chain. Furthermore, sirtuins are able to directly deacetylate and activate different complexes of the respiratory chain. This prompted us to analyse sirtuin levels in skin fibroblasts from patients with cytochrome c-oxidase (COX) deficiency and to test the impact of different pharmaceutical activators of sirtuins (SRT1720, paeonol) to modulate sirtuins and possibly respiratory chain enzymes in patient cells in vitro. Methods We assayed intracellular levels of sirtuin 1 and the mitochondrial sirtuins SIRT3 and SIRT4 in human fibroblasts from patients with COX- deficiency. Furthermore, sirtuins were measured after inhibiting complex IV in healthy control fibroblasts by cyanide and after incubation with activators SRT1720 and paeonol. To determine the effect of sirtuin inhibition at the cellular level we measured total cellular acetylation (control and patient cells, with and without treatment) by Western blot. Results We observed a significant decrease in cellular levels of all three sirtuins at the activity, protein and transcriptional level (by 15% to 50%) in COX-deficient cells. Additionally, the intracellular concentration of NAD+ was reduced in patient cells. We mimicked the biochemical phenotype of COX- deficiency by incubating healthy fibroblasts with cyanide and observed reduced sirtuin levels. A pharmacological activation of sirtuins resulted in normalized sirtuin levels in patient cells. Hyper acetylation was also reversible after treatment with sirtuin activators. Pharmacological modulation of sirtuins resulted in altered respiratory chain complex activities. Conclusions We found inhibition of situins 1, 3 and 4 at activity, protein and transcriptional levels in fibroblasts from patient with COX-deficiency. Pharmacological activators were able to restore reduced sirtuin levels and thereby modulate respiratory chain activities.

The present study was conducted to determine the effect of sodium cyanide on oxidative metabolism, alterations in the activities of lactate dehydrogenase (LDH) and succinate dehydrogenase (SDH), behavior, respiratory rate and metabolites (lactic and pyruvic acid) of the fingerling of Clarias gariepinus. The fingerlings were exposed to a sublethal concentration (0.75 mgL-1) of sodium cyanide. Fish showed a gradual decrease in respiratory rate, increase in LDH and decrease in SDH levels, and lactic acid followed a similar trend with pyruvate in an eight-day trial. The changes in the levels of these enzyme activities may be as a result of impaired oxidative metabolism and cellular damage, which had effect on the release of these enzymes. Elevation in the level of lactic acid and decrease in pyruvic acid was due to shift from aerobic to anaerobic metabolism which resulted to a severe drop in the respiratory rate of the fish. It may be as a result of blockage of electron transfer from cytochrome-c oxidase to molecular oxygen which might lead to cellular hypoxia even in the presence of normal oxygenation of hemoglobin. Thus, inhibition of oxidative metabolism by sodium cyanide in C. garipenus was reconfirmed. Behavioral changes caused by sodium cyanide exposure in the fish were probably due to the combination of lactate acidosis with cytotoxic hypoxia, which might depress the central nervous system.Key words: Succinate dehydrogenase (SDH), lactate dehydrogenase (LDH), lactic acid, pyruvate, behavior, enzymes.

A new series of 4′- C -cyano and 4′- C -cyano-4′-deoxy pyrimidine pyranonucleosides has been designed and synthesized. Commercially available 1,2,3,4,6-penta- O -acetyl- d -mannopyranose ( 1 ) was condensed with silylated 5-fluorouracil, uracil, and thymine, respectively to afford after deacetylation 1-(α- d -mannopyranosyl)nucleosides ( 2a–c ). Subjecting 2a–c to the sequence of specific acetalation, selective protection of the primary hydroxyl group and oxidation, the 4′-ketonucleosides 6a–c and 7c were obtained. Reaction of compounds 6a , b, and 7c with sodium cyanide and subsequent deprotection gave the target 1-(4′- C -cyano-α- d -mannopyranosyl)nucleosides 12a–c . Deoxygenation at the 4′-position of cyanohydrins 8a , b, and 11c followed by deprotection led to the desired 1-(4′- C -cyano-4′-deoxy-α- d -talopyranosyl)nucleosides ( 15a–c ). The newly synthesized compounds were evaluated for their potential antiviral and cytostatic activities in cell culture.

