Biomedical subjects

A R Clarke

Publications and source records attributed to A R Clarke.

At least 19 recordsLinked to original sources

PubMed2002

Low frequency ultrasonic propagation through fibre reinforced, polymer composites.

This paper concentrates upon mesoscale variations observed in the time-of-flight (TOF) area scans of shear wave propagation through 'identically processed', injection moulded, glass fibre reinforced, polypropylene plaques. The effect of these structural variations on the derived 3D stiffness constants is discussed. Hence the random measurement errors associated with the stiffness constant measurements are differentiated from the intrinsic process-induced spatial variations. Interesting correlations between TOF and received amplitudes of shear wave propagation have been found and our tentative interpretation of these data in terms of mesostructural variations in the reinforcing fibre locations and fibre orientations is presented.

Journal Article↗

PubMed2002

Automated reconstruction of curvilinear fibres from 3D datasets acquired by X-ray microtomography.

The characterization of fibrous structures is important in both composites and textiles research for relating to the bulk properties of the material. However, the microscopic nature of the fibres and their high densities make them very difficult to characterize. Many techniques have been developed for the measurement and characterization of fibrous structures but they tend to be restricted to measurements on the sample surface or within physical cross-sections. X-ray microtomography can be used to non-destructively probe the internal structure of a range of fibrous materials, providing large amounts of 3D data. A technique has been developed for tracing fibres within 3D datasets acquired by X-ray microtomography and this has been applied to a glass fibre reinforced composite and also a non-woven textile sample. The 3D fibrous structures of both samples were successfully reconstructed and their fibre orientation distributions calculated. This technique enables novel characterizations, such as the through-thickness variation of fibre orientation in non-wovens.

Journal Article↗

PubMed2001

Suppression of intestinal and mammary neoplasia by lifetime administration of aspirin in Apc(Min/+) and Apc(Min/+), Msh2(-/-) mice.

Numerous studies have indicated that exposure to nonsteroidal anti-inflammatory drugs is associated with a lowered risk of colorectal cancer. However, analyses of the effect of aspirin upon tumorigenesis in Apc(Min/+) mice have yielded contrasting results. We show that adult dietary exposure to aspirin does not suppress intestinal tumorigenesis in Apc(Min/+) mice, but that continual exposure from the point of conception does. To test whether this regime could suppress the phenotype of murine models of hereditary nonpolyposis colorectal cancer, Msh2-deficient mice were exposed to aspirin. This did not modify the mutator phenotype of Msh2(-/-) mice, but weakly extended survival. Finally, we analyzed (Apc(Min/+), Msh2(-/-)) mice and found that lifetime aspirin exposure significantly delayed the onset of both intestinal and mammary neoplasia. Thus embryonic and perinatal exposure to aspirin suppresses neoplasia specifically associated with the loss of Apc function, opening a potential window of opportunity for nonsteroidal anti-inflammatory drug intervention.

Animals↗

PubMed2001

Excess beta activity in children with attention-deficit/hyperactivity disorder: an atypical electrophysiological group.

Studies of children with attention deficit/hyperactivity disorder (ADHD) have typically found elevated levels of slow wave activity in their EEGs, but in two of our previous studies, a small subset of ADHD children with excess beta activity in the EEG was identified. The aim of this study was to determine whether children with excess beta activity represent a distinct electrophysiological subtype of ADHD, to quantify the differences in their EEGs, and to determine if this group of children with ADHD have behavioural profiles different from other children with ADHD. Results indicated that children with excess beta represent a small independent subset of children diagnosed with ADHD, which primarily consists of children with a diagnosis of ADHD combined type. Behaviourally, this group was similar to other children with ADHD, although the excess-beta group were more prone to temper tantrums and to be moody. The excess in beta activity was found primarily in the frontal regions and may be associated with frontal lobe self-regulation and inhibition control.

Attention Deficit Disorder with Hyperactivity↗

PubMed2001

XAFS study of the high-affinity copper-binding site of human PrP(91-231) and its low-resolution structure in solution.

Here, we describe the structure of a C-terminal high-affinity copper-binding site within a truncated recombinant human PrP containing residues 91-231, which lacks the octapeptide repeat region. We show that at least two extra co-ordinating groups are involved in binding this copper(II) ion in conjunction with histidine residues 96 and 111 in a region of the molecule known to be critical in conferring strain type. In addition, using X-ray solution scattering, a low-resolution shape of PrP(91-231) is provided. The restored molecular envelope is consistent with the picture where the N-terminal segment, residues 91-120, extends out from the previously known globular domain containing residues 121-231.

