Switching the mode of metabolism in the yeast Saccharomyces cerevisiae

The biochemistry of most metabolic pathways is conserved from bacteria to humans, although the control mechanisms are adapted to the needs of each cell type. Oxygen depletion commonly controls the switch from respiration to fermentation. However, Saccharomyces cerevisiae also controls that switch in response to the external glucose level. We have generated an S. cerevisiae strain in which glucose uptake is dependent on a chimeric hexose transporter mediating reduced sugar uptake. This strain shows a fully respiratory metabolism also at high glucose levels as seen for aerobic organisms, and switches to fermentation only when oxygen is lacking. These observations illustrate that manipulating a single step can alter the mode of metabolism. The novel yeast strain is an excellent tool to study the mechanisms underlying glucose-induced signal transduction. © 2004 European Molecular Biology Organization.

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.

Investigation of the metabolism of N, N-Dimethylformamide and its metabolites

The industrial solvent N, N-dimethylformamide (DMF) causes liver damage in humans. The hepatotoxicity of N-alkylformamides seems to be linked to their metabolism to N-alkylcarbamic acid thioesters. To clarify the role of metabolism in DMF hepatotoxicity, the metabolic fate of DMF was investigated in rodents. DMF was rapidly metabolised and excreted in the urine as N-hydroxymethyl-N-methyl-formamide (HMMF), N-acetyl-S-(N-methylcarbamoyl) cysteine (AMCC) and a metabolite measured as formamide by GLC. At high doses (0.7 and 7.0mmo1/kg) a small proportion of the dose was excreted unchanged. AMCC, measured by GLC after derivatisation to ethyl N-methylcarbamate, was a minor metabolite. Only 5.2% of the dose (0.1mmo1/kg) in rats or 1.2% in mice was excreted as AMCC. The minor extent of this metabolic pathway in rodents might account for the marginal liver damage induced by DMF in these species. In a collaborative study, volunteers were shown to metabolise DMF to AMCC to a greater extent than rodents. Nearly 15% of the inhaled dose (0.049mmo1/kg) was excreted as AMCC. This result suggests that the metabolic pathway leading to AMCC is more important in humans than in rodents. Consequently the risk associated with exposure to DMF might be higher in humans than in rodents. The metabolism of formamides to S-(N-alkylcarbamoyl) glutathione, the metabolic precursor of the thioester mercapturates, was studied using mouse, rat and human hepatic microsomes. The metabolism of NMF (10mM) to S-(N-methylcarbanoyl)glutathione (SMG) required the presence of GSH, NADPH and air. Generation of S-(N-methyl-carbamoyl)glutathione (SMG) was inhibited when incubations were conducted in an atmosphere of CO:air (1:1) or when SKF 525-A (3.0mM) was included in the incubations. Pre-treatment of mice with phenobarbitone (PB, 80mg/kg for 4 days) or beta-naphthoflavone (BNF, 50mg/kg for 4 days) failed to increase the microsomal formation of SMG from NMF. This result suggests that the oxidation of NMF is catalysed by a cytochrome P-450 isozyme which is unaffected by PB or BNF. Microsomal incubations with DMF (5 or 10mM) failed to generate measurable amounts of SMG although DMF was metabolised to HMMF. Incubations of microsomes with HMMF resulted in the generation of a small amount of SMG which was affected by inhibitors of microsomal enzymes in the same way as in the case of NMF. HMMF was metabolised to AMCC by rodents in vivo. This result suggests that HMMF is a major intermediate in the metabolic activation of DMF.

