Parallels between embryo and cancer cell metabolism.

A key characteristic of cancer cells is the ability to switch from a predominantly oxidative metabolism to glycolysis and the production of lactate even when oxygen is plentiful. This metabolic switch, known as the Warburg effect, was first described in the 1920s, and has fascinated and puzzled researchers ever since. However, a dramatic increase in glycolysis in the presence of oxygen is one of the hallmarks of the development of the early mammalian embryo; a metabolic switch with many parallels to the Warburg effect of cancers. The present review provides a brief overview of this and other similarities between the metabolism in tumours and early embryos and proposes whether knowledge of early embryo metabolism can help us to understand metabolic regulation in cancer cells.

The redox chemistry of the Alzheimer's disease amyloid beta peptide.

There is a growing body of evidence to support a role for oxidative stress in Alzheimer's disease (AD), with increased levels of lipid peroxidation, DNA and protein oxidation products (HNE, 8-HO-guanidine and protein carbonyls respectively) in AD brains. The brain is a highly oxidative organ consuming 20% of the body's oxygen despite accounting for only 2% of the total body weight. With normal ageing the brain accumulates metals ions such iron (Fe), zinc (Zn) and copper (Cu). Consequently the brain is abundant in antioxidants to control and prevent the detrimental formation of reactive oxygen species (ROS) generated via Fenton chemistry involving redox active metal ion reduction and activation of molecular oxygen. In AD there is an over accumulation of the Amyloid beta peptide (Abeta), this is the result of either an elevated generation from amyloid precursor protein (APP) or inefficient clearance of Abeta from the brain. Abeta can efficiently generate reactive oxygen species in the presence of the transition metals copper and iron in vitro. Under oxidative conditions Abeta will form stable dityrosine cross-linked dimers which are generated from free radical attack on the tyrosine residue at position 10. There are elevated levels of urea and SDS resistant stable linked Abeta oligomers as well as dityrosine cross-linked peptides and proteins in AD brain. Since soluble Abeta levels correlate best with the degree of degeneration [C.A. McLean, R.A. Cherny, F.W. Fraser, S.J. Fuller, M.J. Smith, K. Beyreuther, A.I. Bush, C.L. Masters, Soluble pool of Abeta amyloid as a determinant of severity of neurodegeneration in Alzheimer's disease, Ann. Neurol. 46 (1999) 860-866] we suggest that the toxic Abeta species corresponds to a soluble dityrosine cross-linked oligomer. Current therapeutic strategies using metal chelators such as clioquinol and desferrioxamine have had some success in altering the progression of AD symptoms. Similarly, natural antioxidants curcumin and ginkgo extract have modest but positive effects in slowing AD development. Therefore, drugs that target the oxidative pathways in AD could have genuine therapeutic efficacy.

Model studies of cholesterol and ascorbate oxidation by copper complexes: relevance to Alzheimer's disease beta-amyloid metallochemistry.

The neurotoxicity of the amyloid-beta peptide (Abeta) is causally linked to Alzheimer's disease (AD) and may be related to the redox chemistry associated with its interactions with copper ions and cholesterol in brain tissue. We have used density functional theory (DFT) calculations to study the mechanism controlling the Abeta/Cu catalyzed oxidation reactions of cholesterol and ascorbate using a model system. The computed results based on a binuclear Cu complex predict that oxidation of cholesterol (yielding 4-cholesten-3-one as a specific product) proceeds at a slow rate when catalyzed by a Abeta/Cu(II)|His-|Cu(II)/Abeta) aggregate. The computed results also suggest that monomeric Abeta/Cu(II) is not able to oxidize cholesterol. DFT also predicted that Abeta will cross-link via covalent dityrosine formation during the oxidation of ascorbate but not during the oxidation of cholesterol. Experimental data were consistent with these predictions.

Expression of the kynurenine pathway enzymes in human microglia and macrophages.

