Effects of lithium and valproic acid on gene expression and phenotypic markers in an NT2 neurosphere model of neural development

Mood stabilising drugs such as lithium (LiCl) and valproic acid (VPA) are the first line agents for treating conditions such as Bipolar disorder and Epilepsy. However, these drugs have potential developmental effects that are not fully understood. This study explores the use of a simple human neurosphere-based in vitro model to characterise the pharmacological and toxicological effects of LiCl and VPA using gene expression changes linked to phenotypic alterations in cells. Treatment with VPA and LiCl resulted in the differential expression of 331 and 164 genes respectively. In the subset of VPA targeted genes, 114 were downregulated whilst 217 genes were upregulated. In the subset of LiCl targeted genes, 73 were downregulated and 91 were upregulated. Gene ontology (GO) term enrichment analysis was used to highlight the most relevant GO terms associated with a given gene list following toxin exposure. In addition, in order to phenotypically anchor the gene expression data, changes in the heterogeneity of cell subtype populations and cell cycle phase were monitored using flow cytometry. Whilst LiCl exposure did not significantly alter the proportion of cells expressing markers for stem cells/undifferentiated cells (Oct4, SSEA4), neurons (Neurofilament M), astrocytes (GFAP) or cell cycle phase, the drug caused a 1.4-fold increase in total cell number. In contrast, exposure to VPA resulted in significant upregulation of Oct4, SSEA, Neurofilament M and GFAP with significant decreases in both G2/M phase cells and cell number. This neurosphere model might provide the basis of a human-based cellular approach for the regulatory exploration of developmental impact of potential toxic chemicals.

Angiotensin II directly induces muscle protein catabolism through the ubiquitin-proteasome proteolytic pathway and may play a role in cancer cachexia

The ability of angiotensin I (Ang I) and II (Ang II) to induce directly protein degradation in skeletal muscle has been studied in murine myotubes. Angiotensin I stimulated protein degradation with a parabolic dose-response curve and with a maximal effect between 0.05 and 0.1 μM. The effect was attenuated by coincubation with the angiotensin-converting enzyme (ACE) inhibitor imidaprilat, suggesting that angiotensin I stimulated protein degradation through conversion to Ang II. Angiotensin II also stimulated protein breakdown with a similar dose-response curve, and with a maximal effect between 1 and 2.5 μM. Total protein degradation, induced by both Ang I and Ang II, was attenuated by the proteasome inhibitors lactacystin (5 μM) and MG132 (10 μM), suggesting that the effect was mediated through upregulation of the ubiquitin-proteasome proteolytic pathway. Both Ang I and Ang II stimulated an increased proteasome 'chymotrypsin-like' enzyme activity as well as an increase in protein expression of 20S proteasome α-subunits, the 19S subunits MSSI and p42, at the same concentrations as those inducing protein degradation. The effect of Ang I was attenuated by imidaprilat, confirming that it arose from conversion to Ang II. These results suggest that Ang II stimulates protein degradation in myotubes through induction of the ubiquitin-proteasome pathway. Protein degradation induced by Ang II was inhibited by insulin-like growth factor and by the polyunsaturated fatty acid, eicosapentaenoic acid. These results suggest that Ang II has the potential to cause muscle atrophy through an increase in protein degradation. The highly lipophilic ACE inhibitor imidapril (Vitor™) (30 mg kg-1) attenuated the development of weight loss in mice bearing the MAC16 tumour, suggesting that Ang II may play a role in the development of cachexia in this model. © 2005 Cancer Research.

Blockade of interleukin-6 signaling inhibits the classic pathway and promotes an alternative pathway of macrophage activation after spinal cord injury in mice

