The Influence of Metabolic Inhibitors, Antibiotics, and Microgravity on Intact Cell MALDI-TOF Mass Spectra of the Cyanobacterium Synechococcus Sp. UPOC S4.

The aim and novelty of this paper are found in assessing the influence of inhibitors and antibiotics on intact cell MALDI-TOF mass spectra of the cyanobacterium Synechococcus sp. UPOC S4 and to check the impact on reliability of identification. Defining the limits of this method is important for its use in biology and applied science. The compounds included inhibitors of respiration, glycolysis, citrate cycle, and proteosynthesis. They were used at 1-10 µM concentrations and different periods of up to 3 weeks. Cells were also grown without inhibitors in a microgravity because of expected strong effects. Mass spectra were evaluated using controls and interpreted in terms of differential peaks and their assignment to protein sequences by mass. Antibiotics, azide, and bromopyruvate had the greatest impact. The spectral patterns were markedly altered after a prolonged incubation at higher concentrations, which precluded identification in the database of reference spectra. The incubation in microgravity showed a similar effect. These differences were evident in dendrograms constructed from the spectral data. Enzyme inhibitors affected the spectra to a smaller extent. This study shows that only a long-term presence of antibiotics and strong metabolic inhibitors in the medium at 10-5 M concentrations hinders the correct identification of cyanobacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF).

Ethylmalonic acid impairs bioenergetics by disturbing succinate and glutamate oxidation and induces mitochondrial permeability transition pore opening in rat cerebellum.

Tissue accumulation and high urinary excretion of ethylmalonic acid (EMA) are found in ethylmalonic encephalopathy (EE), an inherited disorder associated with cerebral and cerebellar atrophy whose pathogenesis is poorly established. The in vitro and in vivo effects of EMA on bioenergetics and redox homeostasis were investigated in rat cerebellum. For the in vitro studies, cerebellum preparations were exposed to EMA, whereas intracerebellar injection of EMA was used for the in vivo evaluation. EMA reduced state 3 and uncoupled respiration in vitro in succinate-, glutamate-, and malate-supported mitochondria, whereas decreased state 4 respiration was observed using glutamate and malate. Furthermore, mitochondria permeabilization and succinate supplementation diminished the decrease in state 3 with succinate. EMA also inhibited the activity of KGDH, an enzyme necessary for glutamate oxidation, in a mixed manner and augmented mitochondrial efflux of α-ketoglutarate. ATP levels were markedly reduced by EMA, reflecting a severe bioenergetic disruption. Docking simulations also indicated interactions between EMA and KGDH and a competition with glutamate and succinate for their mitochondrial transporters. In vitro findings also showed that EMA decreased mitochondrial membrane potential and Ca2+ retention capacity, and induced swelling in the presence of Ca2+ , which were prevented by cyclosporine A and ADP and ruthenium red, indicating mitochondrial permeability transition (MPT). Moreover, EMA, at high concentrations, mildly increased ROS levels and altered antioxidant defenses in vitro and in vivo. Our data indicate that EMA-induced impairment of glutamate and succinate oxidation and MPT may contribute to the pathogenesis of the cerebellum abnormalities in EE.

Caffeic acid and caffeine attenuate toxicity associated with malonic or methylmalonic acid exposure in Drosophila melanogaster.

The deficiency in the activity of the mitochondrial enzyme methylmalonyl-CoA mutase (MCM, EC 5.4.99.2) leads to a condition called methylmalonic academia, which is characterised by the accumulation of methylmalonic (MMA), malonic (MA) or other organic acids. Importantly, we have recently found that supplementation with Ilex paraguariensis aqueous extract offered protection against toxicity associated with MMA or MA exposure to Drosophila melanogaster. Of note, caffeic acid (CA) and caffeine (CAF) were the major phytochemicals found in our Ilex paraguariensis crude extract. Therefore, here, we have exploited CA and/or CAF to test the hypothesis that supplementation with the isolated compounds (either alone or combined) could exert a protective effect against MMA or MA-induced toxicity in flies. Therefore, flies were exposed to MA (5 mM) or MMA (5 mM) and concomitantly treated with CA (1.39 µg/mL), CAF (1.27 µg/mL) or CA + CAF for 10 days for survival, and for 4 days for behavioural and biochemical assays. CA, CAF and CA + CAF treatments completely abolished the mortality associated with either MMA or MA exposure. Moreover, CA and CAF, either alone or combined, completely abolished behavioural changes, and completely protect against changes in thiobarbituric acid reactive substances (TBARS) levels, catalase (CAT) activity and MTT reduction ability, associated with MA or MMA exposure. In turn, CAF restored SOD activity in the head of flies exposed to MA or MMA. However, CA and CAF (either alone or combined) significantly decreased acetylcholinesterase (AChE) activity per se, while CAF alone protected from changes in AChE activity (in head tissue) associated with MA or MMA. Finally, CA and/or CAF were able to protect from a decrease in glucose and triglyceride levels associated with both MA and MMA exposures in haemolymph. Together, our data confirm the hypothesis that supplementation with CA and/or CAF offers protection against detrimental changes associated with MMA or MA exposure in flies, being responsible, at least in part, for the protective effect of I. paraguariensis crude extract which was reported previously.