Studies of the stability and metabolism of tumour inhibitory tetrazinones

Temozolomide is an imidazotetrazinone with antineoplastic properties. It is structurally related to dacarbazine. Temozolomide was not metabolized in vitro by liver fractions. Chemical decomposition appears to play an important r^ole in its in vitro and in vivo disposition. In contrast, 3-methylbenzotriazinone, a structural analogue, was metabolized by hepatic microsomes to afford benzotriazinone and a hydrophilic metabolite. The cytotoxicity of temozolomide, dacarbazine, 5-[3-(hydroxy-methyl-3-methyl-triazen-1-yl]imidazole-5-carboxamide (HMMTIC) and 3-monomethyl-(triazen-1-yl)imidazole-4-carboxamide (MTIC) were investigated in TLX5 murine lymphoma cells. Unlike dacarbazine, which was not toxic, MTIC, HMMTIC and temozolomide were cytotoxic in the absence of microsomes. Decarbazine was only cytotoxic in the presence of microsomes. The formation of MTIC from dacarbazine, HMMTIC and temozolomide was determined by reversed phase high performance liquid chromatography in mixtures incubated under conditions identical to those described before. MTIC was generated chemically from temozolomide and HMMTIC metabolically from dacarbazine. Using [14C]temozolomide, it was found that, in mice, the major route of excretion of the drug is via the kidneys. An acidic metabolite (metabolite I) was found in the urine of mice which had received temozolomide but its identity has not been established. 1H NMR, UV and chemical analyses revealed that Metabolite I possesses an intact NNN linkage and the site of metabolism is at the N3 methyl group. A further acidic metabolite (metabolite II) was found in the urine of patients. Metabolite II was unambiguously identified as the 8-carboxylic acid derivative of temozolomide. In vitro cytotoxicity assay showed that ony metabolite II is cytotoxic but not metabolite I. Pharmacokinetic studies of temozolomide and MTIC in vivo were performed on mice bearing TLX5 tumour. Temozolomide was eliminated from the plasma monophasically with a t1/2 of 0.7hr. MTIC was identified as a product of decomposition. MTIC was eliminated rapidly with a t1/2 of 2min. Though temozolomide shares many biochemical and biological similarities with clinically used dacarbazine, the results obtained in this study show that it differs markedly in its pharmacokinetic properties from dacarbazine, as temozolomide produced relatively sustained plasma levels which were reflected by drug concentrations in the tumour.

Effects of syllable structure in aphasic errors:implications for a new model of speech production

Current models of word production assume that words are stored as linear sequences of phonemes which are structured into syllables only at the moment of production. This is because syllable structure is always recoverable from the sequence of phonemes. In contrast, we present theoretical and empirical evidence that syllable structure is lexically represented. Storing syllable structure would have the advantage of making representations more stable and resistant to damage. On the other hand, re-syllabifications affect only a minimal part of phonological representations and occur only in some languages and depending on speech register. Evidence for these claims comes from analyses of aphasic errors which not only respect phonotactic constraints, but also avoid transformations which move the syllabic structure of the word further away from the original structure, even when equating for segmental complexity. This is true across tasks, types of errors, and, crucially, types of patients. The same syllabic effects are shown by apraxic patients and by phonological patients who have more central difficulties in retrieving phonological representations. If syllable structure was only computed after phoneme retrieval, it would have no way to influence the errors of phonological patients. Our results have implications for psycholinguistic and computational models of language as well as for clinical and educational practices.

Sampling and measurement methods for a study of childhood refractive error in a UK population

Background There is a paucity of data describing the prevalence of childhood refractive error in the United Kingdom. The Northern Ireland Childhood Errors of Refraction study, along with its sister study the Aston Eye Study, are the first population-based surveys of children using both random cluster sampling and cycloplegic autorefraction to quantify levels of refractive error in the United Kingdom. Methods Children aged 6–7 years and 12–13 years were recruited from a stratified random sample of primary and post-primary schools, representative of the population of Northern Ireland as a whole. Measurements included assessment of visual acuity, oculomotor balance, ocular biometry and cycloplegic binocular open-field autorefraction. Questionnaires were used to identify putative risk factors for refractive error. Results 399 (57%) of 6–7 years and 669 (60%) of 12–13 years participated. School participation rates did not vary statistically significantly with the size of the school, whether the school is urban or rural, or whether it is in a deprived/non-deprived area. The gender balance, ethnicity and type of schooling of participants are reflective of the Northern Ireland population. Conclusions The study design, sample size and methodology will ensure accurate measures of the prevalence of refractive errors in the target population and will facilitate comparisons with other population-based refractive data.

