The effects of some neurotoxins on tetrahydrobiopterin metabolism

Previous studies in man have shown that following dosing with L--3,4-dihydroxyphenylalanine (L-DOPA) and cotrimoxazole, plasma biopterins were raised. By analogy with dihydropteridine reductase deficient children in whom plasma biopterins are greatly elevated and the observations that these preparations were dihydropteridine reductase inhibitors, it was assumed that these raised plasma levels were due to increased efflux from tissues which resulted in tissue depletion of biopterins. In some human disease states such as senile dementia of the Alzheimer type lowered plasma biopterins were observed; by analogy with tetrahydrobiopterin synthesis deficient children these reduced plasma biopterins were attributed to lowered tetrahydrobiopterin synthesis and concomitant low tissue biopterin levels. Because of ethical considerations it was not possible to measure directly the tissue biopterins changes in either case. The Wistar rat was used as a model for human tetrahydrobiopterin metabolism, since tissues not normally accessible for study in humans, such as the brain and liver, could be examined for their effects on tetrahydrobiopterin metabolism after administration of the various agents. Plasma total biopterins in normal conditions were found to be much higher than in healthy humans. The elevation of plasma total biopterins concentration following the administration of dihydropteridine reductase inhibitors to humans, such as L-DOPA and cotrimoxazole was not observed in the rat. However, the administration of inhibitors of de novo tetrahydrobiopterin biosynthesis, such as diaminohydroxypyrimidine (DAHP) and bromocriptine was shown to decrease plasma biopterins concentration. In general, hepatic biopterins were decreased after administration of both dihydropteridine reductase inhibitors and de novo biosynthesis inhibitors. Drugs which are direct (bromocriptine) or indirect (L-DOPA and Sinemet Plus) agonists at dopamine receptors were investigated and were shown to decrease hepatic total biopterins concentration, but had no effect on brain biopterins. Bromocriptine was demonstrated as a potent inhibitor of de novo tetrahydrobiopterin biosynthesis in vivo and in vitro. Cotrimoxazole decreased brain tetrahydrobiopterin concentration. DAHP was effective in causing hyperphenylalaninaemia due to tetrahydrobiopterin deficiency in the rat. p-hydroxyphenylacetate was shown to be an effective inhibitor of dihydropteridine reductase in vivo. Phenylacetate administration had no observable effect on tetrahydrobiopterin metabolism, but did cause tyrosinaemia. It is proposed that scopolamine reduces tetrahydrobiopterin turnover. Lead and aluminium exposure caused deranged tetrahydrobiopterin metabolism. Aluminium, but not lead decreased brain choline acetyltransferase activity. Phenylalanine loading in normal human subjects was followed by an elevation in plasma biopterins which was not observed after tyrosine loading. Plasma N : B ratios correlated well with VEP latencies after tyrosine loading, but not after phenylalanine loading in healthy subjects. The use of derived pterin measurements as an indicator of tetrahydrobiopterin turnover or tetrahydrofolate status is discussed in the text.

Tetrahydrobiopterin Metabolism

Tetrahydrobiopterin is the cofactor for the hydroxylation of phenylalanine, tyrosine and tryptophan and is therefore essential for the production of monoamine neurotransmitters. Neopterin, a biosynthetic precusor of tetrahydrobiopterin, and biopterin appear in urine. In normal subjects the urinary neopterin to biopterin ratio has been found to be about 1.00. In patients suffering from Alzheimer's disease, Down's syndrome and depression the urinary neopterin to biopterin ratio has been found to be elevated. In some Alzheimer's and depressed patients the increased urinary neopterin to biopterin ratio is proportional to the severity of the disease. Folates were found not to increase tetrahydrobiopterin biosynthesis in the rat as previously thought. Methotrexate was found to reduce liver biopterin levels and increas_ urinary biopterin levels in the rat. Methotrexate also reduced brain pterin levels but had no influence on liver pterin. Urinary isoxanthopterin, found in some patients, was found to be derived from biopterin and neopterin in the rat. Isoxanthopterin is proposed as an indicator of the levels of tetrahydrobiopterin turnover.