Borrelia burgdorferi, the causative agent of Lyme disease, exists in nature through a complex life cycle involving ticks of the Ixodes genus and mammalian hosts. During its life cycle, B. burgdorferi experiences fluctuations in oxygen tension and may encounter reactive oxygen species (ROS). The key metalloenzyme to degrade ROS in B. burgdorferi is SodA. Although previous work suggests that B. burgdorferi SodA is an iron-dependent superoxide dismutase (SOD), later work demonstrates that B. burgdorferi is unable to transport iron and contains an extremely low intracellular concentration of iron. Consequently, the metal cofactor for SodA has been postulated to be manganese. However, experimental evidence to support this hypothesis remains lacking. In this study, we provide biochemical and genetic data showing that SodA is a manganese-dependent enzyme. First, B. burgdorferi contained SOD activity that is resistant to H(2)O(2) and NaCN, characteristics associated with Mn-SODs. Second, the addition of manganese to the Chelex-treated BSK-II enhanced SodA expression. Third, disruption of the manganese transporter gene bmtA, which significantly lowers the intracellular manganese, greatly reduced SOD activity and SodA expression, suggesting that manganese regulates the level of SodA. In addition, we show that B. burgdorferi is resistant to streptonigrin, a metal-dependent redox cycling compound that produces ROS, and that SodA plays a protective role against the streptonigrin. Taken together, our data demonstrate the Lyme disease spirochete encodes a manganese-dependent SOD that contributes to B. burgdorferi defense against intracellular superoxide.

In a previous study, we found that magnesium deficiency stimulated prostacyclin production and suggested that this stimulation resulted from an enhanced Ca2+ influx induced by magnesium deficiency. In this study, we further examined prostacyclin generation in cultured human umbilical vein endothelial cells after intracellular free calcium content ([Ca2+]i) was altered by addition of diltiazem, nifedipine, verapamil, sodium cyanide (NaCN), ruthenium red or quinidine to a low magnesium medium. The results showed that diltiazem, nifedipine, verapamil and ruthenium red inhibited 45Ca2+ influx, and NaCN and quinidine had no effect on 45Ca2+ influx. However, all of these compounds decreased [Ca2+]i, [3H]arachidonic acid release and prostacyclin production. The reduced [3H]arachidonic acid content in cellular phospholipids caused by low magnesium treatment was not altered by the added compounds. We suggested that arachidonic acid release and prostacyclin production was calcium-dependent in cultured endothelial cells.

Peroxidases catalyze the oxidation of nitrite to nitrate in the presence of hydrogen peroxide. Two pathways may occur: one entailing the intermediate formation of NO2 and the other implying the generation of peroxynitrite. The products of nitrite (NO2−) oxidation by salivary peroxidase (SPO) and commercial bovine lactoperoxidase (LPO) are studied by utilizing an electrochemical assay that allows the direct, continuous monitoring of NO and/or NO2 and by HPLC to assess nitrates at the end of the reaction. Dialyzed saliva and LPO, in the presence of H2O2, convert nitrite into nitrate and form some NO, with a molar ratio of 103. In our experimental conditions, no NO2 was detectable among the products of nitrite oxidation. SCN− inhibits NO formation and so does I−, although at higher concentrations. No effects are observed with Cl− or Br−. We conclude that SPO and LPO transform NO2− into nitrate-forming small amounts of NO in the presence of H2O2 as an intermediate or a by-product, synthesized through the peroxynitrite pathway. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:87–93, 2012; View this article online at wileyonlinelibrary.com. DOI 10.1002/jbt.21407