A Reproducible New Model of Focal Ischemic Injury in the Marmoset Monkey: MRI and Behavioural Follow-Up.

Ischemic stroke mostly affects the primary motor cortex and descending motor fibres, with consequent motor impairment. Pre-clinical models of stroke with reproducible and long-lasting sensorimotor deficits in higher-order animals are lacking. We describe a new method to induce focal brain damage targeting the motor cortex to study damage to the descending motor tracts in the non-human primate. Stereotaxic injection of malonate into the primary motor cortex produced a focal lesion in middle-aged marmosets (Callithrix jacchus). Assessment of sensorimotor function using a neurological scale and testing of forelimb dexterity and strength lasted a minimum of 12 weeks. Lesion evolution was followed by magnetic resonance imaging (MRI) at 24 h, 1 week, 4 and 12 weeks post-injury and before sacrifice for immunohistochemistry. Our model produced consistent lesions of the motor cortex, subcortical white matter and caudate nucleus. All animals displayed partial spontaneous recovery with long lasting motor deficits of force (54% loss) and dexterity (≈ 70% loss). Clearly visible T2 hypointensity in the white matter was observed with MRI and corresponded to areas of chronic gliosis in the internal capsule and lenticular fasciculus. We describe a straightforward procedure to reproducibly injure the motor cortex in the marmoset monkey, causing long-lasting motor deficits. The MRI signature reflects Wallerian degeneration and remote injury of corticospinal and corticopontine tracts, as well as subcortical motor loops. Our model may be suitable for the testing of therapies for post-stroke recovery, particularly in the chronic phase.

Ilex paraguariensis Attenuates Changes in Mortality, Behavioral and Biochemical Parameters Associated to Methyl Malonate or Malonate Exposure in Drosophila melanogaster.

Methylmalonic acidemia is a genetic disease characterized by accumulation of organic acids, such as methylmalonic (MMA) and malonic (MA) acids. Considering that the accumulation of MMA and MA causes several damages due to oxidative stress, antioxidants are thought to play a pivotal role in preventing deleterious effects associated with exposure to such compounds. Ilex paraguariensis (IP) was used here to test the hypothesis that supplementation with the aqueous extract of this plant could exert protective effect against MMA or MA induced mortality, behavioral and/or biochemical changes in Drosophila melanogaster (DM). Initially, a curve time- and dose-response to MMA (1-10 mM), MA (1-10 mM) and IP (63-500 µM) was performed. Thereafter, flies were concomitantly exposed to MA (5 mM), MMA (5 mM) and/or IP (250 µg/mL) during 15 days for survival assay, and for 48 hs to MA (1 or 5 mM), MMA (1 or 5 mM) and/or IP (250 µg/mL) for subsequent investigations. Both MMA and MA exposure resulted in higher incidence of mortality, a worse performance in the negative geotaxis assay and increased locomotion in open-field test as compared with control group. Furthermore, a marked increase in non-protein thiol (NPSH) and in thiobarbituric acid reactive substances (TBARS) levels, decrease in superoxide dismutase (SOD), catalase and acetylcholinesterase (AChE) activities, and decrease in MTT and resazurin reduction were noted in MMA or MA treated groups. IP treatment offered significant protection against all alterations associated to MMA or MA exposure. This study confirm the hypothesis that supplementation with IP offers protection against changes associated to MMA or MA exposure in DM, due, at least in part, to its antioxidant effect.