Non-invasive phakometric measurement of corneal and crystalline lens alignment in human eyes

We describe a non-invasive phakometric method for determining corneal axis rotation relative to the visual axis (β) together with crystalline lens axis tilt (α) and decentration (d) relative to the corneal axis. This does not require corneal contact A-scan ultrasonography for the measurement of intraocular surface separations. Theoretical inherent errors of the method, evaluated by ray tracing through schematic eyes incorporating the full range of human ocular component variations, were found to be larger than the measurement errors (β < 0.67°, α < 0.72° and d < 0.08 mm) observed in nine human eyes with known ocular component dimensions. Intersubject variations (mean ± S.D.: β = 6.2 ± 3.4° temporal, α = 0.2 ± 1.8° temporal and d = 0.1 ± 0.1 mm temporal) and repeatability (1.96 × S.D. of difference between repeat readings: β ± 2.0°, α ± 1.8° and d ± 0.2 mm) were studied by measuring the left eyes of 45 subjects (aged 18-42 years, 29 females and 16 males, 15 Caucasians, 29 Indian Asians, one African, refractive error range -7.25 to +1.25 D mean spherical equivalent) on two occasions. © 2005 The College of Optometrists.

Sensitivity of peptide conformational dynamics on clustering of a classical molecular dynamics trajectory

We investigate the sensitivity of a Markov model with states and transition probabilities obtained from clustering a molecular dynamics trajectory. We have examined a 500 ns molecular dynamics trajectory of the peptide valine-proline-alanine-leucine in explicit water. The sensitivity is quantified by varying the boundaries of the clusters and investigating the resulting variation in transition probabilities and the average transition time between states. In this way, we represent the effect of clustering using different clustering algorithms. It is found that in terms of the investigated quantities, the peptide dynamics described by the Markov model is sensitive to the clustering; in particular, the average transition times are found to vary up to 46%. Moreover, inclusion of nonphysical sparsely populated clusters can lead to serious errors of up to 814%. In the investigation, the time step used in the transition matrix is determined by the minimum time scale on which the system behaves approximately Markovian. This time step is found to be about 100 ps. It is concluded that the description of peptide dynamics with transition matrices should be performed with care, and that using standard clustering algorithms to obtain states and transition probabilities may not always produce reliable results.

Phonological–lexical activation:a lexical component or anoutput buffer? Evidence from aphasic errors

Single word production requires that phoneme activation is maintained while articulatory conversion is taking place. Word serial recall, connected speech and non-word production (repetition and spelling) are all assumed to involve a phonological output buffer. A crucial question is whether the same memory resources are also involved in single word production. We investigate this question by assessing length and positional effects in the single word repetition and reading of six aphasic patients. We expect a damaged buffer to result in error rates per phoneme which increase with word length and in position effects. Although our patients had trouble with phoneme activation (they made mainly errors of phoneme selection), they did not show the effects expected from a buffer impairment. These results show that phoneme activation cannot be automatically equated with a buffer. We hypothesize that the phonemes of existing words are kept active though permanent links to the word node. Thus, the sustained activation needed for their articulation will come from the lexicon and will have different characteristics from the activation needed for the short-term retention of an unbound set of units. We conclude that there is no need and no evidence for a phonological buffer in single word production.

Studies on the generalisation of Gaussian processes and Bayesian neural networks

The assessment of the reliability of systems which learn from data is a key issue to investigate thoroughly before the actual application of information processing techniques to real-world problems. Over the recent years Gaussian processes and Bayesian neural networks have come to the fore and in this thesis their generalisation capabilities are analysed from theoretical and empirical perspectives. Upper and lower bounds on the learning curve of Gaussian processes are investigated in order to estimate the amount of data required to guarantee a certain level of generalisation performance. In this thesis we analyse the effects on the bounds and the learning curve induced by the smoothness of stochastic processes described by four different covariance functions. We also explain the early, linearly-decreasing behaviour of the curves and we investigate the asymptotic behaviour of the upper bounds. The effect of the noise and the characteristic lengthscale of the stochastic process on the tightness of the bounds are also discussed. The analysis is supported by several numerical simulations. The generalisation error of a Gaussian process is affected by the dimension of the input vector and may be decreased by input-variable reduction techniques. In conventional approaches to Gaussian process regression, the positive definite matrix estimating the distance between input points is often taken diagonal. In this thesis we show that a general distance matrix is able to estimate the effective dimensionality of the regression problem as well as to discover the linear transformation from the manifest variables to the hidden-feature space, with a significant reduction of the input dimension. Numerical simulations confirm the significant superiority of the general distance matrix with respect to the diagonal one.In the thesis we also present an empirical investigation of the generalisation errors of neural networks trained by two Bayesian algorithms, the Markov Chain Monte Carlo method and the evidence framework; the neural networks have been trained on the task of labelling segmented outdoor images.