The aim of our study was to analyze a distribution of metabolic flux controls of all mitochondrial complexes of ATP-Synthasome and mitochondrial creatine kinase (MtCK) in situ in permeabilized cardiac cells. For this we used their specific inhibitors to measure flux control coefficients ( C vi JATP ) in two different systems: A) direct stimulation of respiration by ADP and B) activation of respiration by coupled MtCK reaction in the presence of MgATP and creatine. In isolated mitochondria the C vi JATP were for system A: Complex I - 0.19, Complex III – 0.06, Complex IV 0.18, adenine nucleotide translocase (ANT) – 0.11, ATP synthase – 0.01, Pi carrier - 0.20, and the sum of C vi JATP was 0.75. In the presence of 10 mM creatine (system B) the C vi JATP were 0.38 for ANT and 0.80 for MtCK. In the permeabilized cardiomyocytes inhibitors had to be added in much higher final concentration, and the following values of C vi JATP were determined for condition A and B, respectively: Complex I - 0.20 and 0.64, Complex III - 0.41 and 0.40, Complex IV - 0.40 and 0.49, ANT - 0.20 and 0.92, ATP synthase - 0.065 and 0.38, Pi carrier - 0.06 and 0.06, MtCK 0.95. The sum of C vi JATP was 1.33 and 3.84, respectively. Thus, C vi JATP were specifically increased under conditions B only for steps involved in ADP turnover and for Complex I in permeabilized cardiomyocytes within Mitochondrial Interactosome, a supercomplex consisting of MtCK, ATP-Synthasome, voltage dependent anion channel associated with tubulin βII which restricts permeability of the mitochondrial outer membrane.

Previous work has shown that the carotid body glomus cells can function as glucose sensors. The activation of these chemoreceptors, and of its afferent nucleus in the brainstem (solitary tract nucleus - STn), induces rapid changes in blood glucose levels and brain glucose retention. Nitric oxide (NO) in STn has been suggested to play a key role in the processing of baroreceptor signaling initiated in the carotid sinus. However, the relationship between changes in NO in STn and carotid body induced glycemic changes has not been studied. Here we investigated in anesthetized rats how changes in brain glucose retention, induced by the local stimulation of carotid body chemoreceptors with sodium cyanide (NaCN), were affected by modulation of NO levels in STn. We found that NO donor sodium nitroprusside (SNP) micro-injected into STn completely blocked the brain glucose retention reflex induced by NaCN chemoreceptor stimulation. In contrast, NOS inhibitor N(ω)-nitro-L-arginine methyl ester (L-NAME) increased brain glucose retention reflex compared to controls or to SNP rats. Interestingly, carotid body stimulation doubled the expression of nNOS in STn, but had no effect in iNOS. NO in STn could function to terminate brain glucose retention induced by carotid body stimulation. The work indicates that NO and STn play key roles in the regulation of brain glucose retention.

background: There has been an ongoing debate in the reproductive field about whether mammalian spermatozoa rely on glycolysis, oxidative phosphorylation or both for their energy production. Recent studies have proposed that human spermatozoa depend mainly on glucose for motility and fertilization but the mechanism behind an efficient glycolysis in human spermatozoa is not well understood. Here, we demonstrate how human spermatozoa utilize exogenous pyruvate to enhance glycolytic ATP production, motility, hyperactivation and capacitation, events that are crucial for male fertility. methods: Purified human spermatozoa from healthy donors were incubated under capacitating conditions (including albumin, bicarbonate and glucose) and tested for changes in ATP levels, motility, hyperactivation and tyrosine phosphorylation after treatment with pyruvate. The experiments were repeated in the presence of sodium cyanide in order to assess the contribution from mitochondrial respiration. The metabolism of 13 C labeled glucose and pyruvate was traced by a combination of liquid chromatography and mass spectrometry. results: The treatment of human spermatozoa with exogenous pyruvate increased intracellular ATP levels, progressive motility and hyperactivation by 56, 21 and 130%, respectively. In addition, added pyruvate induced a significant increase in tyrosine phosphorylation levels. Blocking of the electron transport chain did not markedly affect the results, indicating that the mechanism is independent of oxidative phosphorylation. However, the observed effects could be counteracted by oxamate, an inhibitor of lactate dehydrogenase (LDH). Metabolic tracing experiments revealed that the observed rise in ATP concentration resulted from an enhanced glycolytic flux, which was increased by more than 50% in the presence of exogenous pyruvate. Moreover, all consumed 13 C labeled pyruvate added was converted to lactate rather than oxidized in the tricarboxylic acid cycle. conclusions: Human spermatozoa seem to rely mainly, if not entirely, on glycolysis as the source of ATP fueling the energydemanding processes of motility and capacitation. The efficient glycolysis is dependent on exogenous pyruvate, which indirectly feeds the accelerated glycolysis with NAD + through the LDH-mediated conversion of pyruvate to lactate. Pyruvate is present in the human female reproductive tract at concentrations in accordance with our results. As seen in other mammals, the motility and fertility of human spermatozoa seem to be dictated by the available energy substrates present in the conspecific female.