Background Recent in vivo and in vitro studies in non-neuronal and neuronal tissues have shown that different pathways of macrophage activation result in cells with different properties. Interleukin (IL)-6 triggers the classically activated inflammatory macrophages (M1 phenotype), whereas the alternatively activated macrophages (M2 phenotype) are anti-inflammatory. The objective of this study was to clarify the effects of a temporal blockade of IL-6/IL-6 receptor (IL-6R) engagement, using an anti-mouse IL-6R monoclonal antibody (MR16-1), on macrophage activation and the inflammatory response in the acute phase after spinal cord injury (SCI) in mice. Methods MR16-1 antibodies versus isotype control antibodies or saline alone were administered immediately after thoracic SCI in mice. SC tissue repair was compared between the two groups by Luxol fast blue (LFB) staining for myelination and immunoreactivity for the neuronal markers growth-associated protein (GAP)-43 and neurofilament heavy 200 kDa (NF-H) and for locomotor function. The expression of T helper (Th)1 cytokines (interferon (IFN)-? and tumor necrosis factor-a) and Th2 cytokines (IL-4, IL-13) was determined by immunoblot analysis. The presence of M1 (inducible nitric oxide synthase (iNOS)-positive, CD16/32-positive) and M2 (arginase 1-positive, CD206-positive) macrophages was determined by immunohistology. Using flow cytometry, we also quantified IFN-? and IL-4 levels in neutrophils, microglia, and macrophages, and Mac-2 (macrophage antigen-2) and Mac-3 in M2 macrophages and microglia. Results LFB-positive spared myelin was increased in the MR16-1-treated group compared with the controls, and this increase correlated with enhanced positivity for GAP-43 or NF-H, and improved locomotor Basso Mouse Scale scores. Immunoblot analysis of the MR16-1-treated samples identified downregulation of Th1 and upregulation of Th2 cytokines. Whereas iNOS-positive, CD16/32-positive M1 macrophages were the predominant phenotype in the injured SC of non-treated control mice, MR16-1 treatment promoted arginase 1-positive, CD206-positive M2 macrophages, with preferential localization of these cells at the injury site. MR16-1 treatment suppressed the number of IFN-?-positive neutrophils, and increased the number of microglia present and their positivity for IL-4. Among the arginase 1-positive M2 macrophages, MR16-1 treatment increased positivity for Mac-2 and Mac-3, suggestive of increased phagocytic behavior. Conclusion The results suggest that temporal blockade of IL-6 signaling after SCI abrogates damaging inflammatory activity and promotes functional recovery by promoting the formation of alternatively activated M2 macrophages.

Expression of the ubiquitin-proteasome pathway and muscle loss in experimental cancer cachexia

Muscle protein degradation is thought to play a major role in muscle atrophy in cancer cachexia. To investigate the importance of the ubiquitin-proteasome pathway, which has been suggested to be the main degradative pathway mediating progressive protein loss in cachexia, the expression of mRNA for proteasome subunits C2 and C5 as well as the ubiquitin-conjugating enzyme, E2(14k), has been determined in gastrocnemius and pectoral muscles of mice bearing the MAC16 adenocarcinoma, using competitive quantitative reverse transcriptase polymerase chain reaction. Protein levels of proteasome subunits and E2(14k) were determined by immunoblotting, to ensure changes in mRNA were reflected in changes in protein expression. Muscle weights correlated linearly with weight loss during the course of the study. There was a good correlation between expression of C2 and E2(14k) mRNA and protein levels in gastrocnemius muscle with increases of 6-8-fold for C2 and two-fold for E2(14k) between 12 and 20% weight loss, followed by a decrease in expression at weight losses of 25-27%, although loss of muscle protein continued. In contrast, expression of C5 mRNA only increased two-fold and was elevated similarly at all weight losses between 7.5 and 27%. Both proteasome functional activity, and proteasome-specific tyrosine release as a measure of total protein degradation was also maximal at 18-20% weight loss and decreased at higher weight loss. Proteasome expression in pectoral muscle followed a different pattern with increases in C2 and C5 and E2(14k) mRNA only being seen at weight losses above 17%, although muscle loss increased progressively with increasing weight loss. These results suggest that activation of the ubiquitin-proteasome pathway plays a major role in protein loss in gastrocnemius muscle, up to 20% weight loss, but that other factors such as depression in protein synthesis may play a more important role at higher weight loss. © 2005 Cancer Research.

Effects of glutathione, N-acetyl-cysteine, α-lipoic acid and dihydrolipoic acid on the cytotoxicity of a 2-pyridylcarboxamidrazone antimycobacterial agent in human mononuclear leucocytes in vitro