Studies of the metabolism and the toxicity of N-Methylformamide and related amides

The hepatotoxicity of the industrial solvent and investigational anti-tumour agent N-methylformamide (NMF, HOCNHCH3) and several structural analogues was assessed in mice. NMF and its ethyl analogue (NEF) were equipotent hepatotoxins causing extensive centrilobular necrosis and damage to the gall bladder. Pretreatment of mice with SKF525A did not influence the toxicity of these N-alkylformamides. Replacement of the formyl hydrogen of NMF with deuterium or methyl significantly reduced its hepatotoxicity. An in vitro model for the study of the toxicity and metabolism of N-alkylformamides was developed using isolated mouse hepatocytes. The cytotoxicity of NMF in vitro was concentration-dependent with maximal toxicity being achieved at concentrations of 5mM or above. The cytotoxic potential of related amides correlated well with their in vivo hepatotoxic potential. Pretreatment of mice with buthionine sulphoximine (BSO), which depleted hepatocytic levels of glutathione to 15% of control values, exacerbated the cytotoxicity of NMF towards the hepatocytes. NMF (1mM or above), incubated with isolated mouse hepatocytes, depleted intracellular glutathione levels to 26% of control values within 4h. Depletion of glutathione was quantitatively matched by the formation of a carbamoylating metabolite. Metabolism was dependent on the concentration of NMF and was drastically reduced in incubations of hepatocytes isolated from mice pretreated with BSO. The carbamoylating metabolite, S-(N-methylcarbamoyl)-glutathione (SMG), was identified in vitro using FAB-MS. The generation of SMG was subject to a large primary H/D kinetic isotope effect when the formyl hydrogen was replaced with deuterium. Likewise, glutathione depletion and metabolite formation were reduced or abolished by the deuteration or methylation of the formyl moiety of NMF. NEF, like NMF, depleted hepatocytic glutathione levels and was metabolised to a carbamoylating metabolite. Radioactivity derived from 14C-NMF and 14C-NEF, labelled in the alkyl moieties, was found to be irreversibly associated with microsomal protein on incubation in vitro. Binding was dependent on the presence of NADPH and was mostly abolished in the presence of reduced glutathione. SKF525A failed to influence the binding.

Studies of the mechanism of antitumour activity of N-methylformamide

NMF induces the terminal differentiation or acquisition of more benign characteristics in certain malignant cells in vitro and has good antitumour activity against murine tumours in vivo. This study was concerned with a comparison of the mechanism of antitumour activity of NMF in vitro and in vivo against the murine TLX5 lymphoma, which is sensitive to NMF in vivo. TLX5 cells incubated continuously with NMF in vitro showed a concentration and time dependent decrease in cell growth rate, which was associated with an increase in membrane permeability, a decrease in cell size and at the higher NMF concentrations, cell death. Analysis of the cell cycle after incubation with NMF indicated an early G1 phase arrest. TLX5 cells were incubated with NMF and washed free of the drug. Analysis of clonogenicity and tumourigenicity showed that all viable cells retained their proliferative potential and malignancy. Therefore, TLX5 cells exposed to NMF in vitro are not terminally differentiated, but reside in a quiescent substate which was reversed on drug removal. The intracellular GSH levels of TLX5 cells was decreased in a concentration and time dependent fashion by NMF. GSH depletion of TLX5 cells was not however a prerequisite for growth arrest, unlike the reported data for human colon carcinoma cell lines. A single administration of NMF caused a dose dependent regression of the TLX5 lymphoma in tumour bearing mice. Cell death occurred by apoptosis and necrosis. The antitumour activity of NMF was dependent on formyl C-H bond fission, with the parent drug or metabolites reaching all parts of the tumour 4h after dosing. There was a non-dose dependent increase in the S phase population, which was due to an increase in DNA synthesis, 24h after administration of NMF. NMF administration caused a decrease in GSH levels of the TLX5 lymphoma, which did not correlate with the antitumour response. However, the GSH depleting agent, BSO, marginally increased the antitumour activity of NMF.

