Interaction of hydrogen with actinide dioxide (111) surfaces

The interaction of atomic and molecular hydrogen with actinide dioxide (AnO2, An = U, Np, Pu) (111) surfaces has been investigated by DFT+U, where noncollinear 3k antiferromagnetic behaviour and spin-orbit interactions are considered. The adsorption of atomic hydrogen forms a hydroxide group, coupled to the reduction of an actinide ion. The energy of atomic hydrogen adsorption on the UO2 (0.82 eV), NpO2 (−0.10 eV), and PuO2 (−1.25 eV) surfaces has been calculated. The dissociation of molecular hydrogen is not observed, shown to be due to kinetic rather than thermodynamic factors. As a barrier to the formation of a second hydroxyl group, an unusual charge distribution has been shown. This could be a limitation of a (1·1) unit cell method or an artefact of the systems. The recombination of hydrogen ions on the AnO2 (111) surfaces is favoured over hydroxide formation

Interaction of hydrogen with actinide dioxide (011) surfaces

The corrosion and oxidation of actinide metals, leading to the formation of metal-oxide surface layers with the catalytic evolution of hydrogen, impacts the management of nuclear materials. Here, the interaction of hydrogen with actinide dioxide (AnO2, An = U, Np, or Pu) (011) surfaces by Hubbard corrected density functional theory (PBEsol+U) has been studied, including spin–orbit interactions and non-collinear 3k anti-ferromagnetic behavior. The actinide dioxides crystalize in the fluorite-type structure, and although the (111) surface dominates the crystal morphology, the (011) surface energetics may lead to more significant interaction with hydrogen. The dissociative adsorption of hydrogen on the UO2 (0.44 eV), NpO2 (−0.47 eV), and PuO2 (−1.71 eV) (011) surfaces has been calculated. It is found that hydrogen dissociates on the PuO2 (011) surface; however, UO2 (011) and NpO2 (011) surfaces are relatively inert. Recombination of hydrogen ions is likely to occur on the UO2 (011) and NpO2 (011) surfaces, whereas hydroxide formation is shown to occur on the PuO2 (011) surface, which distorts the surface structure

Magnetic structure of UO2 and NpO2 by first-principle methods

The magnetic structure of the actinide dioxides (AnO2) remains a field of intense research. A low-temperature experimental investigation of the magnetic ground-state is complicated by thermal energy released from the radioactive decay of the actinide nuclei. To establish the magnetic ground-state, we have employed high-accuracy computational methods to systematically probe different magnetic structures. A transverse 1k antiferromagnetic ground-state with Fmmm (No. 69) crystal symmetry has been established for UO2, whereas a ferromagnetic (111) ground-state with R[3 with combining macron]m (No. 166) has been established for NpO2. Band structure calculations have been performed to analyse these results

Heterogeneity of O6-alkylguanine DNA-alkyltransferase expression in human breast tumours

An important determinant of cellular resistance to chemotherapeutic O6-alkylating agents, which comprise methylating and chloroethylating agents, is the ability of cells to repair alkylation damage at the O6-position of guanine in DNA. This is achieved by a specific DNA repair enzyme O6-alkylguanine DNA-alkyltransferase. In this study O6-alkylguanine DNA-alkyltransferase expression was measured in human breast tumours using both biochemical and immunohistochemical techniques. O6-alkylguanine DNA-alkyltransferase activity was then compared with known clinical prognostic indices to assess the potential role of O6-alkylguanine DNA-alkyltransferase in predicting the behaviour of this common malignancy. The application of both biochemical and immunohistochemical techniques was feasible and practical. Most breast tumours expressed high levels of O6-alkylguanine DNA-alkyltransferase. Immunohistochemical analysis showed marked variation in expression not only between individuals but also within individual tumours, and in the same patient, between metastases and between primary tumour and metastatic site. O6-alkylguanine DNA-alkyltransferase activity in tissue extracts significantly correlated not only with immunohistochemical staining intensity determined by subjective quantitation, but also with measures of protein levels using a computerised image analysis system including mean grey (P<0.001), percentage of cells positive for O6-alkylguanine DNA-alkyltransferase (P<0.001), and integrated optical density (P<0.001). O6-alkylguanine DNA-alkyltransferase expression did not correlate with any of the established clinical prognostic indicators for current treatment regimens. However, immunohistochemical offers a rapid and convenient method for assessing potential utility of O6-alkylating agents or O6-alkylguanine DNA-alkyltransferase inactivating agents in future studies of breast cancer treatment