A series of antioxidants was used to explore the cytotoxicity of one particularly toxic antimycobacterial 2-pyridylcarboxamidrazone anti-tuberculosis agent against human mononuclear leucocytes (MNL), in comparison with isoniazid (INH) to aid future compound design. INH caused a significant reduction of nearly 40% in cell recovery compared with control (P < 0.0001), although the co-incubation with either glutathione (GSH, 1 mM) or (NAC, 1 mM) showed abolition of INH toxicity. In contrast, the addition of GSH or NAC 1 h after INH failed to protect the cells from INH toxicity (P < 0.0001). The 2-pyridyl-carboxamidrazone 'Compound 1' caused a 50% reduction in cell recovery compared with control (P < 0.001), although this was abolished by the presence of either GSH or NAC. A 1 h post incubation with either NAC or GSH after Compound 1 addition failed to protect the cells from toxicity (P < 0.001). Co-administration of lipoic acid (LA) abolished Compound 1-mediated toxicity, although again, this effect did not occur after LA addition 1 h post incubation with Compound 1 (P < 0.001). However, co-administration of dihydrolipoic acid (DHLA) prevented Compound 1-mediated cell death when incubated with the compound and also after 1 h of Compound 1 alone. Pre-treatment with GSH, then removal of the antioxidant resulted in abolition of Compound 1 toxicity (vehicle control, 63.6 ± 16.7 versus Compound 1 alone 26.1 ± 13.6% versus GSH pre-treatment, 65.7 ± 7.3%). In a cell-free incubation, NMR analysis revealed that GSH does not react with Compound 1, indicating that this agent is not likely to directly deplete membrane thiols. Compound 1's MNL toxicity is more likely to be linked with changes in cell membrane conformation, which may induce consequent thiol depletion that is reversible by exogenous thiols. © 2004 Elsevier B.V. All rights reserved.

The influence of bird droppings and uric acid on the radial growth of five species of saxicolous lichens

Bird droppings were applied over 1 year as a thick paste and as a suspension in deionized water to five species of lichens with different distributions on and off bird perching stones. The paste and suspension increased the radial growth of Parmelia conspersa while the paste increased the growth of Xanthoria parietina and reduced the growth and caused loss of colour in Parmelia glabratula ssp. fuliginosa. There were no statistically significant effects of paste or suspension on the growth of Physcia grisea or Parmelia saxatilis. In P. conspersa and X. parietina the growth responses were similar through the year but in P. glabratula the inhibitory effect of the paste was significant after 8 months growth. Application of a suspension of uric acid over 1 year had no statistically significant effects on the growth of P. conspersa, P. glabratula or X. parietina and was unlikely to be responsible for the effects of bird droppings on growth. The growth responses of the five species agreed well with their distributions in the field.

Effects of lipoic acid and dihydrolipoic acid on total erythrocytic thiols under conditions of restricted glucose in vitro

The effects of lipoic acid and dihydrolipoic acid were explored on total thiol maintenance in diabetic and non-diabetic human erythrocytes in vitro over 22 hr in a 37°C incubation system with no added glucose. Over 18-22.5 hr after treatment in both non-diabetic and diabetic cells, lipoic acid (1 mM) was associated with greater loss of cellular thiols than dihydrolipoic acid (1 mM), compared to respective control values. At 0.1 mM, in non-diabetic cells, although lipoic acid-treated cells' thiol levels were significantly lower than control, there was no significant difference between dihydrolipoic acid-treated cells and control cells regarding thiol levels. In addition, at 0.1 mM, dihydrolipoic acid-treated diabetic cells showed a reduction in thiol levels compared to control. At 0.01 mM, lipoic acid-treated cells had significantly lower measured thiol levels compared with diabetic cells exposed to dihydrolipoic acid, whereas in non-diabetic cells, dihydrolipoic acid-treated erythrocytic thiol levels were significantly greater than those treated with lipoic acid, although there were no other significant differences between the groups. At 22.5 hr, control values of methaemoglobin rose to 6.4 ± 1.1% in diabetic cells and 3.6 ± 2.1% in non-diabetic cells. Lipoic acid (1 mM) showed greater methaemoglobin formation in diabetic rather than non-diabetic cells (13.6 ± 1.5% versus 11.6 ± 1.5%), whereas dihydrolipoic acid-treated diabetic and non-diabetic cells were less potent in methaemoglobin generation (8.5 ± 2.4% and 8.4 ± 1.4%, respectively). These studies suggest that in certain circumstances such as hypoglycaemia, lipoic acid administration may actually be detrimental to cellular oxidant protection status. © 2006 The Authors.