There is good evidence that the kynurenine pathway (KP) and one of its products, quinolinic acid (QUIN) play a role in the pathogenesis of neurological diseases. Monocytic cells are known to be the major producers of QUIN. However, macrophages have the ability to produce approximately 20 to 30-fold more QUIN than microglia. The molecular origin of this difference has not been clarified yet. Using unstimulated and IFN-gamma-stimulated cultures of human fcetal microglia and adult macrophages, we assayed mRNA expression of 8 key enzymes of the KP using RT-PCR and QUIN production using GC-MS. We found that after stimulation with IFN-gamma microglia produced de novo 20-fold less QUIN than macrophages. This quantitative difference in the ability to produce QUIN appears to be associated with a lower expression of 3 important enzymes of the KP in microglia: indoleamine 2,3-dioxygenase (IDO), kynureninase (KYNase) and kynurenine hydroxylase (KYN(OH)ase). These results suggest that activated infiltrating macrophages are the most potent QUIN producers during brain inflammatory diseases with playing a lesser role.

Quinolinic acid in the pathogenesis of Alzheimer's disease.

We propose that the tryptophan catabolites produced through the kynurenine pathway (KP), and more particularly quinolinic acid (QUIN), may play an important role in the pathogenesis of Alzheimer's disease (AD). In this study, we demonstrated that after 72 hours amyloid peptide (Abeta) 1-42 induced indoleamine 2,3-dioxygenase (IDO) expression and in a significant increase in production of QUIN by human macrophages and microglia. In contrast, Abeta11-40 and Prion peptide (PrP) 106-126 did not induce any significant increase in QUIN production. We also investigated the potential modulatory effect of QUIN and kynurenic acid (KYNA) on Abeta11-42 and Abeta1-40 aggregation. After 24 and 120 hours, we did not observe any significant difference in the level of aggregation compared to the control (Abeta alone). Abeta has been shown to induce IL1-beta mRNA expression by human foetal astrocytes and macrophages. We demonstrate that QUIN has the same effect. Interestingly, IL-1beta has been found in association with plaques in AD. All together these data imply that QUIN may be, locally, one of the factors involved in the pathogenesis of neuronal damage in AD.

Oxidative stress and therapeutic opportunities: focus on the Ewing's sarcoma family of tumors.

Reactive oxygen species (ROS) are highly reactive by-products of energy production that can have detrimental as well as beneficial effects. Unchecked, high levels of ROS result in an imbalance of cellular redox state and oxidative stress. High levels of ROS have been detected in most cancers, where they promote tumor development and progression. Many anticancer agents work by further increasing cellular levels of ROS, to overcome the antioxidant detoxification capacity of the cancer cell and induce cell death. However, adaptation of the level of cellular antioxidants can lead to drug resistance. The challenge for the design of effective cancer therapeutics exploiting oxidative stress is to tip the cellular redox balance to induce ROS-dependent cell death but without increasing the antioxidant activity of the cancer cell or inducing toxicity in normal cells.

Stereospecific interactions are necessary for Alzheimer disease amyloid-ß toxicity.

Previous studies suggest membrane binding is a key determinant of amyloid ß (Aß) neurotoxicity. However, it is unclear whether this interaction is receptor driven. To address this issue, a D-handed enantiomer of Aß42 (D-Aß42) was synthesized and its biophysical and neurotoxic properties were compared to the wild-type Aß42 (L-Aß42). The results showed D- and L-Aß42 are chemically equivalent with respect to copper binding, generation of reactive oxygen species and aggregation profiles. Cell binding studies show both peptides bound to cultured cortical neurons. However, only L-Aß42 was neurotoxic and inhibited long term potentiation indicating L-Aß42 requires a stereospecific target to mediate toxicity. We identified the lipid phosphatidylserine, as a potential target. Annexin V, which has very high affinity for externalized phosphatidylserine, significantly inhibited L-Aß42 but not D-Aß42 binding to the cultured cortical neurons and significantly rescued L-Aß42 neurotoxicity. This suggests that Aß mediated toxicity in Alzheimer disease is dependent upon Aß binding to phosphatidylserine on neuronal cells.

Histidine 14 modulates membrane binding and neurotoxicity of the Alzheimer's disease amyloid-beta peptide.