Respiratory complex II in mitochondrial dysfunction-mediated cytotoxicity: Insight from cadmium.

In the present work we studied action of several inhibitors of respiratory complex II (CII) of mitochondrial electron transport chain, namely malonate and thenoyltrifluoroacetone (TTFA) on Cd2+-induced toxicity and cell mortality, using two rat cell lines, pheochromocytoma PC12 and ascites hepatoma AS-30D and isolated rat liver mitochondria (RLM). It was shown that malonate, an endogenous competitive inhibitor of dicarboxylate-binding site of CII, restored in part RLM respiratory function disturbed by Cd2+. In particular, malonate increased both phosphorylating and maximally uncoupled respiration rates in KCl medium in the presence of CI substrates as well as palliated changes in basal and resting state respiration rates produced by the heavy metal on the mitochondria energized by CI or CII substrates. Notably, malonate enhanced Cd2+-induced swelling of the mitochondria energized by CI substrates in KCl and, in a much lesser extent and at higher [Cd2+], in sucrose media but did not influence on the Cd2+ effects in NaCl medium. Besides, malonate did not affect swelling in sucrose media of RLM energized by CIV substrates under using of Cd2+ or Ca2+ whereas it strongly increased the mitochondrial swelling produced by selenite. In addition, malonate produced some protection against Cd2+-promoted necrotic death of AS-30D and PC12 cells and reduced intracellular reactive oxygen species (ROS) formation evoked by Cd2+ in PC12 cells. Importantly, TTFA, an irreversible competitive inhibitor of Q-binding site of CII, per se induced apoptosis of AS-30D cells which was inhibited by co-treatment with Cd2+ as well as decreased the Cd2+-enhanced intracellular ROS formation. In turn, decylubiquinone (dUb) at low µM concentrations did not protect AS-30D cells against the Cd2+-induced necrosis and enhanced the Cd2+-induced apoptosis of the cells. High µM concentrations of dUb were highly toxic for the cells. As consequence, the findings give new evidence indicative of critical involvement of CII in mechanism(s) of Cd2+-produced cytotoxicity and support the notion on CII as a perspective pharmacological target in mitochondria dysfunction-mediated conditions and diseases.

Chemical warfare agent simulants for human volunteer trials of emergency decontamination: A systematic review.

Incidents involving the release of chemical agents can pose significant risks to public health. In such an event, emergency decontamination of affected casualties may need to be undertaken to reduce injury and possible loss of life. To ensure these methods are effective, human volunteer trials (HVTs) of decontamination protocols, using simulant contaminants, have been conducted. Simulants must be used to mimic the physicochemical properties of more harmful chemicals, while remaining non-toxic at the dose applied. This review focuses on studies that employed chemical warfare agent simulants in decontamination contexts, to identify those simulants most suitable for use in HVTs of emergency decontamination. Twenty-two simulants were identified, of which 17 were determined unsuitable for use in HVTs. The remaining simulants (n = 5) were further scrutinized for potential suitability according to toxicity, physicochemical properties and similarities to their equivalent toxic counterparts. Three suitable simulants, for use in HVTs were identified; methyl salicylate (simulant for sulphur mustard), diethyl malonate (simulant for soman) and malathion (simulant for VX or toxic industrial chemicals). All have been safely used in previous HVTs, and have a range of physicochemical properties that would allow useful inference to more toxic chemicals when employed in future studies of emergency decontamination systems.

Comparison of glutathione levels measured using optimized monochlorobimane assay with those from ortho-phthalaldehyde assay in intact cells.

Fluorometric glutathione assays have been generally preferred for their high specificity and sensitivity. An additional advantage offered by fluorescent bimane dyes is their ability to penetrate inside the cell. Their ability to react with glutathione within intact cells is frequently useful in flow cytometry and microscopy. Hence, the aims of our study were to use monochlorobimane for optimizing a spectrofluorometric glutathione assay in cells and then to compare that assay with the frequently used ortho-phthalaldehyde assay. We used glutathione-depleting agents (e.g., cisplatin and diethylmalonate) to induce cell impairment. For glutathione assessment, monochlorobimane (40µM) was added to cells and fluorescence was detected at 394/490nm. In addition to the regularly used calculation of glutathione levels from fluorescence change after 60min, we used an optimized calculation from the linear part of the fluorescence curve after 10min of measurement. We found that 10min treatment of cells with monochlorobimane is sufficient for evaluating cellular glutathione concentration and provides results entirely comparable with those from the standard ortho-phthalaldehyde assay. In contrast, the results obtained by the standardly used evaluation after 60min of monochlorobimane treatment provided higher glutathione values. We conclude that measuring glutathione using monochlorobimane with the here-described optimized evaluation of fluorescence signal could be a simple and useful method for routine and rapid assessment of glutathione within intact cells in large numbers of samples.