Studies on the anti-inflammatory effect of S-Adenosyl-L-methionine

1. S-adenosyl-L-methionine (SAMe) had no effect on cytochrome C reduction by superoxide generated from xanthine oxidase except at high concentrations. This was due to direct inhibition of the enzyme. 2. SAMe inhibited the neutrophil respiratory burst , measured by luminol enhanced chemiluminescence, to FMLP and zymosan A but not to PMA. 3. Adenosine and methylthioadenosine (MTA) inhibited the respiratory burst elicited by FMLP. 4. SAMe inhibited the phagocytosis of latex particles by neutrophils at high concentrations but methionine and S-adenosyl L-homocysteine had no effect. 5. Treatment with SAMe had no effect on cell infiltration or PGE2 production in 6-day air pouches. 6. Treatment with SAMe at the optimum dose of 50mg/kg inhibited the early phases of carrageenan induced rat hind paw inflammation but had a lesser effect on the secondary response. The antiinflammatory effect was sustained after inhibiton of polyamine synthesis. 7. SAMe increased liver putrescine levels in the presence and absence of inflammation Spermidine levels were increased in the presence of inflammation but spermine levels were unaffected by any of the treatments. 8. MT A and adenosine increased liver putrescine and spermidine levels 9. Treatment with SAMe had no effect on the polyamine status of blood. lO.Treatment with SAMe had no effect on the levels of glutathione in liver or blood. 11.SAMe and MTA inhibited histamine and platelet-activating factor (PAF) induced hind paw inflammation but had no effect on inflammation induced by dextran, zymosan, compound 48/80, 5-hydroxytryptamine, arachidonic acid or glucose oxidase. MTA was more effective than SAMe. 12. PAP-induced rat hind paw inflammation was inhibited by isoprenaline and verapamil. Combinations of these drugs with SAMe or MT A had no further enhancement of effect. 13. Incubation of rat PMNLs with [14c ] SAMe increased the intracellular levels of S-adenosyl-L-homocysteine in a dose dependent manner, but had no effect on the intracellular levels of SAMe, adenosine or MT A. 14. Pharmacokinetic studies of plasma SAMe following a single dose of the drug (50mg/kg) i.p. demonstrated that SAMe is rapidly absorbed and metabolised

The role of metabolism in the toxicity of N-alkylformamides

Using ionspray tandem mass spectrometry the glutathione conjugate SMG was identified as a biliary metabolite of DMF in rats (0.003% of a dose of 5OOmg/kg DMF i.p.). Formation of this metabolite was increased five fold after induction of CYP2E1 by acetone, and was inhibited to 20% of control values following pretreatment with disulfrram. Generation of SMG from DMF in vivo was shown to exhibit a large kinetic deuterium isotope effect (KWKD=10.1 ± 1.3), which most likely represents the product of 2 discrete isotope effects on N-demethylation and formyl oxidation reactions.The industrial solvent N,N-dimethylformamide (DMF) and the investigational anti-tumour agent N-methylformamide (NMF) cause liver damage in rodents and humans. The hepatotoxicity of N-alkylformamides is linked to their metabolism to N-alkylcarbamic acid thioesters. The enzymatic details of this pathway were investigated. Hepatocytes isolated from BALB/c mice which had been pretreated with acetone, an inducer of the cytochrome P-450 isozyme CYP2E1, were incubated with NMF (10mM). NMF caused extensive toxicity (> 90% ) as determined by lactate dehydrogenase (LDH) release, compared to cells from untreated animals. Incubation of liver cells with NMF for 6 hrs caused 60±17% LDH release whilst in the presence of DMSO (10mM), an alternative substrate for CYP2E1, LDH release was reduced to 20±10% . The metabolism of NMF to S-(N-methylcarbamoyl)glutathione (SMG) was measured in incubates with liver microsomes from mice, rats or humans. Metabolism of NMF was elevated in microsomes isolated from rats and mice pretreated with acetone, by 339% and 183% respectively. Pretreatment of animals with 4-methylpyrazole induced the metabolism of NMF to 280% by rat microsomes, but was without effect on NMF metabolism by mouse microsomes. The CYP2E1 inhibitors or alternative substrates diethyl dithiocarbamate (DEDTC), p-nitrophenol (PNP) and dimethyl sulphoxide (DMSO) strongly inhibited the metabolism of NMF in suspensions of rat liver microsomes, at concentrations which did not effect aminopyrine N-demethylation. The rate of metabolism of NMF to SMG in human microsomes correlated (r> 0.8) with the rate of metabolism of chlorzoxazone, a CYP2E1 probe. A polyclonal antibody against rat CYP2E1 (10mg/nmol P-450) inhibited NMF metabolism in microsomes from rats and humans by 75% and 80% , respectively. The amount of immunoblottable enzyme in human microsomes, determined using an anti-rat CYP2E1 antibody, correlated with the rate of NMF metabolism (r> 0.8). Purified rat CYP2E1 catalysed the generation of SMG from NMF. Formation of the DMF metabolite N-hydroxymethyl-N-methylformamide (HMMF) in incubations with rat liver microsomes was elevated by 200% following pretreatment of animals with acetone. Co-incubation with DEDTC (100μM) inhibited HMMF generation from DMF by 88% . Co-incubation of DMF (10mM) with NMF (1mM) inhibited the formation of SMG by 95% . A polyclonal antibody against rat CYP2E1 (10mg/nmol P-450) inhibited generation of HMMF in incubates with rat and human liver microsomes by 68.4% and 67.5% , respectively. Purified rat CYP2E1 catalysed the generation of HMMF from DMF. Using ionspray tandem mass spectrometry the glutathione conjugate SMG was identified as a biliary metabolite of DMF in rats (0.003% of a dose of 5OOmg/kg DMF i.p.). Formation of this metabolite was increased five fold after induction of CYP2E1 by acetone, and was inhibited to 20% of control values following pretreatment with disulfrram. Generation of SMG from DMF in vivo was shown to exhibit a large kinetic deuterium isotope effect (KHKD=10.1 ± 1.3), which most likely represents the product of 2 discrete isotope effects on N-demethylation and formyl oxidation reactions.