Binding Sites↗

PubMed2001

Protochlorophyllide oxidoreductase: a homology model examined by site-directed mutagenesis.

An homology model of protochlorophyllide reductase (POR) from Synechocystis sp. was constructed on a template from the tyrosine-dependent oxidoreductase family. The model showed characteristics appropriate to a globular, soluble protein and was used to generate a structure of the ternary complex of POR, nicotinamide adenine dinucleotide phosphate (NADPH), and protochlorophyllide. The POR ternary model was validated by mutagenesis experiments involving predicted coenzyme-binding residues and by chemical modification experiments. A core tryptophan residue was shown to be responsible for much of the protein's fluorescence. Both quenching of this residue by coenzyme and fluorescence resonance energy transfer (FRET) from the protein to the coenzyme allowed the binding constant of NADPH to be determined. Replacement of this residue by Tyr gave an active mutant with approximately halved fluorescence and a negligible FRET signal, consistent with the role of this residue in energy transfer to the NADPH at the active site and with the model. The mechanism of the enzyme is discussed in the context of the model and semiempirical molecular orbital calculations.

Amino Acid Sequence↗

PubMed2001

Novel mechanism of hydrolysis of therapeutic beta-lactams by Stenotrophomonas maltophilia L1 metallo-beta-lactamase.

Stopped-flow tryptophan fluorescence under single turnover and pseudo-first-order conditions has been used to investigate the kinetic mechanism of beta-lactam hydrolysis by the Stenotrophomonas maltophilia L1 metallo-beta-lactamase. For the cephalosporin substrates nitrocefin and cefaclor and the carbapenem meropenem, a substantial quench of fluorescence is observed on association of substrate with enzyme. We have assigned this to a rearrangement event subsequent to formation of an initial collision complex. For the colorimetric compound nitrocefin, decay of this dark inter- mediate represents the overall rate-determining step for the reaction and is equivalent to decay of a previously observed state in which the beta-lactam amide bond has already been cleaved. For both cefaclor and meropenem, the rate-determining step for hydrolysis is loss of a second, less quenched state, in which, however, the beta-lactam amide bond remains intact. We suggest, therefore, that the mechanism of hydrolysis of nitrocefin by binuclear metallo-beta-lactamases may be atypical and that cleavage of the beta-lactam amide bond is the rate-determining step for breakdown of the majority of beta-lactam substrates by the L1 enzyme.

Catalysis↗

PubMed2001

Location and properties of metal-binding sites on the human prion protein.

Although a functional role in copper binding has been suggested for the prion protein, evidence for binding at affinities characteristic of authentic metal-binding proteins has been lacking. By presentation of copper(II) ions in the presence of the weak chelator glycine, we have now characterized two high-affinity binding sites for divalent transition metals within the human prion protein. One is in the N-terminal octapeptide-repeat segment and has a K(d) for copper(II) of 10(-14) M, with other metals (Ni(2+), Zn(2+), and Mn(2+)) binding three or more orders of magnitude more weakly. However, NMR and fluorescence data reveal a previously unreported second site around histidines 96 and 111, a region of the molecule known to be crucial for prion propagation. The K(d) for copper(II) at this site is 4 x 10(-14) M, whereas nickel(II), zinc(II), and manganese(II) bind 6, 7, and 10 orders of magnitude more weakly, respectively, regardless of whether the protein is in its oxidized alpha-helical (alpha-PrP) or reduced beta-sheet (beta-PrP) conformation. A role for prion protein (PrP) in copper metabolism or transport seems likely and disturbance of this function may be involved in prion-related neurotoxicity.

Amino Acid Sequence↗

PubMed2001

The A245K mutation exposes another stage of the bacterial L-lactate dehydrogenase reaction mechanism.

The A245K mutant of Bacillus stearothermophilus L-lactate dehydrogenase has been expressed in Escherichia coli and purified. A qualitative change in the reaction mechanism prior to the hydride transfer step in the reverse direction in the mutant is revealed. Both transient and steady state characteristics of the mutant are presented and show in contrast to the wild-type enzyme where a rearrangement of an enzyme-NADH-pyruvate complex is rate-limiting that in the mutant the rearrangement is much faster and hydride transfer is the first slow step. The steady state is limited by a new second slower conformation change involving an NAD+ complex. The mutation may provide a valuable framework for inhibitor and drug design research.

Alanine↗

PubMed2001

Msh-2 suppresses in vivo mutation in a gene dose and lesion dependent manner.