A five-step synthesis of 4-chloromethyl- and 4-mesyloxymethyl-5-tosyl-1,2,3,4-tetrahydropyrimidin-2-ones has been developed. The reaction of N-[(2-benzoyloxy-1-tosyl)ethyl]urea with sodium enolates of α-tosylketones followed by cyclization–dehydration, and debenzoylation gave 4-hydroxymethyl-5-tosyl-1,2,3,4-tetrahydropyrimidin-2-ones, which were transformed into the 4-chloromethyl- or 4-mesyloxymethyl-derivatives. Treatment of the latter with nucleophilic reagents, such as sodium cyanide, sodium diethyl malonate, sodium thiophenolate, or potassium phthalimide, afforded the corresponding 4,7-disubstituted 6-tosyl-2,3,4,5-tetrahydro-1H-1,3-diazepin-2-ones as a result of ring expansion. The effect of the leaving group and the substitution at the position C6 on the reactivity of the pyrimidines is discussed.

Chiral macrocyclic V(V) salen complexes 1a – f derived from macrocyclic ligands obtained by the reaction of 1 R ,2 R -(−) diaminocyclohexane/(1 R ,2 R )-(+)-1,2-diphenylethylenediamine with bis-aldehydes 2 and 3 were synthesized and used as efficient catalysts in asymmetric cyanation reactions. The V(V) catalysts demonstrated excellent performance (product yields and ees up to 99%) with potassium cyanide (KCN) and sodium cyanide (NaCN). The catalytic system also performed very well with a safer source of cyanide-ethyl cyanoformate to give cyanohydrin carbonates in excellent yield and ee (up to 97%). The V(V) macrocyclic salen complex 1b retained its performance at multi-gram level and was conveniently recycled for a number of times.

The effects of exposure to one-third and one-fifth sublethal concentrations (0.106 and 0.064 mg/L) of sodium cyanide on protein metabolism on freshwater carp, Labeo rohita, was studied. Three functionally different tissues, namely, the liver, muscle, and gills, were studied after 5, 10, and 15 days. Exposures produced marked changes in protein metabolic profile in all tissues studied. These changes were more pronounced in the one-third sublethal concentration, suggesting a cumulative action of toxicant. This investigation revealed that the total, structural, and soluble proteins and urea content in all the three tissues were decreased, whereas free amino acids, ammonia, and enzyme activity (i.e., protease, alanine aminotransferase, and aspartate aminotransferase) exhibited elevated levels at both sublethal concentrations. Variation in protein metabolism in the fish, induced by sodium cyanide, demonstrated its toxic effects on cellular metabolism, thereby leading to impaired protein synthetic machinery. The results of the present study indicate that a mechanism of impaired energy transformation has direct action on the fish, L. rohita, and its impact is clearly evident from the change in the nutritional content of the fish.

Present study concerns the effect of sodium cyanide on the Indian major carp, Labeo rohita. Fishes were exposed to lethal (0.32 mg/L) and sublethal (0.064 mg/L) concentrations of sodium cyanide. The effect of intoxication was studied on Na(+)K(+)ATPase, Mg(+2)ATPase and Ca(2+)ATPase in various physiological tissues (gill, liver, and muscle) at the end of 1, 2, 3 and 4 days of lethal and 5, 10 and 15 days of sublethal exposure periods. Sodium cyanide induced significant inhibitory effects on the ATPase activity of the fish. Inhibition of the ATPase blocked the active transport system of the gill epithelial as well as chloride cells, and thus altered the osmo-regulatory mechanism of the fish. The value of the measured responses as an indicator of stress caused by water contamination discussed. The results confirm that ATPase levels significantly decreased in treated fish, indicating that ATPases could be used as sensitive and useful biomarkers for cyanide pollution.