Profile of anisometropia and aniso-astigmatism in children; prevalence and association with age, ocular biometric measures, and refractive status

Purpose. We describe the profile and associations of anisometropia and aniso-astigmatism in a population-based sample of children. Methods. The Northern Ireland Childhood Errors of Refraction (NICER) study used a stratified random cluster design to recruit a representative sample of children from schools in Northern Ireland. Examinations included cycloplegic (1% cyclopentolate) autorefraction, and measures of axial length, anterior chamber depth, and corneal curvature. ?2 tests were used to assess variations in the prevalence of anisometropia and aniso-astigmatism by age group, with logistic regression used to compare odds of anisometropia and aniso-astigmatism with refractive status (myopia, emmetropia, hyperopia). The Mann-Whitney U test was used to examine interocular differences in ocular biometry. Results. Data from 661 white children aged 12 to 13 years (50.5% male) and 389 white children aged 6 to 7 years (49.6% male) are presented. The prevalence of anisometropia =1 diopters sphere (DS) did not differ statistically significantly between 6- to 7-year-old (8.5%; 95% confidence interval [CI], 3.9–13.1) and 12- to 13-year-old (9.4%; 95% CI, 5.9–12.9) children. The prevalence of aniso-astigmatism =1 diopters cylinder (DC) did not vary statistically significantly between 6- to 7-year-old (7.7%; 95% CI, 4.3–11.2) and 12- to 13-year-old (5.6%; 95% CI, 0.5–8.1) children. Anisometropia and aniso-astigmatism were more common in 12- to 13-year-old children with hyperopia =+2 DS. Anisometropic eyes had greater axial length asymmetry than nonanisometropic eyes. Aniso-astigmatic eyes were more asymmetric in axial length and corneal astigmatism than eyes without aniso-astigmatism. Conclusions. In this population, there is a high prevalence of axial anisometropia and corneal/axial aniso-astigmatism, associated with hyperopia, but whether these relations are causal is unclear. Further work is required to clarify the developmental mechanism behind these associations.

Quantitative analysis of multi-spectral fundus images

We have developed a new technique for extracting histological parameters from multi-spectral images of the ocular fundus. The new method uses a Monte Carlo simulation of the reflectance of the fundus to model how the spectral reflectance of the tissue varies with differing tissue histology. The model is parameterised by the concentrations of the five main absorbers found in the fundus: retinal haemoglobins, choroidal haemoglobins, choroidal melanin, RPE melanin and macular pigment. These parameters are shown to give rise to distinct variations in the tissue colouration. We use the results of the Monte Carlo simulations to construct an inverse model which maps tissue colouration onto the model parameters. This allows the concentration and distribution of the five main absorbers to be determined from suitable multi-spectral images. We propose the use of "image quotients" to allow this information to be extracted from uncalibrated image data. The filters used to acquire the images are selected to ensure a one-to-one mapping between model parameters and image quotients. To recover five model parameters uniquely, images must be acquired in six distinct spectral bands. Theoretical investigations suggest that retinal haemoglobins and macular pigment can be recovered with RMS errors of less than 10%. We present parametric maps showing the variation of these parameters across the posterior pole of the fundus. The results are in agreement with known tissue histology for normal healthy subjects. We also present an early result which suggests that, with further development, the technique could be used to successfully detect retinal haemorrhages.

Measurement and compensation of pivot point positional error on five-axis machines

Measuring and compensating the pivot points of five-axis machine tools is always challenging and very time consuming. This paper presents a newly developed approach for automatic measurement and compensation of pivot point positional errors on five-axis machine tools. Machine rotary axis errors are measured using a circular test. This method has been tested on five-axis machine tools with swivel table configuration. Results show that up to 99% of the positional errors of the rotary axis can be compensated by using this approach.

Using monthly returns to model conditional heteroscedasticity

This empirical study examines the extent of non-linearity in a multivariate model of monthly financial series. To capture the conditional heteroscedasticity in the series, both the GARCH(1,1) and GARCH(1,1)-in-mean models are employed. The conditional errors are assumed to follow the normal and Student-t distributions. The non-linearity in the residuals of a standard OLS regression are also assessed. It is found that the OLS residuals as well as conditional errors of the GARCH models exhibit strong non-linearity. Under the Student density, the extent of non-linearity in the GARCH conditional errors was generally similar to those of the standard OLS. The GARCH-in-mean regression generated the worse out-of-sample forecasts.