Evaluation of absorbent materials for use as ad hoc dry decontaminants during mass casualty incidents as part of the UK's Initial Operational Response (IOR).

The UK's Initial Operational Response (IOR) is a revised process for the medical management of mass casualties potentially contaminated with hazardous materials. A critical element of the IOR is the introduction of immediate, on-scene disrobing and decontamination of casualties to limit the adverse health effects of exposure. Ad hoc cleansing of the skin with dry absorbent materials has previously been identified as a potential means of facilitating emergency decontamination. The purpose of this study was to evaluate the in vitro oil and water absorbency of a range of materials commonly found in the domestic and clinical environments and to determine the effectiveness of a small, but representative selection of such materials in skin decontamination, using an established ex vivo model. Five contaminants were used in the study: methyl salicylate, parathion, diethyl malonate, phorate and potassium cyanide. In vitro measurements of water and oil absorbency did not correlate with ex vivo measurements of skin decontamination. When measured ex vivo, dry decontamination was consistently more effective than a standard wet decontamination method ("rinse-wipe-rinse") for removing liquid contaminants. However, dry decontamination was ineffective against particulate contamination. Collectively, these data confirm that absorbent materials such as wound dressings and tissue paper provide an effective, generic capability for emergency removal of liquid contaminants from the skin surface, but that wet decontamination should be used for non-liquid contaminants.

Caenorhabditis elegans chronically exposed to a Mn/Zn ethylene-bis-dithiocarbamate fungicide show mitochondrial Complex I inhibition and increased reactive oxygen species.

Reports have linked human exposure to Mn/Zn ethylene-bis-dithiocarbamate (Mn/Zn-EBDC) fungicides with multiple pathologies, from dermatitis to central nervous system dysfunction. Although members of this family of agrochemicals have been available for over 50 years, their mechanism of toxicity in humans is still unclear. Since mitochondrial inhibition and oxidative stress are implicated in a wide variety of diseases, we hypothesized that Caenorhabditis elegans (C. elegans) exposed to a commercially-available formulation of an Mn/Zn-EBDC-containing fungicide (Manzate; MZ) would also show these endpoints. Thus, worms were treated chronically (24h) with various MZ concentrations and assayed for reduced mitochondrial function and increased levels of reactive oxygen species (ROS). Oxygen consumption studies suggested Complex I inhibition in all treatment groups compared to controls (**p<0.01). In order to verify these findings, assays specific for Complex II or Complex IV activity were also completed. Data analysis from these studies indicated that neither complex was adversely affected by MZ treatment. Additional data from ATP assays indicated a statistically significant decrease (***p<0.001) in ATP levels in all treatment groups when compared to control worms. Further studies were completed to determine if exposure of C. elegans to MZ also resulted in increased ROS concentrations. Studies demonstrated that hydrogen peroxide, but not superoxide or hydroxyl radical, levels were statistically significantly increased (*p<0.05). Since hydrogen peroxide is known to up-regulate glutathione-S-transferase (GST), we used a GST:green fluorescent protein transgenic worm strain to test this hypothesis. Results from these studies indicated a statistically significant increase (***p<0.001) in green pixel number following MZ exposure. Taken together, these data indicate that C. elegans treated with MZ concentrations to which humans are exposed show mitochondrial Complex I inhibition with concomitant hydrogen peroxide production. Since these mechanisms are associated with numerous human diseases, we suggest further studies to determine if MZ exposure induces similar toxic mechanisms in mammals.

Composites of malonic acid diamides and phospholipids--Impact of lipoplex stability on transfection efficiency.