Ecto-5′-nucleotidase (NT5E), a predominant enzyme that produces extracellular adenosine from AMP, plays an important role in a variety of physiological and pathophysiological processes. This study was performed to identify agents that affect NT5E activity using C6 glioma cells. When cells were incubated with sodium nitroprusside (SNP), phorbol 12-myristate 13- acetate, forskolin, lipopolysaccharide, or interferon-γ, only SNP inhibited NT5E activity in a time- and concentration-dependent manner (IC50 = 1.2 μM). The inhibitory effect of SNP was long-lasting even after SNP washout; and its action was not mimicked by nitric oxide generating agents, 8-bromo cyclic GMP, ferricyanide, ferrocyanide, or sodium cyanide. SNP did not change NT5E mRNA level or membrane surface protein expression. Similar to SNP, Fe2+ inhibited NT5E activity, but to a lesser extent. Although Fe2+ is known to increase oxidative stress, Fe2+-mediated oxidative stress was not involved in SNP inhibition of NT5E because the inhibition of NT5E by SNP was not affected by superoxide dismutase and catalase. In contrast, addition of Zn2+, an essential metal co-factor of NT5E activity, prevented SNP from inhibiting NT5E. These results suggest that SNP disrupts a critical Zn2+-dependent enzyme activity and might be useful as a pharmacological tool for inhibiting NT5E. Keywords:: ecto-5′-nucleotidase, sodium nitroprusside, Zn2+, Fe2+, C6 glioma cell

Methyl-CpG-binding protein 2 (MeCP2) deficiency causes Rett syndrome (RTT), a neurodevelopmental disorder characterized by severe cognitive impairment, synaptic dysfunction, and hyperexcitability. Previously we reported that the hippocampus of MeCP2-deficient mice (Mecp2(-/y)), a mouse model for RTT, is more susceptible to hypoxia. To identify the underlying mechanisms we now focused on the anoxic responses of wildtype (WT) and Mecp2(-/y) CA1 neurons in acute hippocampal slices. Intracellular recordings revealed that Mecp2(-/y) neurons show only reduced or no hyperpolarizations early during cyanide-induced anoxia, suggesting potassium channel (K(+) channel) dysfunction. Blocking adenosine-5'-triphosphate-sensitive K(+) channels (K(ATP-)) and big-conductance Ca(2+)-activated K(+) channels (BK-channels) did not affect the early anoxic hyperpolarization in either genotype. However, blocking Ca(2+) release from the endoplasmic reticulum almost abolished the anoxic hyperpolarizations in Mecp2(-/y) neurons. Single-channel recordings confirmed that neither K(ATP)- nor BK-channels are the sole mediators of the early anoxic hyperpolarization. Instead, anoxia Ca(2+)-dependently activated various small/intermediate-conductance K(+) channels in WT neurons, which was less evident in Mecp2(-/y) neurons. Yet, pharmacologically increasing the Ca(2+) sensitivity of small/intermediate-conductance K(Ca) channels fully restored the anoxic hyperpolarization in Mecp2(-/y) neurons. Furthermore, Ca(2+) imaging unveiled lower intracellular Ca(2+) levels in resting Mecp2(-/y) neurons and reduced anoxic Ca(2+) transients with diminished Ca(2+) release from intracellular stores. In conclusion, the enhanced hypoxia susceptibility of Mecp2(-/y) hippocampus is primarily associated with disturbed Ca(2+) homeostasis and diminished Ca(2+) rises during anoxia. This secondarily attenuates the activation of K(Ca) channels and thereby increases the hypoxia susceptibility of Mecp2(-/y) neuronal networks. Since cytosolic Ca(2+) levels also determine neuronal excitability and synaptic plasticity, Ca(2+) homeostasis may constitute a promising target for pharmacotherapy in RTT.

Carp fingerlings exposed to a sublethal concentration (0.5 mg L−1) of sodium cyanide showed a steady decrement over a 7-day period in respiratory rate, rise in lactate dehydrogenase (LDH), and fall in succinate dehydrogenase (SDH) activities followed by variations in lactic and pyruvate levels. Changes in these enzyme activities might be due to impaired oxidative metabolism and severe cellular damage leading to the release of these enzymes. Decline in the activities of SDH and LDH clearly represents a shift from aerobic to anaerobic metabolism as evidenced by elevated lactate and decline in pyruvate levels. The shift to anaerobic metabolism is also reflected by severe drop in the respiratory rate of the fish. This may be a consequence of the blockage of electron transfer from cytochrome c oxidase to molecular oxygen, thus ceasing cellular respiration and it can lead to cellular hypoxia even in the presence of normal hemoglobin oxygenation. Hence, we indirectly reconfirm the inhibition of oxidative metabol...