Effects of lipoic acid and dihydrolipoic acid on 4-aminophenol-mediated erythrocytic toxicity in vitro

The effects of the antioxidant lipoic acid and its reduced form, dihydrolipoic acid (DHLA), were studied on the process of the erythrocytic toxicity of 4-aminophenol in human erythrocytes in vitro. 4-Aminophenol alone caused a stepwise increase in methaemoglobin formation, along with a commensurate decrease in total thiols. At 10 min., in the presence of lipoic acid alone and the thiol depletor 1-chloro-2,4-dinitrobenzene (CDNB) alone, 4-aminophenol-mediated methaemoglobin formation was significantly increased, whilst thiol levels were significantly reduced compared with the 4-aminophenol alone. At 10 min., with DHLA and CDNB alone, 4-aminophenol was associated with significantly increased methaemoglobin formation. However, thiol levels were not significantly different in the presence of DHLA compared with 4-aminophenol alone, although thiol levels were different compared with control (4-aminophenol alone) in the incubations with CDNB alone. At 15 min., only CDNB/4-aminophenol methaemoglobin formation differed from control, whilst thiol levels were significantly lower in the presence of CDNB alone compared with 4-aminophenol alone. Lipoic acid enhanced the toxicity of 4-aminophenol in terms of increased methaemoglobin formation coupled with increased thiol depletion, whilst DHLA showed increased 4-aminophenol-mediated methaemoglobin formation without thiol depletion. Lipoic acid, and to a lesser extent its reduced derivative DHLA, acted as a prooxidant in the presence of 4-aminophenol, enhancing the oxidative stress effects of the amine in human erythrocytes. © Basic & Clinical Pharmacology & Toxicology 2006.

Kinetic study of the pyrolysis of miscanthus and its acid hydrolysis residue by thermogravimetric analysis

The kinetic parameters of the pyrolysis of miscanthus and its acid hydrolysis residue (AHR) were determined using thermogravimetric analysis (TGA). The AHR was produced at the University of Limerick by treating miscanthus with 5 wt.% sulphuric acid at 175 °C as representative of a lignocellulosic acid hydrolysis product. For the TGA experiments, 3 to 6 g of sample, milled and sieved to a particle size below 250 μm, were placed in the TGA ceramic crucible. The experiments were carried out under non-isothermal conditions heating the samples from 50 to 900 °C at heating rates of 2.5, 5, 10, 17 and 25 °C/min. The activation energy (EA) of the decomposition process was determined from the TGA data by differential analysis (Friedman) and three isoconversional methods of integral analysis (Kissinger–Akahira–Sunose, Ozawa–Flynn–Wall, Vyazovkin). The activation energy ranged from 129 to 156 kJ/mol for miscanthus and from 200 to 376 kJ/mol for AHR increasing with increasing conversion. The reaction model was selected using the non-linear least squares method and the pre-exponential factor was calculated from the Arrhenius approximation. The results showed that the best fitting reaction model was the third order reaction for both feedstocks. The pre-exponential factor was in the range of 5.6 × 1010 to 3.9 × 10+ 13 min− 1 for miscanthus and 2.1 × 1016 to 7.7 × 1025 min− 1 for AHR.

Thermal processing of miscanthus, sugarcane bagasse, sugarcane trash and their acid hydrolysis residues

The research presented in this thesis was developed as part of DIBANET, an EC funded project aiming to develop an energetically self-sustainable process for the production of diesel miscible biofuels (i.e. ethyl levulinate) via acid hydrolysis of selected biomass feedstocks. Three thermal conversion technologies, pyrolysis, gasification and combustion, were evaluated in the present work with the aim of recovering the energy stored in the acid hydrolysis solid residue (AHR). Mainly consisting of lignin and humins, the AHR can contain up to 80% of the energy in the original feedstock. Pyrolysis of AHR proved unsatisfactory, so attention focussed on gasification and combustion with the aim of producing heat and/or power to supply the energy demanded by the ethyl levulinate production process. A thermal processing rig consisting on a Laminar Entrained Flow Reactor (LEFR) equipped with solid and liquid collection and online gas analysis systems was designed and built to explore pyrolysis, gasification and air-blown combustion of AHR. Maximum liquid yield for pyrolysis of AHR was 30wt% with volatile conversion of 80%. Gas yield for AHR gasification was 78wt%, with 8wt% tar yields and conversion of volatiles close to 100%. 90wt% of the AHR was transformed into gas by combustion, with volatile conversions above 90%. 5volO2%-95vol%N2 gasification resulted in a nitrogen diluted, low heating value gas (2MJ/m3). Steam and oxygen-blown gasification of AHR were additionally investigated in a batch gasifier at KTH in Sweden. Steam promoted the formation of hydrogen (25vol%) and methane (14vol%) improving the gas heating value to 10MJ/m3, below the typical for steam gasification due to equipment limitations. Arrhenius kinetic parameters were calculated using data collected with the LEFR to provide reaction rate information for process design and optimisation. Activation energy (EA) and pre-exponential factor (ko in s-1) for pyrolysis (EA=80kJ/mol, lnko=14), gasification (EA=69kJ/mol, lnko=13) and combustion (EA=42kJ/mol, lnko=8) were calculated after linearly fitting the data using the random pore model. Kinetic parameters for pyrolysis and combustion were also determined by dynamic thermogravimetric analysis (TGA), including studies of the original biomass feedstocks for comparison. Results obtained by differential and integral isoconversional methods for activation energy determination were compared. Activation energy calculated by the Vyazovkin method was 103-204kJ/mol for pyrolysis of untreated feedstocks and 185-387kJ/mol for AHRs. Combustion activation energy was 138-163kJ/mol for biomass and 119-158 for AHRs. The non-linear least squares method was used to determine reaction model and pre-exponential factor. Pyrolysis and combustion of biomass were best modelled by a combination of third order reaction and 3 dimensional diffusion models, while AHR decomposed following the third order reaction for pyrolysis and the 3 dimensional diffusion for combustion.