O6-methylguanine-DNA-methyltransferase expression and gene polymorphisms in relation to chemotherapeutic response in metastatic melanoma

In a retrospective study, O6-methylguanine-DNA-methyltransferase (MGMT) expression was analysed by immunohistochemistry using monoclonal human anti-MGMT antibody in melanoma metastases in patients receiving dacarbazine (DTIC) as single-drug therapy or as part of combination chemotherapy with DTIC–vindesine or DTIC–vindesine–cisplatin. The correlation of MGMT expression levels with clinical response to chemotherapy was investigated in 79 patients with metastatic melanoma. There was an inverse relationship between MGMT expression and clinical response to DTIC-based chemotherapy (P=0.05). Polymorphisms in the coding region of the MGMT gene were also investigated in tumours from 52 melanoma patients by PCR/SSCP and nucleotide sequence analyses. Single-nucleotide polymorphisms (SNPs) in exon 3 (L53L and L84F) and in exon 5 (I143V/K178R) were identified. There were no differences in the frequencies of these polymorphisms between these melanoma patients and patients with familial melanoma or healthy Swedish individuals. Functional analysis of variants MGMT-I143V and -I143V/K178R was performed by in vitro mutagenesis in Escherichia coli. There was no evidence that these variants decreased the MGMT DNA repair activity compared to the wild-type protein. All melanoma patients with the MGMT 53/84 polymorphism except one had tumours with high MGMT expression. There was no significant correlation between any of the MGMT polymorphisms and clinical response to chemotherapy, although an indication of a lower response rate in patients with SNPs in exon 5 was obtained. Thus, MGMT expression appears to be more related to response to chemotherapy than MGMT polymorphisms in patients with metastatic melanoma