Gingipains from Porphyromonas gingivalis increase the chemotactic and respiratory burst-priming properties of the 77-amino-acid interleukin-8 variant

Porphyromonas gingivalis, a gram-negative anaerobe which is implicated in the etiology of active periodontitis, secretes degradative enzymes (gingipains) and sheds proinflammatory mediators (e.g., lipopolysaccharides [LPS]). LPS triggers the secretion of interleukin-8 (IL-8) from immune (72-amino-acid [aa] variant [IL-8(72aa)]) and nonimmune (IL-8(77aa)) cells. IL-8(77aa) has low chemotactic and respiratory burst-inducing activity but is susceptible to cleavage by gingipains. This study shows that both R- and K-gingipain treatments of IL-8(77aa) significantly enhance burst activation by fMLP and chemotactic activity (P < 0.05) but decrease burst activation and chemotactic activity of IL-8(72aa) toward neutrophil-like HL60 cells and primary neutrophils (P < 0.05). Using tandem mass spectrometry, we have demonstrated that R-gingipain cleaves 5- and 11-aa peptides from the N-terminal portion of IL-8(77aa) and the resultant peptides are biologically active, while K-gingipain removes an 8-aa N-terminal peptide yielding a 69-aa isoform of IL-8 that shows enhanced biological activity. During periodontitis, secreted gingipains may differentially affect neutrophil chemotaxis and activation in response to IL-8 according to the cellular source of the chemokine.

Investigating polymer-peptide conjugates and electrospinning for the production of advanced materials

This thesis describes the production of advanced materials comprising a wide array of polymer-based building blocks. These materials include bio-hybrid polymer-peptide conjugates, based on phenylalanine and poly(ethylene oxide), and polymers with intrinsic microporosity (PIMs). Polymer-peptides conjugates were previously synthesised using click chemistry. Due to the inherent disadvantages of the reported synthesis, a new, simpler, inexpensive protocol was sought. Three synthetic methods based on amidation chemistry were investigated for both oligopeptide and polymerpeptide coupling. The resulting conjugates produced were then assessed by various analytical techniques, and the new synthesis was compared with the established protocol. An investigation was also carried out focussing on polymer-peptide coupling via ester chemistry, involving deprotection of the carboxyl terminus of the peptide. Polymer-peptide conjugates were also assessed for their propensity to self-assemble into thixotropic gels in an array of solvent mixtures. Determination of the rules governing this particular self-assembly (gelation) was required. Initial work suggested that at least four phenylalanine peptide units were necessary for self-assembly, due to favourable hydrogen bond interactions. Quantitative analysis was carried out using three analytical techniques (namely rheology, FTIR, and confocal microscopy) to probe the microstructure of the material and provided further information on the conditions for self-assembly. Several polymers were electrospun in order to produce nanofibres. These included novel materials such as PIMs and the aforementioned bio-hybrid conjugates. An investigation of the parameters governing successful fibre production was carried out for PIMs, polymer-peptide conjugates, and for nanoparticle cages coupled to a polymer scaffold. SEM analysis was carried out on all material produced during these electrospinning experiments.