The development of a sustainable agricultural industry through the application of biochar and pyroligneous acid

This paper marks the first in a series of studies into the potential use of pyrolysis products in the development of more sustainable practices within the agricultural industry. In this study, the immediate benefits of the application of biochar to crop yields of Raphanus sativus (radishes) are assessed. Furthermore, the study reports on the preliminary findings into the potential application of pyroligneous acid (wood vinegar) as a biocidal agent against crop disease. Although germination tests undertaken on biochar/compost blends of up to 1: 2, by weight, showed no significant adverse effect from the addition of the nutrient rich carbonaceous solid, evidence of substantial increases in crop yield through the addition of biochar were not observed. In sharp contrast, zones of inhibition were observed at 3-10 vol. % upon application of pyroligneous acid to two causal agents responsible for certain diseases in vegetable and fruit crops, i.e. Rhizobium radiobacter (agrobacterium tumefaciens) and Xanthomonas campestris, highlighting the versatility in the application of pyrolysis products and avenues for exploration in the development of this biomass conversion technology.

Exploratory catalyst screening studies on the base free conversion of glycerol to lactic acid and glyceric acid in water using bimetallic Au-Pt nanoparticles on acidic zeolites

The base free oxidation of glycerol with molecular oxygen in water using bimetallic Au-Pt catalysts on three different acidic zeolite supports (H-mordenite, H-β and H-USY) was explored in a batch setup. At temperatures between 140 and 180 °C, lactic acid formation was significant and highest selectivity (60 % lactic acid at 80 % glycerol conversion) was obtained using Au-Pt/USY-600 (180 °C). A selectivity switch to glyceric acid (GLYA) was observed when the reactions were performed at 100 °C. Highest conversion and selectivity towards GLYA were obtained with Au-Pt/H-β as the catalyst (68 % selectivity at 68 % conversion).

Effect of eicosapentaenoic acid, protein and amino acids on protein synthesis and degradation in skeletal muscle of cachectic mice

Atrophy of skeletal muscle reduces both the quality and quantity of life of patients with cancer cachexia. Loss of muscle mass is thought to arise from a reduction in protein synthesis combined with an enhanced rate of protein degradation, and few treatments are available to counteract this process. Eicosapentaenoic acid (EPA) has been shown to attenuate the enhanced protein degradation, but to have no effect on protein synthesis. This study examines the effect of EPA combined with a protein and amino-acid supplementation on protein synthesis and degradation in gastrocnemius muscle of mice bearing the cachexia-inducing MAC16 tumour. Muscles from cachectic mice showed an 80% reduction in protein synthesis and about a 50-fold increase in protein degradation compared with muscles from nontumour-bearing mice of the same age and weight. Treatment with EPA (1 g kg-1) daily reduced protein degradation by 88%, but had no effect on protein synthesis. Combination of EPA with casein (5.35 g kg-1) also had no effect on protein synthesis, but when combined with the amino acids leucine, arginine and methionine there was almost a doubling of protein synthesis. The addition of carbohydrate (10.7 g kg-1) to stimulate insulin release had no additional effect. The combination involving the amino acids produced almost a doubling of the ratio of protein synthesis to protein degradation in gastrocnemius muscle over that of EPA alone. No treatment had a significant effect on tumour growth rate, but the inclusion of amino acids had a more significant effect on weight loss induced by the MAC16 tumour than that of EPA alone. The results suggest that combination therapy of cancer cachexia involving both inhibition of the enhanced protein degradation and stimulation of the reduced protein synthesis may be more effective than either treatment alone. © 2004 Cancer Research UK.