One-carbon metabolism and epigenetic programming of mammalian development

One-carbon (1C) metabolism comprises a series of integrated metabolic pathways, including the linked methionine-folate cycles, that provide methyl groups for the synthesis of biomolecules and the epigenetic regulation of gene expression via chromatin methylation. Most of the research investigating the function of 1C metabolism pertains to studies undertaken in the rodent liver. Comparatively little is known about the function of 1C metabolism in reproductive and embryonic cells, particularly in domestic ruminant species. Periconceptional dietary deficiencies in 1C substrates and cofactors are known to lead to epigenetic alterations in DNA methylation in genes that regulate key developmental processes in the embryo. Such modifications can have negative implications on the subsequent development, metabolism and health of offspring. This thesis sought to improve current understanding of the regulation of 1C metabolism in the ruminant liver, ovary and preimplantation embryo through in vivo and in vitro nutritional supplementation experiments coupled with metabolomic, transcriptomic and epigenetic analyses. The first part of this thesis (Chapter 2) assessed the metabolic consequences of dietary methyl deficiency using novel mass spectrometry–based methods that were developed for the quantification of B vitamins, folates and 1C-related amines in sheep liver. This study provided the first comparison of the relative abundance of bioactive 1C metabolites in liver harvested from methyl deficient sheep relative to a control study population of abattoir derived sheep. Relevant reductions in dietary methyl availability led to significant alterations in hepatic 1C metabolite concentrations. Large natural variations in the hepatic concentrations of individual metabolites in both sheep study populations reflected the dietary and genetic variation in our chosen outbred model species. These metabolomics platforms will be useful for investigating 1C metabolism and linked biochemical pathways in order to facilitate future dietary and genetic studies of metabolic health and epigenetic regulation of gene expression. Based on the absence of methionine cycle enzyme transcripts (e.g. MAT1A and BHMT) in the bovine ovary and preimplantation embryo, the second part of this thesis (Chapter 3 and Chapter 4) addressed the hypothesis that ruminant reproductive and embryonic cells are highly sensitive to methyl group availability and, therefore, epigenetic programming during the periconceptional period. Transcript analyses confirmed MAT2A expression in the bovine liver, ovary and at each stage of preimplantation embryo development assessed to Day 8. Transcripts for BHMT isoforms (BHMT and BHMT2) were detected in the bovine ovary but were weak or absent in embryos, highlighting a key difference in methionine metabolism between hepatic and reproductive cells. Bovine embryos were produced in vitro using custom-made media containing 0 (nonphysiological), 10 (low physiological), 50 (high physiological), and 500 µmol/L (supraphysiological) added methionine (Chapter 3). Gross morphological assessments of embryo stage, grade, cell lineage allocation and primary sex ratio revealed that culture in non- and supraphysiological methionine concentrations was detrimental for embryo development, whilst culture in the high physiological concentration appeared to be best. Reduced representation bisulphite sequencing (RRBS) of inner cell mass (ICM) and trophectoderm (TE) cells immunodissected from Day 8 blastocysts demonstrated that culturing embryos in low physiological methionine led to global hypomethylation within both cell lineages. Bioinformatic analyses of differentially methylated genes included gene set enrichment analyses (GSEA). Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that were enriched within the ICM were associated with protein catabolism and autophagy, and significant terms and pathways enriched within the TE were associated with cellular transport. Of particular biological interest was the loss of methylation within regulatory region (DMR2) of the paternally imprinted gene, IGF2R, in the TE following culture in low physiological methionine. Transcript analysis found no significant effect of methionine concentration on the expression of IGF2R or the antisense transcript, AIRN, in the primary cell lineages of the Day 8 bovine preimplantation embryo. Hypomethylation of IGF2R DMR2 has been associated with aberrant IGF2R expression and large offspring syndrome (LOS) in cattle and sheep that were subjected to embryo manipulation during assisted reproductive technology (ART) procedures, such as somatic cell nuclear transfer (SCNT) or non-physiological in vitro embryo culture environments. Chapter 5 sought to evaluate the effect of somatic donor cell type on epigenetic reprogramming via DNA methylation in hepatocytes isolated from cloned sheep. RRBS facilitated the comparison of methylation reprogramming between Finn Dorset (D) clone hepatocytes and their mammary epithelial (OP5) donor cell line; and, Lleyn (L) clone hepatocytes and their Lleyn fetal fibroblast (LFF4) donor cell line. Methylation was most closely correlated between D and L clone hepatocytes than between clones and their respective donor cell lines. In general, hepatocytes were hypomethylated relative to their somatic donor cell nuclei. GSEA identified genes that encoded transcription factor proteins enriched within the ‘Sequence-specific DNA binding’ term (GO:0043565) as differentially methylated between clone hepatocytes and their donor cell lines. In addition, imprinted genes, including IGF2R, were differentially methylated in clone hepatocytes relative to somatic cell nuclei. In summary, this thesis promotes and supports the importance of an optimal methyl balance to support periconceptional development in mammals. The experiments detailed herein provide an insight into the metabolic consequences of dietary methyl deficiency (and excess) in outbred populations of domestic ruminants, with a specific focus on the liver, ovary and preimplantation embryo. The results demonstrate that tissue- and species-specific features of 1C metabolism render ruminant embryonic cells sensitive to methionine inputs within a physiological range. The observation that in vitro embryo culture and manipulation techniques, such as somatic cell nuclear transfer, can cause epigenetic alterations to DNA methylation during preimplantation development provides a basis for further study into the safety and efficacy of emerging assisted reproductive technologies

Effect of antiandrogen flutamide on measures of hepatic regeneration in rats

Male rat liver undergoes a process of demasculinization during hepatic regeneration following partial hepatectomy. The possibility that antiandrogens might potentiate this demasculinization process and in so doing augment the hepatic regenerative response was investigated. Adult male Wistar rats were treated with the antiandrogen flutamide (2 mg/rat/day or 5 mg/rat/day subcutaneously) or vehicle for three days prior to and daily after a 70% partial hepatectomy. At various times after hepatectomy, the liver remnants were removed and weighed. Rates of DNA and polyamine synthesis were assessed by measuring thymidine kinase and ornithine decarboxylase activities, respectively. Hepatic estrogen receptor status and the activity of alcohol dehydrogenase, an androgen-sensitive protein, were measured. Prior to surgery, the administration of 5 mg/day flutamide reduced the hepatic cytosolic androgen receptor activity by 98% and hepatic cytosolic estrogen receptor content by 92% compared to that present in vehicle-treated controls. After hepatectomy, however, all differences in sex hormone receptor activity between the treatment groups were abolished. The rate of liver growth after partial hepatectomy in the three groups was identical. Moreover, hepatectomy-induced increases in ornithine decarboxylase activity and thymidine kinase activity were comparable. These data demonstrate that, although flutamide administration initially alters the sex hormone receptor status of the liver, these affects have no effect on the hepatic regenerative response following a partial hepatectomy. © 1989 Plenum Publishing Corporation

Influence of O6-benzylguanine on the anti-tumour activity and normal tissue toxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea and molecular combinations of 5-fluorouracil and 2-chloroethyl-1-nitrosourea in mice