Specific recognition of CN(-) in sodium cyanide solution was achieved using two imidazole-based receptors (A and B). Visually detectable color changes were associated with the formation of hydrogen bonded adducts, A.CN(-) and B.CN(-). Ratiometric fluorescence response was achieved for receptor A on binding to CN(-), and this reagent was used for imaging bacterial cells pre-exposed to 1.42 microM CN(-) solution.

In one series of experiments, heart frequency (f (H)), blood pressure (P (a)), gill ventilation frequency (f ( R )), ventilation amplitude (V (AMP)) and total gill ventilation (V (TOT)) were measured in intact jeju (Hoplerythrinus unitaeniatus) and jeju with progressive denervation of the branchial branches of cranial nerves IX (glossopharyngeal) and X (vagus) without access to air. When these fish were submitted to graded hypoxia (water PO(2) approximately 140, normoxia to 17 mmHg, severe hypoxia), they increased f ( R ), V (AMP), V (TOT) and P (a) and decreased f (H). In a second series of experiments, air-breathing frequency (f (RA)), measured in fish with access to the surface, increased with graded hypoxia. In both series, bilateral denervation of all gill arches eliminated the responses to graded hypoxia. Based on the effects of internal (caudal vein, 150 microg NaCN in 0.2 mL saline) and external (buccal) injections of NaCN (500 microg NaCN in 1.0 mL water) on f (R), V (AMP), V (TOT), P (a) and f (H) we conclude that the O(2) receptors involved in eliciting changes in gill ventilation and associated cardiovascular responses are present on all gill arches and monitor the O(2) levels of both inspired water and blood perfusing the gills. We also conclude that air breathing arises solely from stimulation of branchial chemoreceptors and support the hypothesis that internal hypoxaemia is the primary drive to air breathing.

Quantitative and qualitative changes in the content of elements in the solid and liquid phases occurred as the pulp moved through fermenters during biooxidation of an ore flotation concentrate. The association of microorganisms were adapted for utilizing sulfur-containing substrates; however, the rate of their oxidation was insufficient, which led to an increase in the amount of sodium cyanide required for gold recovery. The replacement of one-fourth of the liquid phase of the pulp (density, 13%) with a mineral medium without an energy source, the fractional addition of FeSO4 x 7H2O (1 g/l per day), and the improvement of pulp aeration made it possible to increase the content of SO4(2-) by 80.7, 86.2, and 58.5%, respectively. When one-fourth of the liquid phasa of the pulp (density, 24%) was replaced with a mineral medium without an energy source, the rate of additional oxidation of sulfide minerals increased, which increased the efficiency of gold extraction into solution and gold recovery on charcoal by 3.4 and 3.6%, respectively, and reduced sodium cyanide consumption by 3 kg/ton.

BACKGROUND: Previous research on insecticidal formate esters in flies and mosquitoes has documented toxicity profiles, metabolism characteristics and neurological impacts. The research presented here investigated mitochondrial impacts of insecticidal formate esters and their hydrolyzed metabolite formic acid in the model dipteran insect Drosophila melanogaster Meig. These studies compared two Drosophila strains: an insecticide-susceptible strain (Canton-S) and a strain resistant by cytochrome P450 overexpression (Hikone-R). RESULTS: In initial studies investigating inhibition of mitochondrial cytochrome c oxidase, two proven insecticidal materials (hydramethylnon and sodium cyanide) caused significant inhibition. However, for insecticidal formate esters and formic acid, no significant inhibition was identified in either fly strain. Mitochondrial impacts of formate esters were then investigated further by tracking toxicant-induced cytochrome c release from mitochondria into the cytoplasm, a biomarker of apoptosis and neurological dysfunction. Formic acid and three positive control treatments (rotenone, antimycin A and sodium cyanide) induced cytochrome c release, verifying that formic acid is capable of causing mitochondrial disruption. However, when comparing formate ester hydrolysis and cytochrome c release between Drosophila strains, formic acid liberation was only weakly correlated with cytochrome c release in the susceptible Canton-S strain (r2 = 0.70). The resistant Hikone-R strain showed no correlation (r2 < 0.0001) between formate ester hydrolysis and cytochrome c release. CONCLUSION: The findings of this study provide confirmation of mitochondrial impacts by insecticidal formate esters and suggest links between mitochondrial disruption, respiratory inhibition, apoptosis and formate-ester-induced neurotoxicity. Copyright © 2009 Society of Chemical Industry