Amyloid-beta peptide (Abeta) toxicity is thought to be responsible for the neurodegeneration associated with Alzheimer's disease. While the mechanism(s) that modulate this toxicity are still widely debated, it has previously been demonstrated that modifications to the three histidine residues (6, 13, and 14) of Abeta are able to modulate the toxicity. Therefore to further elucidate the potential role of the histidine (H) residues in Abeta toxicity, we synthesized Abeta peptides with single alanine substitutions for each of the three histidine residues and ascertained how these substitutions affect peptide aggregation, metal binding, redox chemistry, and cell membrane interactions, factors which have previously been shown to modulate Abeta toxicity. Abeta{42} H13A and Abeta{42} H6A modified peptides were able to induce significant cell toxicity in primary cortical cell cultures at levels similar to the wild-type peptide. However, Abeta{42} H14A did not induce any measurable toxicity in the same cultures. This lack of toxicity correlated with the inability of the Abeta{42} H14A to bind to cell membranes. The interaction of Abeta with cell membranes has previously been shown to be dependent on electrostatic interactions between Abeta and the negatively charged head group of phosphatidylserine. Our data suggests that it is the imidazole sidechain of histidine 14 that modulates this interaction and strategies inhibiting this interaction may have therapeutic potential for Alzheimer's disease.

Copper-mediated amyloid-beta toxicity is associated with an intermolecular histidine bridge.

Amyloid-beta peptide (Abeta) is pivotal to the pathogenesis of Alzheimer disease. Here we report the formation of a toxic Abeta-Cu2+ complex formed via a histidine-bridged dimer, as observed at Cu2+/peptide ratios of >0.6:1 by EPR spectroscopy. The toxicity of the Abeta-Cu2+ complex to cultured primary cortical neurons was attenuated when either the pi -or tau-nitrogen of the imidazole side chains of His were methylated, thereby inhibiting formation of the His bridge. Toxicity did not correlate with the ability to form amyloid or perturb the acyl-chain region of a lipid membrane as measured by diphenyl-1,3,5-hexatriene anisotropy, but did correlate with lipid peroxidation and dityrosine formation. 31P magic angle spinning solid-state NMR showed that Abeta and Abeta-Cu2+ complexes interacted at the surface of a lipid membrane. These findings indicate that the generation of the Abeta toxic species is modulated by the Cu2+ concentration and the ability to form an intermolecular His bridge.

Methylation of the imidazole side chains of the Alzheimer disease amyloid-beta peptide results in abolition of superoxide dismutase-like structures and inhibition of neurotoxicity.

The toxicity of the amyloid-beta peptide (Abeta) is thought to be responsible for the neurodegeneration associated with Alzheimer disease. Generation of hydrogen peroxide has been implicated as a key step in the toxic pathway. Abeta coordinates the redox active metal ion Cu2+ to catalytically generate H2O2. Structural studies on the interaction of Abeta with Cu have suggested that the coordination sphere about the Cu2+ resembles the active site of superoxide dismutase 1. To investigate the potential role for such structures in the toxicity of Abeta, two novel Abeta40 peptides, Abeta40(HistauMe) and Abeta40(HispiMe), have been prepared, in which the histidine residues 6, 13, and 14 have been substituted with modified histidines where either the pi- or tau-nitrogen of the imidazole side chain is methylated to prevent the formation of bridging histidine moieties. These modifications did not inhibit the ability of these peptides to form fibrils. However, the modified peptides were four times more effective at generating H2O2 than the native sequence. Despite the ability to generate more H2O2, these peptides were not neurotoxic. Whereas the modifications to the peptide altered the metal binding properties, they also inhibited the interaction between the peptides and cell surface membranes. This is consistent with the notion that Abeta-membrane interactions are important for neurotoxicity and that inhibiting these interactions has therapeutic potential.

Metal ions, pH, and cholesterol regulate the interactions of Alzheimer's disease amyloid-beta peptide with membrane lipid.