Vitamin E-stabilised UHMWPE for orthopaedic implants:quantitative determination of vitamin E and characterisation of its transformation products

The fate of vitamin E and the formation and identification of its transformation products were investigated at different stages of the manufacturing process of commercially produced cross-linked (by γ-irradiation) UHMWPE stabilised with vitamin E (vitamin E infused-post irradiation) used for tibia-components (as articulating surfaces) in total knee arthroplasty (total knee replacement). Vitamin E (α-tocopherol) and its transformation products were extracted from microtomed Tibia films and the different products were separated, isolated, purified using high performance liquid chromatography (HPLC), and characterised by spectroscopic methods and LC-MS. The amount of vitamin E and that of the products formed in the different Tibia samples and in their extracts were also quantified using FTIR and HPLC analysis and calibration curves. Thorough analysis of the Tibia extracts has shown that a number of vitamin E transformation products were formed at different concentrations at two selected stages of the implant manufacturing process that is before and after sterilisation by γ-irradiation. The identified products were found to correspond mainly to different stereoisomeric forms of a small number of vitamin E transformation products. Most of the observed products were of dimeric and trimeric nature with their identity confirmed through a detailed study of their spectral and chromatographic characteristics. It was found that the products of vitamin E, prior to the sterilisation step but after the crosslinking and doping of vitamin E, were mainly the dihydroxydimers and trimers (Tibia samples at this stage are referred to as “Tibia-VEPE”). After sterilisation and completion of the manufacturing process, additional dimers of vitamin E were also formed (Tibia samples at this stage are referred to as ‘Tibia-VEPE-Sterile’), Furthermore, two tocopherol-derived aldehydes (aldehyde 5-formyl-γ-tocopherol and aldehyde 7-formyl-γ-tocopherol) were also formed but at very low concentrations especially in the Tibia-VEPE-Sterile samples. The question of whether vitamin E becomes chemically reacted (grafted) onto the polymer matrix during the manufacturing process of the Tibia is also addressed.

Luminescent techniques for studying free radical production and cell viability in relation to cardiovascular disease and HRT

Epidemiological studies have suggested that hormone replacement therapy (HRT) offers protection from atherosclerosis, a precursor of cardiovascular disease (CVD), in postmenopausal women. There is good evidence that oxidation of low-density lipoprotein (LDL) by leucocyte-derived reactive oxygen species plays a key role in development of an atherosclerotic plaque. Therefore we have investigated whether the possible protection against CVD by HRT could be due to immunomodulation, specifically of free radical production. The study involves 2 approaches: I) analysing the production of free radicals by leucocytes from women on HRT, 2) investigating the effect of I7p-oestradiol and progesterone on cultured myeloid cells (HL60 and U937). Free radical production by leucocytes was determined using a recently developed bioluminescent assay. In the assay, Pholasin® emits light in the presence of free radicals produced by the NADPH oxidase system of leucocytes stimulated with PMA or fMLP. Cell viability was also investigated using a bioluminescent assay (Cell Titer-Glo®) in which cytosolic ATP levels were measured by the production of luminescence in the presence of Luciferin/Luciferase reagent. Studies of leucocytes from HRT patients showed considerable variation in free radical production, which appeared to be dependent on HRT regime. Studies on the cultured cells showed that there was no cell proliferation at low hormone concentrations, while high concentrations caused cytotoxicity. The effect of hormones on free radical production in this in vitro model system is currently being investigated. The results show that the effects of the hormones on cells of the immune system are very dose dependent, and that both beneficial and adverse effects may occur. In conclusion, luminescent techniques offer a valuable and sensitive approach to studying inflammatory and oxidative processes both in vivo and in vitro.