The use of cationic lipids as gene delivery systems is a basic method in gene therapy. Through ongoing research, lipofection is currently the leader of non-viral vectors in clinical trials. However, in order to unleash the full potential of lipofection further intensive investigations are indispensable. In this study, various lipoplex formulations were compared regarding their ability to bind DNA. To obtain information about a possible premature release of DNA at the cell surface, heparin and chondroitin dependent lipoplex destabilization experiments were carried out. Complementary investigations in cell culture were performed to quantify DNA outside the cell. Additionally, DNase I stability was investigated. In this regard a multitude of methods, namely confocal laser scanning microscopy (CLSM), polymerase chain reaction (PCR), cell culture experiments, ethidium bromide assay, gel electrophoresis, Langmuir-isotherm experiments, infrared reflection absorption spectroscopy (IRRAS), Brewster angle microscopy (BAM), zeta-(ζ)-potential measurements, and dynamic light scattering (DLS), were applied. Although the complexation of DNA is a fundamental step, we show that the DNA release by biological agents (proteoglycans) and an unsuccessful cell attachment are major transfection limiting parameters.

Strength and fine dexterity recovery profiles after a primary motor cortex insult and effect of a neuronal cell graft.

The aim of this study was to set up (a) a large primary motor cortex (M1) lesion in rodent and (b) the conditions for evaluating a long-lasting motor deficit in order to propose a valid model to test neuronal replacement therapies aimed at improving motor deficit recovery. A mitochondrial toxin, malonate, was injected to induce extensive destruction of the forelimb M1 cortex. Three key motor functions that are usually evaluated following cerebral lesion in the clinic-strength, target reaching, and fine dexterity-were assessed in rats by 2 tests, a forelimb grip strength test and a skilled reaching task (staircase) for reaching and dexterity. The potential enhancement of postlesion recovery induced by a neuronal cell transplantation was then explored and confirmed by histological analyses. Both tests showed a severe functional impairment 2 days post lesion, however, reaching remained intact. Deficits in forelimb strength were long lasting (up to 3 months) but spontaneously recovered despite the extensive lesion size. This natural grip strength recovery could be enhanced by cell therapy. Histological analyses confirmed the presence of grafted cells 3 months postgraft and showed partial tissue reconstruction with some living neuronal cells in the graft. In contrast, fine dexterity never recovered in the staircase test even after grafting. These results suggest that cell replacement was only partially effective and that the forelimb M1 area may be a node of the sensorimotor network, where compensation from secondary pathways could account for strength recovery but recovery of forelimb fine dexterity requires extensive tissue reconstruction.

The inhibition of 2-arachidonoyl-glycerol (2-AG) biosynthesis, rather than enhancing striatal damage, protects striatal neurons from malonate-induced death: a potential role of cyclooxygenase-2-dependent metabolism of 2-AG.

The cannabinoid CB2 receptor, which is activated by the endocannabinoid 2-arachidonoyl-glycerol (2-AG), protects striatal neurons from apoptotic death caused by the local administration of malonate, a rat model of Huntington's disease (HD). In the present study, we investigated whether endocannabinoids provide tonic neuroprotection in this HD model, by examining the effect of O-3841, an inhibitor of diacylglycerol lipases, the enzymes that catalyse 2-AG biosynthesis, and JZL184 or OMDM169, two inhibitors of 2-AG inactivation by monoacylglycerol lipase (MAGL). The inhibitors were injected in rats with the striatum lesioned with malonate, and several biochemical and morphological parameters were measured in this brain area. Similar experiments were also conducted in vitro in cultured M-213 cells, which have the phenotypic characteristics of striatal neurons. O-3841 produced a significant reduction in the striatal levels of 2-AG in animals lesioned with malonate. However, surprisingly, the inhibitor attenuated malonate-induced GABA and BDNF deficiencies and the reduction in Nissl staining, as well as the increase in GFAP immunostaining. In contrast, JZL184 exacerbated malonate-induced striatal damage. Cyclooxygenase-2 (COX-2) was induced in the striatum 24 h after the lesion simultaneously with other pro-inflammatory responses. The COX-2-derived 2-AG metabolite, prostaglandin E2 glyceryl ester (PGE2-G), exacerbated neurotoxicity, and this effect was antagonized by the blockade of PGE2-G action with AGN220675. In M-213 cells exposed to malonate, in which COX-2 was also upregulated, JZL184 worsened neurotoxicity, and this effect was attenuated by the COX-2 inhibitor celecoxib or AGN220675. OMDM169 also worsened neurotoxicity and produced measurable levels of PGE2-G. In conclusion, the inhibition of 2-AG biosynthesis is neuroprotective in rats lesioned with malonate, possibly through the counteraction of the formation of pro-neuroinflammatory PGE2-G, formed from COX-2-mediated oxygenation of 2-AG. Accordingly, MAGL inhibition or the administration of PGE2-G aggravates the malonate toxicity.