Iron-grown Acidithiobacillus ferrooxidans MON-1 cells are highly resistant to organomercurial compounds as well as mercuric chloride (HgCl2). Existence of a novel Hg2+-reducing enzyme system, in which mercury resistant aa3-type cytochrome c oxidase catalyzes the reduction of Hg2+ with reduced mammalian cytochrome c or Fe2+ as an electron donor to give Hg0, has been shown in iron-grown MON-1 cells. There has been no reports on the mechanism of Hg2+ reduction by sulfur-grown A. ferrooxidans cells. The level of mercury resistance in sulfur-grown A. ferrooxidans MON-1 cells was compared with that of iron-grown MON-1 cells. Strain MON-1 was able to grow in 1% elemental sulfur medium (pH 2.5) containing 10 μM of Hg2+ or 0.2 μM phenylmercury acetate (PMA), suggesting that the levels of mercury resistance to inorganic and organic mercurial compounds are nearly the same in iron- and sulfur-grown MON-1 cells. Activity levels of Hg0 volatilization from HgCl2, PMA, and methylmercury chloride (MMC) were also nearly the same in iron- and sulfur-grown cells and these activities were markedly activated by 100 mM of Fe2+, but strongly inhibited by 1 mM of sodium cyanide, indicating that sulfur-grown MON-1 cells has the activity of ferrous iron-dependent mercury reducing enzyme system containing aa3-type cytochrome oxidase. aa3-type cytochrome c oxidase purified partially from sulfur-grown MON-1 cells showed both the iron oxidase and mercury reductase activities in the presence, but not in the absence, of rusticyanin and c-type cytochromes (Cyc1 and Cyc2) partially purified from iron-grown MON-1 cells.

Treatment of hexacyclo[7.4.2.01,9.03,7.04,14.06,15]pentadecane-10,12-diene-2,8-dione with aqueous sodium cyanide produced 2,8-dihydroxy-hexacyclo[7.4.2.01,9.03,7.04,14.06,15]pentadecane-10,12-diene-2,8-lactam and with sodium cyanide, ammonium chloride and ammonium hydroxide, 2-amino-8-hydroxy-hexacyclo [7.4.2.01,9.03,7.04,14.06,15]pentadecane-10,12-diene-2,8-lactam was obtained. 10-Hydroxy-hexacyclo [10.2.1.02,11.04,10.04,14.09,13]pentadecane-5,7-diene-3-one was converted into the corresponding aminonitrile and hexacyclo[10.2.1.02,11.04,10.04,14.09,13]pentadecane-5,7-diene-10-hydroxy-3-spiro-5′-hydantoin. Treatment of the latter with barium hydroxide produced exo-3-amino-10-hydroxy-hexacyclo [10.2.1.02,11.04,10.04,14.09,13]pentadecane-5,7-diene-endo-3-carboxylic acid. The isomeric endo-3-amino-10-hydroxy-hexacyclo[10.2.1.02,11.04,10.04,14.09,13]pentadecane-5,7-diene-exo-3-carboxylic acid was obtained from 3-cyano-3-ureido-hexacyclo[10.2.1.02,11.04,10.04,14.09,13]pentadecane-5,7-diene-10-ol.