Characterization of neutrophil function in Papillon-Lefèvre syndrome

Papillon-Lefévre syndrome is a rare, inherited, autosomal-recessive disease, characterized by palmoplantar keratosis and severe prepubertal periodontitis, leading to premature loss of all teeth. Papillon-Lefévre syndrome is caused by a mutation in the cathepsin C gene, resulting in complete loss of activity and subsequent failure to activate immune response proteins. Periodontitis in Papillon-Lefévre syndrome is thought to arise from failure to eliminate periodontal pathogens as a result of cathepsin C deficiency, although mechanistic pathways remain to be elucidated. The aim of this study was to characterize comprehensively neutrophil function in Papillon-Lefévre syndrome. Peripheral blood neutrophils were isolated from 5 patients with Papillon-Lefévre syndrome, alongside matched healthy control subjects. For directional chemotactic accuracy, neutrophils were exposed to the chemoattractants MIP-1α and fMLP and tracked by real-time videomicroscopy. Reactive oxygen species generation was measured by chemiluminescence. Neutrophil extracellular trap formation was assayed fluorometrically, and proinflammatory cytokine release was measured following overnight culture of neutrophils with relevant stimuli. Neutrophil serine protease deficiencies resulted in a reduced ability of neutrophils to chemotax efficiently and an inability to generate neutrophil extracellular traps. Neutrophil extracellular trap-bound proteins were also absent in Papillon-Lefévre syndrome, and Papillon-Lefévre syndrome neutrophils released higher levels of proinflammatory cytokines in unstimulated and stimulated conditions, and plasma cytokines were elevated. Notably, neutrophil chemoattractants MIP-1α and CXCL8 were elevated in Papillon-Lefévre syndrome neutrophils, as was reactive oxygen species formation. We propose that relentless recruitment and accumulation of hyperactive/reactive neutrophils (cytokines, reactive oxygen species) with increased tissue transit times into periodontal tissues, alongside a reduced antimicrobial capacity, create a locally destructive chronic inflammatory cycle in Papillon-Lefévre syndrome.

Mechanisms of chloroperoxidase-catalyzed enantioselective reactions as probed by site-directed mutagenesis and isotopic labeling

Chloroperoxidase (CPO) is a heme-containing glycoprotein secreted by the marine fungus Caldariomyces fumago. Chloroperoxidase contains one ferriprotoporphyrin IX prosthetic group per molecule and catalyzes a variety of reactions, such as halogenation, peroxidation and epoxidation. The versatile catalytic activities of CPO coupled with the increasing demands for chiral synthesis have attracted an escalating interest in understanding the mechanistic and structural properties of this enzyme. In order to better understand the mechanisms of CPO-catalyzed enantioselective reactions and to fine-tune the catalytic properties of chloroperoxidase, asparagine 74 (N74) located in the narrow substrate access channel of CPO was replaced by a bulky, nonpolar valine and a polar glutamine using site-directed mutagenesis. The CPO N74 mutants displayed significantly enhanced activity toward nonpolar substrates compared to wild-type CPO as a result of changes in space and polarity of the heme distal environment. More interestingly, N74 mutants showed dramatically decreased chlorination and catalase activity but significantly enhanced epoxidation activity as a consequence of improved kinetic perfection introduced by the mutation as reflected by the favorable changes in k cat and kcat/KM of these reactions. It is also noted that the N74V mutant is capable of decomposing cyanide, the most notorious poison for many hemoproteins, as judged by the unique binding behavior of N74V with potassium cyanide. Histidine 105 (H105) was replaced by a nonpolar amino acid alanine using site-directed mutagenesis. The CPO H105 mutant (H105A) displayed dramatically decreased chlorination and catalase activity possibly because of the decreased polarity in the heme distal environment and loss of the hydrogen bonds between histidine 105 and glutamic acid 183. However, significantly increased enantioselectivity was observed for the epoxidation of bulky styrene derivatives. Furthermore, my study provides strong evidence for the proposed histidine/cysteine ligand switch in chloroperoxidase, providing experimental support for the structure of the 420-nm absorption maximum for a number of carbon monoxide complexes of heme-thiolate proteins. For the NMR study, [dCPO(heme)] was produced using 90% deuterated growth medium with excess heme precursors and [dCPO(Phe)] was grown in the same highly deuterated medium that had been supplemented with excess natural phenylalanine. To make complete heme proton assignments, NMR spectroscopy has been performed for high-resolution structural characterization of [dCPO(heme)] and [dCPO(Phe)] to achieve unambiguous and complete heme proton assignments, which also allows important amino acids close to the heme active center to be determined.