Previous studies have demonstrated that novel molecular combinations of 5-fluorouracil (5FU) and 2-chloroethyl-1-nitrosourea (CNU) have good preclinical activity and may exert less myelotoxicity than the clinically used nitrosoureas such as 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). This study examined the effect of O6-alkylguanine-DNA-alkyltransferase (ATase) depletion by the pseudosubstrate O6-benzylguanine (BG) on the anti-tumour activity and normal tissue toxicity in mice of three such molecular combinations, in comparison with BCNU. When used as single agents at their maximum tolerated dose, all three novel compounds produced a significant growth retardation of BCNU-resistant murine colon and human breast xenografts. This in vivo anti-tumour effect was potentiated by BG, but was accompanied by severe myelotoxicity as judged by spleen colony forming assays. However, while tumour resistance to BCNU was overcome using BG, this was at the expense of enhanced bone marrow, gut and liver toxicity. Therefore, although this ATase-depletion approach resulted in improved anti-tumour activity for all three 5-FU:CNU molecular combinations, the potentiated toxicities in already dose-limiting tissues indicate that these types of agents offer no therapeutic advantage over BCNU when they are used together with BG. © 1999 Cancer Research Campaig

Mathematical modelling of polyamine metabolism in bloodstream-form trypanosoma brucei: An application to drug target identification

© 2013 Gu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedThis article has been made available through the Brunel Open Access Publishing Fund.We present the first computational kinetic model of polyamine metabolism in bloodstream-form Trypanosoma brucei, the causative agent of human African trypanosomiasis. We systematically extracted the polyamine pathway from the complete metabolic network while still maintaining the predictive capability of the pathway. The kinetic model is constructed on the basis of information gleaned from the experimental biology literature and defined as a set of ordinary differential equations. We applied Michaelis-Menten kinetics featuring regulatory factors to describe enzymatic activities that are well defined. Uncharacterised enzyme kinetics were approximated and justified with available physiological properties of the system. Optimisation-based dynamic simulations were performed to train the model with experimental data and inconsistent predictions prompted an iterative procedure of model refinement. Good agreement between simulation results and measured data reported in various experimental conditions shows that the model has good applicability in spite of there being gaps in the required data. With this kinetic model, the relative importance of the individual pathway enzymes was assessed. We observed that, at low-to-moderate levels of inhibition, enzymes catalysing reactions of de novo AdoMet (MAT) and ornithine production (OrnPt) have more efficient inhibitory effect on total trypanothione content in comparison to other enzymes in the pathway. In our model, prozyme and TSHSyn (the production catalyst of total trypanothione) were also found to exhibit potent control on total trypanothione content but only when they were strongly inhibited. Different chemotherapeutic strategies against T. brucei were investigated using this model and interruption of polyamine synthesis via joint inhibition of MAT or OrnPt together with other polyamine enzymes was identified as an optimal therapeutic strategy.The work was carried out under a PhD programme partly funded by Prof. Ray Welland, School of Computing Science, University of Glasgo

Polyamine metabolism is involved in adipogenesis of 3T3-L1 cells

Polyamines spermidine and spermine are known to be required for mammalian cell proliferation and for embryonic development. Alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC) a limiting enzyme of polyamine biosynthesis, depleted the cellular polyamines and prevented triglyceride accumulation and differentiation in 3T3-L1 cells. In this study, to explore the function of polyamines in adipogenesis, we examined the effect of polyamine biosynthesis inhibitors on adipocyte differentiation and lipid accumulation of 3T3-L1 cells. The spermidine synthase inhibitor trans-4-methylcyclohexylamine (MCHA) increased spermine/spermidine ratios, whereas the spermine synthase inhibitor N-(3-aminopropyl)-cyclohexylamine (APCHA) decreased the ratios in the cells. MCHA was found to decrease lipid accumulation and GPDH activity during differentiation, while APCHA increased lipid accumulation and GPDH activity indicating the enhancement of differentiation. The polyamine-acetylating enzyme, spermidine/spermine N1-acetyltransferase (SSAT) activity was increased within a few hours after stimulus for differentiation, and was found to be elevated by APCHA. In mature adipocytes APCHA decreased lipid accumulation while MCHA had the opposite effect. An acetylpolyamine oxidase and spermine oxidase inhibitor MDL72527 or an antioxidant N-acetylcysteine prevented the promoting effect of APCHA on adipogenesis. These results suggest that not only spermine/spermidine ratios but also polyamine catabolic enzyme activity may contribute to adipogenesis