Inhibition of calpain-regulated p35/cdk5 plays a central role in sildenafil-induced protection against chemical hypoxia produced by malonate.

Phosphodiesterase 5 (PDE5) inhibitors have recently been reported to exert beneficial effects against ischemia-reperfusion injury in several organs but their neuroprotective effects in brain stroke models are scarce. The present study was undertaken to assess the effects of sildenafil against cell death caused by intrastriatal injection of malonate, an inhibitor of succinate dehydrogenase; which produces both energy depletion and lesions similar to those seen in cerebral ischemia. Our data demonstrate that sildenafil (1.5mg/kg by mouth (p.o.)), given 30min before malonate (1.5µmol/2µL), significantly decreased the lesion volume caused by this toxin. This protective effect can be probably related to the inhibition of excitotoxic pathways. Thus, malonate induced the activation of the calcium-dependent protease, calpain and the cyclin-dependent kinase 5, cdk5; which resulted in the hyperphosphorylation of tau and the cleavage of the protective transcription factor, myocyte enhancer factor 2, MEF2. All these effects were also significantly reduced by sildenafil pre-treatment, suggesting that sildenafil protects against malonate-induced cell death through the regulation of the calpain/p25/cdk5 signaling pathway. Similar findings were obtained using inhibitors of calpain or cdk5, further supporting our contention. Sildenafil also increased MEF2 phosphorylation and Bcl-2/Bax and Bcl-xL/Bax ratios, effects that might as well contribute to prevent cell death. Finally, sildenafil neuroprotection was extended not only to rat hippocampal slices subjected to oxygen and glucose deprivation when added at the time of reoxygenation, but also, in vivo when administered after malonate injection. Thus, the therapeutic window for sildenafil against malonate-induced hypoxia was set at 3h.

Sativex-like combination of phytocannabinoids is neuroprotective in malonate-lesioned rats, an inflammatory model of Huntington's disease: role of CB1 and CB2 receptors.

We have investigated whether a 1:1 combination of botanical extracts enriched in either Δ(9)-tetrahydrocannabinol (Δ(9)-THC) or cannabidiol (CBD), which are the main constituents of the cannabis-based medicine Sativex, is neuroprotective in Huntington's disease (HD), using an experimental model of this disease generated by unilateral lesions of the striatum with the mitochondrial complex II inhibitor malonate. This toxin damages striatal neurons by mechanisms that primarily involve apoptosis and microglial activation. We monitored the extent of this damage and the possible preservation of the striatal parenchyma by treatment with a Sativex-like combination of phytocannabinoids using different histological and biochemical markers. Results were as follows: (i) malonate increased the volume of edema measured by in vivo NMR imaging and the Sativex-like combination of phytocannabinoids partially reduced this increase; (ii) malonate reduced the number of Nissl-stained cells, while enhancing the number of degenerating cells stained with FluoroJade-B, and the Sativex-like combination of phytocannabinoids reversed both effects; (iii) malonate caused a strong glial activation (i.e., reactive microglia labeled with Iba-1, and astrogliosis labeled with GFAP) and the Sativex-like combination of phytocannabinoids attenuated both responses; and (iv) malonate increased the expression of inducible nitric oxide synthase and the neurotrophin IGF-1, and both responses were attenuated after the treatment with the Sativex-like combination of phytocannabinoids. We also wanted to establish whether targets within the endocannabinoid system (i.e., CB(1) and CB(2) receptors) are involved in the beneficial effects induced in this model by the Sativex-like combination of phytocannabinoids. This we did using selective antagonists for both receptor types (i.e., SR141716 and AM630) combined with the Sativex-like phytocannabinoid combination. Our results indicated that the effects of this combination are blocked by these antagonists and hence that they do result from an activation of both CB(1) and CB(2) receptors. In summary, this study provides preclinical evidence in support of a beneficial effect of the cannabis-based medicine Sativex as a neuroprotective agent capable of delaying signs of disease progression in a proinflammatory model of HD, which adds to previous data obtained in models priming oxidative mechanisms of striatal injury. However, the interest here is that, in contrast with these previous data, we have now obtained evidence that both CB(1) and CB(2) receptors appear to be involved in the effects produced by a Sativex-like phytocannabinoid combination, thus stressing the broad-spectrum properties of Sativex that may combine activity at the CB(1) and/or CB(2) receptors with cannabinoid receptor-independent actions.