Isoellipticines: novel compounds for leukaemia treatment

Ellipticine, an anticancer agent, has had limited clinical success due to low solubility and toxic side effects. To overcome these limitations, a panel of novel ellipticine isomers were designed and synthesised with the aim of evaluating their anti-cancer effects on selected cancer cell lines. A preliminary NCI 60-cell screen demonstrated that these isoellipticines displayed promising anti-tumour activity across a number of different cell types, particularly leukaemia cell lines. We consequently examined the effect of these derivatives in detail on the Acute Myeloid Leukaemia (AML) cell line, MV4-11. Cell cycle analyses revealed that the compounds had a range of distinctive cell cycle effects on MV4-11 cells. 7-Hydroxyisoellipticine showed the most promise with respect to cytostatic activity. We demonstrated that this compound inhibited proliferation of leukaemia cells by preventing cells from progressing from G2 phase. Our research suggests that this is mediated by an induction of reactive oxygen species (ROS), which in turn activates the DNA damage response pathway. More extensive research on the source of ROS generated by the most potent derivative, 7-formyl-10-methylisoellipticine showed that this compounds cytotoxicity is partially mediated by an induction of mitochondrial derived reactive oxygen species (ROS). We showed that 7-formyl-10-methylisoellipticine has synergistic effects when used in combination with the clinically used AML drug, daunorubicin, as well as DPI, a Nox inhibitor. Additionally, combination experiments with other drugs served to give us a deeper insight into 7- formyl-10-methylisoellipticine mechanism of action. 7-Formyl-10-methylisoellipticine also displayed promising in vivo results. Treatment resulted in a lack of toxicity, as measured by body weight changes and liver enzyme analyses. Most importantly, 7-formyl-10-methylisoellipticine demonstrated potent anti-tumour activity in the in vivo xenograft mouse model, implying the potential of isoellipticines as novel chemotherapeutic agents in the treatment of leukaemia. In summary, this study provides for the first time detailed cellular information on the potential use of isoellipticines as chemotherapeutic agents. Our study documents for the first time, the therapeutic potential of an isoellipticine compound in a subcutaneous AML cell-derived xenograft (CDX) model. By probing the mechanism of action of this novel compound class we have uncovered a potential clinical application in the field of adjuvant therapy. We anticipate that the recent research on ellipticine derivatives, such as this study, will lead the development of an ellipticine analogue that may be employed clinically.

(Table 1) Concentrations of amino acids in sediments from DSDP Site 68-502

In this chapter, we will report on the amino acids in the total acid hydrolysate of eight sediment samples from Leg 68 Site 502. This site was located on a topographic high at a depth of 3051 meters in the Colombian Basin of the western Caribbean Sea. Four holes were cored at the site by means of the hydraulic piston corer to a maximum sediment depth of 218 meters. The composite section is a virtually continuous, undisturbed sediment record covering almost 8 million years from the Holocene to late Miocene. Age estimates for the section are based on excellent magnetostratigraphic and biostratigraphic records. Four lithostratigraphic units (A, B, C, and D) were recognized, based on differences in color and content of clay, ash, foraminifers, and siliceous microfossils (Prell, Gardner, et al., 1980): A, yellowish brown to light brownish gray foraminifer-bearing (> 10%) nannofossil marl; B, gray to olive gray foraminifer-bearing nannofossil marl with occasional ash beds; C, light gray to dark greenish gray calcareous clay and foraminifer-bearing (< 10%) nannofossil marl; D, pale green to grayish green calcareous, ash-bearing clay with siliceous microfossils. The calcium carbonate content of these sediments increases from about 27 to about 49% from late Miocene to middle Pliocene (about 3.6 Ma) and remains uniform at about 48 to 50% from that time throughout the Quaternary. The eight sediment samples for amino acid analyses came from the third (502B) and fourth (502C) holes at Site 502. Samples ranged in sub-bottom depth from 4.3 to 225 meters spanning time from 0.3 to 7.7 Ma.