The interaction of A beta peptides with the lipid matrix of neuronal cell membranes plays an important role in the pathogenesis of Alzheimer's disease. By using EPR and CD spectroscopy, we found that in the presence of Cu(2+) or Zn(2+), pH, cholesterol, and the length of the peptide chain influenced the interaction of these peptides with lipid bilayers. In the presence of Zn(2+), A beta 40 and A beta 42 both inserted into the bilayer over the pH range 5.5-7.5, as did A beta 42 in the presence of Cu(2+). However, A beta 40 only penetrated the lipid bilayer in the presence of Cu(2+) at pH 5.5-6.5; at higher pH there was a change in the Cu(2+) coordination sphere that inhibited membrane insertion. In the absence of the metals, insertion of both peptides only occurred at pH < 5.5. Raising cholesterol to 0.2 mol fraction of the total lipid inhibited insertion of both peptides under all conditions investigated. Membrane insertion was accompanied by the formation of alpha-helical structures. The nature of these structures was the same irrespective of the conditions used, indicating a single low energy structure for A beta in membranes. Peptides that did not insert into the membrane formed beta-sheet structures on the surface of the lipid.

Effector HIV-specific cytotoxic T-lymphocyte activity in long-term nonprogressors: associations with viral replication and progression.

Ex vivo effector cytotoxic T-lymphocyte (CTL) activity was assessed in 27 members of the Australian Long-Term Nonprogressor cohort and correlated with genetic, virological, and immunological markers. The 27 individuals were antiretroviral naive with CD4(+) T-cell counts of >500 cells/ microl for more than 8 years after human immunodeficiency virus type 1 (HIV-1) infection. Effector CTL activity was determined using a standard ex vivo chromium release assay. Individuals with CTL activity (HIV-1 env(IIIB) or pol or gag) were then compared to those without CTL activity in relation to plasma HIV-1 RNA, ICD p24 antigen, beta(2)-microglobulin, CD4 and CD8 T-cell counts, CCR5 and CCR2b genotypes, and progression to CD4 <500 cells/microl or commencement of antiretroviral treatment. Of the 27 individuals examined, 19 had no detectable effector CTL activity. The eight individuals with detectable CTL activity had significantly higher plasma levels of HIV-1 RNA (P = 0.014), immune complex dissociated p24 antigen (P = 0.006), and beta(2)-microglobulin (P = 0.009). There was increased risk of progression within 4 years of study entry in individuals with detectable effector CTL activity, higher plasma levels of HIV-1 RNA, higher beta(2)-microglobulin levels, and higher immune complex dissociated p24 antigen levels at enrollment (P = 0.017, P = 0.004, P = 0.027, P = 0.008 respectively). Multivariate analysis demonstrated viral load remained the strongest predictor of disease progression within this group (P = 0.017). There were no significant associations between CTL response and chemokine receptor genotype. These findings demonstrate the importance of HIV replication in generating an effector CTL response and show that effector CTL activity may be an early predictor of progression in people with long-term asymptomatic HIV infection.

Neurotoxic, redox-competent Alzheimer's beta-amyloid is released from lipid membrane by methionine oxidation.

The amyloid beta peptide is toxic to neurons, and it is believed that this toxicity plays a central role in the progression of Alzheimer's disease. The mechanism of this toxicity is contentious. Here we report that an Abeta peptide with the sulfur atom of Met-35 oxidized to a sulfoxide (Met(O)Abeta) is toxic to neuronal cells, and this toxicity is attenuated by the metal chelator clioquinol and completely rescued by catalase implicating the same toxicity mechanism as reduced Abeta. However, unlike the unoxidized peptide, Met(O)Abeta is unable to penetrate lipid membranes to form ion channel-like structures, and beta-sheet formation is inhibited, phenomena that are central to some theories for Abeta toxicity. Our results show that, like the unoxidized peptide, Met(O)Abeta will coordinate Cu2+ and reduce the oxidation state of the metal and still produce H2O2. We hypothesize that Met(O)Abeta production contributes to the elevation of soluble Abeta seen in the brain in Alzheimer's disease.