An erythromycin process improvement using the diethyl methylmalonate-responsive (Dmr) phenotype of the Saccharopolyspora erythraea mutB strain.

The Saccharopolyspora erythraea mutB knockout strain, FL2281, having a block in the methylmalonyl-CoA mutase reaction, was found to carry a diethyl methylmalonate-responsive (Dmr) phenotype in an oil-based fermentation medium. The Dmr phenotype confers the ability to increase erythromycin A (erythromycin) production from 250-300% when the oil-based medium is supplemented with 15 mM levels of this solvent. Lower concentrations of the solvent stimulated proportionately less erythromycin production, while higher concentrations had no additional benefit. Although the mutB strain is phenotypically a low-level erythromycin producer, diethyl methylmalonate supplementation allowed it to produce up to 30% more erythromycin than the wild-type (control) strain-a strain that does not show the Dmr phenotype. The Dmr phenotype represents a new class of strain improvement phenotype. A theory to explain the biochemical mechanism for the Dmr phenotype is proposed. Other phenotypes found to be associated with the mutB knockout were a growth defect and hyper-pigmentation, both of which were restored to normal by exposure to diethyl methylmalonate. Furthermore, mutB fermentations did not significantly metabolize soybean oil in the presence of diethyl methylmalonate. Finally, a novel method is proposed for the isolation of additional mutants with the Dmr phenotype.

Cystamine and ethyl-eicosapentaenoic acid treatment fail to prevent malonate-induced striatal toxicity in mice.

Cystamine has demonstrated neuroprotective activity in a variety of studies, and is currently being evaluated in a human clinical trial in Huntington's disease (HD). Cystamine treatment of various genetic models of HD demonstrated protection against neurodegeneration and/or improvement in behavior. Given the need for a rapid screening tool for HD therapeutics, we assessed the potential therapeutic benefits of cystamine in a short-term acute toxicity murine model of striatal cell death. Cystamine did not provide neuroprotection against bilateral intrastriatal malonate injections in mice as measured by lesion size, loss of striatal volume, or decreased striatal neuronal counts. Similar results were obtained for treatment with another potential therapeutic agent that was protective in genetic mouse models of HD, the essential fatty acid ethyl-eicosapentaenoic acid. Our findings suggest that this toxic model is not reflective or predictive of findings in genetic mouse models, and may not be useful as a preclinical screen for HD therapeutics.

Malonyl-CoA synthetase, encoded by ACYL ACTIVATING ENZYME13, is essential for growth and development of Arabidopsis.

Malonyl-CoA is the precursor for fatty acid synthesis and elongation. It is also one of the building blocks for the biosynthesis of some phytoalexins, flavonoids, and many malonylated compounds. In plants as well as in animals, malonyl-CoA is almost exclusively derived from acetyl-CoA by acetyl-CoA carboxylase (EC 6.4.1.2). However, previous studies have suggested that malonyl-CoA may also be made directly from malonic acid by malonyl-CoA synthetase (EC 6.2.1.14). Here, we report the cloning of a eukaryotic malonyl-CoA synthetase gene, Acyl Activating Enzyme13 (AAE13; At3g16170), from Arabidopsis thaliana. Recombinant AAE13 protein showed high activity against malonic acid (K(m) = 529.4 ± 98.5 µM; V(m) = 24.0 ± 2.7 µmol/mg/min) but little or no activity against other dicarboxylic or fatty acids tested. Exogenous malonic acid was toxic to Arabidopsis seedlings and caused accumulation of malonic and succinic acids in the seedlings. aae13 null mutants also grew poorly and accumulated malonic and succinic acids. These defects were complemented by an AAE13 transgene or by a bacterial malonyl-CoA synthetase gene under control of the AAE13 promoter. Our results demonstrate that the malonyl-CoA synthetase encoded by AAE13 is essential for healthy growth and development, probably because it is required for the detoxification of malonate.