Mice deficient for the mismatch repair (MMR) gene Msh2 show accelerated tumourigenesis and a reduced apoptotic response to DNA damage of methylation type. Here we examine the effect of mutation for Msh2 on in vivo mutation frequencies in the intestine as determined by loss of function at the Dolichos biflorus (Dlb-1) locus. Spontaneous mutation frequencies were scored in cohorts of ageing mice either wild type or mutant for Msh2. In mice less than 1 year old, mutation frequencies were only elevated in Msh2 null mice. However, beyond this age heterozygous Msh2 mice showed significantly higher mutation frequencies than controls. These findings implicate a gene dose dependent requirement for Msh2 in mutation suppression and prompted an analysis of young Msh2 mutants following exposure to DNA damage. Following exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), Msh2 deficient mice show a reduced apoptotic response and an increase in mutation frequency. Heterozygotes did not differ from controls. Following exposure to cisplatin, no significant elevation was seen in mutation frequencies, even within homozygotes. This is particularly surprising given the association between cisplatin resistance and MMR deficiency. These findings therefore demonstrate a complex reliance upon functional Msh2 in mutation surveillance. We have identified three separate scenarios. First, where retention of both Msh2 alleles over an extended period of time appears critical to the suppression of spontaneous mutation; second, 3 weeks following exposure to MNNG, where only complete loss of Msh2 results in elevated mutation; and finally following cisplatin exposure, where induced levels of mutation are independent of Msh2 status.

Animals↗

PubMed2001

Ab initio protein structure prediction using physicochemical potentials and a simplified off-lattice model.

This study describes a computational method for ab inito protein structure prediction. Protein conformation has been modeled by using six optimized backbone torsion angles and fixed side chains approximating rotationally averaged real side chains. The approximations aim to keep complexity of the structure description to a minimum without seriously compromising the accuracy of the structural representation. An evolutionary Monte Carlo algorithm has been developed to search through this restricted conformational space to locate low-energy protein structures. A simple physicochemical force field has been developed to assess the energies of different conformations within this structural description. The corresponding residue interaction energies are based on hydrophobic, hydrophilic, steric, and hydrogen-bonding potentials. The search procedure has been used to locate native energy minima from primary sequence alone. The 3-D structures of polypeptides up to 38 residues with both beta and alpha secondary structural elements have been accurately predicted. The search procedure has been found to be highly efficient and follows an energetically and structurally plausible pathway to locate native populations. The simple force field described in the study has been compared with a more complex all-atom model and been found to be similarly effective in predicting the structures of proposed independent folding units. Proteins 2001;43:186-202.

Algorithms↗

PubMed2001

A role for mismatch repair in control of DNA ploidy following DNA damage.

Many reports have shown a link between mismatch repair (MMR) deficiency and loss of normal cell cycle control, particularly loss of G2 arrest. However almost all of these studies utilized transformed cell lines, and thus the involvement of other genes in this phenotype cannot be excluded. We have examined the effects of cisplatin treatment on primary embryo fibroblasts (MEFs) derived from mice in which the MMR gene Msh2 had been inactivated (Msh2(-/-)). This analysis determined that both primary Msh2(-/-) and wild type (WT) fibroblasts exhibited an essentially identical G2 arrest following cisplatin treatment. Similarly, we observed a cisplatin-induced G2 arrest in immortalized MMR deficient (Mlh1(-/-) and Pms2(-/-)) and WT MEFs. p53 deficient primary MEFs (p53(-/-)) exhibited both a clear G2 arrest and an increase in cells with a DNA content of 8N in response to cisplatin. When the Msh2 and p53 defects were combined (p53(-/-)/Msh2(-/-)) the G2 arrest was essentially identical to the p53(-/-) fibroblasts. However, the p53(-/-)/Msh2(-/-) fibroblasts demonstrated a further increase in cells with an 8N DNA content, above that seen in the p53(-/-) fibroblasts. These results suggest that loss of MMR on its own is not enough to overcome G2 arrest following exposure to cisplatin but does play a role in preventing polyploidization, or aberrant DNA reduplication, in the absence of functional p53.

Animals↗

PubMed2001

Identification and structure of the nerve growth factor binding site on TrkA.