Hydrogen peroxide (H2O2) is produced during liver transplantation. Ischemia/reperfusion induces oxidation and causes intracellular Ca2+ overload, which harms liver cells. Our goal was to determine the precise mechanisms of these processes.Hepatocytes were extracted from rats. Intracellular Ca2+ concentrations ([Ca2+](i)), inner mitochondrial membrane potentials and NAD(P)H levels were measured using fluorescence imaging. Phospholipase C (PLC) activity was detected using exogenous PIP2. ATP concentrations were measured using the luciferin-luciferase method. Patch-clamp recordings were performed to evaluate membrane currents.H2O2 increased intracellular Ca2+ concentrations ([Ca2+](i)) across two kinetic phases. A low concentration (400 micromol/L) of H2O2 induced a sustained elevation of [Ca2+](i) that was reversed by removing extracellular Ca2+. H2O2 increased membrane currents consistent with intracellular ATP concentrations. The non-selective ATP-sensitive cation channel blocker amiloride inhibited H2O2-induced membrane current increases and [Ca2+](i) elevation. A high concentration (1 mmol/L)of H2O2 induced an additional transient elevation of [Ca2+](i), which was abolished by the specific PLC blocker U73122 but was not eliminated by removal of extracellular Ca2+. PLC activity was increased by 1 mmol/L H2O2 but not by 400 micromol/L H2O2.H2O2 mobilizes Ca2+ through two distinct mechanisms. In one, 400 micromol/L H2O2-induced sustained [Ca2+](i) elevation is mediated via a Ca2+ influx mechanism, under which H2O2 impairs mitochondrial function via oxidative stress,reduces intracellular ATP production, and in turn opens ATP-sensitive, non-specific cation channels, leading to Ca2+ influx.In contrast, 1 mmol/L H2O2-induced transient elevation of [Ca2+](i) is mediated via activation of the PLC signaling pathway and subsequently, by mobilization of Ca2+ from intracellular Ca2+ stores.

Aims Hydrogen sulfide (H 2 S) at low concentrations serves as a physiological endogenous vasodilator molecule, while at higher concentrations it can trigger cytotoxic effects. The aim of our study was to elucidate the potential mechanisms responsible for the effects of H 2 S on vascular tone. Main methods We measured the vascular tone in vitro in precontracted rat thoracic aortic rings and we have tested the effect of different oxygen levels and a variety of inhibitors affecting known vasodilatory pathways. We have also compared the vascular effect of high concentrations of H 2 S to those of pharmacological inhibitors of oxidative phosphorylation. Furthermore, we measured adenosine triphosphate (ATP)-levels in the same vascular tissues. Key findings We have found that in rat aortic rings: (1) H 2 S decreases ATP levels; (2) relaxations to H 2 S depend on the ambient oxygen concentration; (3) prostaglandins do not take part in the H 2 S induced relaxations; (4) the 3':5'-cyclic guanosine monophosphate (cGMP)–nitric oxide (NO) pathway does not have a role in the relaxations (5) the role of K ATP channels is limited, while Cl − /HCO 3 − channels have a role in the relaxations. (6): We have observed that high concentrations of H 2 S relax the aortic rings in a fashion similar to sodium cyanide, and both agents reduce cellular ATP levels to a comparable degree. Significance H 2 S, a new gasotransmitter of emerging importance, leads to relaxation via Cl − /HCO 3 − channels and metabolic inhibition and the interactions of these two factors depend on the oxygen levels of the tissue.

Metabolic dysfunction is well-documented in Huntington’s disease (HD). However, the link between the mutant huntingtin (mHTT) gene and the pathology is unknown. The tricarboxylic acid (TCA) cycle is the main metabolic pathway for the production of NADH for conversion to ATP via the electron transport chain (ETC). The objective of this study was to test for differences in enzyme activities, mRNAs and protein levels related to the TCA cycle between lymphoblasts from healthy subjects and from patients with HD. The experiments utilize the advantages of lymphoblasts to reveal new insights about HD. The large quantity of homogeneous cell populations permits multiple dynamic measures to be made on exactly comparable tissues. The activities of nine enzymes related to the TCA cycle and the expression of twenty-nine mRNAs encoding for these enzymes and enzyme complexes were measured. Cells were studied under baseline conditions and during metabolic stress. The results support our recent findings that the activities of the pyruvate dehydrogenase complex (PDHC) and succinate dehydrogenase (SDH) are elevated in HD. The data also show a large unexpected depression in MDH activities. Furthermore, message levels for isocitrate dehydrogenase 1 (IDH1) were markedly increased in in HD lymphoblasts and were responsive to treatments. The use of lymphoblasts allowed us to clarify that the reported decrease in aconitase activity in HD autopsy brains is likely due to secondary hypoxic effects. These results demonstrate the mRNA and enzymes of the TCA cycle are critical therapeutic targets that have been understudied in HD.