Nerve growth factor (NGF) is involved in the development and maintenance of the nervous system and has been implicated as a possible therapeutic target molecule in a number of neurodegenerative diseases, especially Alzheimer's disease. NGF binds with high affinity to the extracellular region of a tyrosine kinase receptor, TrkA, which comprises three leucine-rich motifs (LRMs), flanked by two cysteine-rich clusters, followed by two immunoglobulin-like (Ig-like) domains. We have expressed the second Ig-like domain as a recombinant protein in E. coli and demonstrate that NGF binds to this domain with similar affinity to the native receptor. This domain (TrkAIg(2)) has the ability to sequester NGF in vitro, preventing NGF-induced neurite outgrowth, and in vivo, inhibiting NGF-induced plasma extravasation. We also present the three-dimensional structure of the TrkAIg(2) domain in a new crystal form, refined to 2.0 A resolution.

Animals↗

PubMed2001

The molecular biology of prion propagation.

Prion diseases such as Creutzfeldt-Jakob disease (CJD) in humans and scrapie and bovine spongiform encephalopathy (BSE) in animals are associated with the accumulation in affected brains of a conformational isomer (PrP(Sc)) of host-derived prion protein (PrP(C)). According to the protein-only hypothesis, PrP(Sc) is the principal or sole component of transmissible prions. The conformational change known to be central to prion propagation, from a predominantly alpha-helical fold to one predominantly comprising beta structure, can now be reproduced in vitro, and the ability of beta-PrP to form fibrillar aggregates provides a plausible molecular mechanism for prion propagation. The existence of multiple prion strains has been difficult to explain in terms of a protein-only infectious agent but recent studies of human prion diseases suggest that strain-specific phenotypes can be encoded by different PrP conformations and glycosylation patterns. The experimental confirmation that a novel form of human prion disease, variant CJD, is caused by the same prion strain as cattle BSE, has highlighted the pressing need to understand the molecular basis of prion propagation and the transmission barriers that limit their passage between mammalian species. These and other advances in the fundamental biology of prion propagation are leading to strategies for the development of rational therapeutics.

Animals↗

PubMed2001

Stability and folding rates of domains spanning the large A-band super-repeat of titin.

Titin is a very large (>3 MDa) protein found in striated muscle where it is believed to participate in myogenesis and passive tension. A prominent feature in the A-band portion of titin is the presence of an 11-domain super-repeat of immunoglobulin superfamily and fibronectin-type-III-like domains. Seven overlapping constructs from human cardiac titin, each consisting of two or three domains and together spanning the entire 11-domain super-repeat, have been expressed in Escherichia coli. Fluorescence unfolding experiments and circular dichroism spectroscopy have been used to measure folding stabilities for each of the constructs and to assign unfolding rates for each super-repeat domain. Immunoglobulin superfamily domains were found to fold correctly only in the presence of their C-terminal fibronectin type II domain, suggesting close and possibly rigid association between these units. The domain stabilities, which range from 8.6 to 42 kJ mol(-1) under physiological conditions, correlate with previously reported mechanical forces required to unfold titin domains. Individual domains vary greatly in their rates of unfolding, with a range of unfolding rate constants between 2.6 x 10(-6) and 1.2 s(-1). This variation in folding behavior is likely to be an important determinant in ensuring independent folding of domains in multi-domain proteins such as titin.

Amino Acid Sequence↗

PubMed2001

Age and sex effects in the EEG: differences in two subtypes of attention-deficit/hyperactivity disorder.

OBJECTIVES: This study investigated age-related changes and sex differences in the EEGs of two groups of children with attention-deficit/hyperactivity disorder (ADHD) combined type and ADHD predominantly inattentive type, in comparison with a control group of normal children. METHODS: Forty boys and forty girls were included in each group. The EEG was recorded during an eyes-closed resting condition and Fourier transformed to provide estimates for total power, absolute and relative power in the delta, theta, alpha and beta bands, and for theta/alpha and theta/beta ratios. RESULTS: Total power, relative alpha, and the theta/alpha and theta/beta ratios were differentiated between all 3 groups. Sex differences between the ADHD subjects and the control group were greater in males than females and matured faster in males. With increasing age, the EEG of the ADHD inattentive group was found to change at a similar rate to the changes found in the normal group, with the differences in power levels remaining constant. In the ADHD combined group, the power was found to change at a greater rate than in the ADHD inattentive group, with power levels of the two ADHD groups becoming similar with age. CONCLUSIONS: These results are supportive of a two-component model of ADHD, with the hyperactive/impulsive component maturing with age and the inattentive component remaining more stable.

Aging↗

PubMed2001

Age and sex effects in the EEG: development of the normal child.

OBJECTIVES: This study investigated age-related changes and sex differences in the EEGs of normal children. METHODS: Forty boys and 40 girls, between the ages of 8 and 12 years, participated in this study. The EEG was recorded during an eyes-closed resting condition and Fourier transformed to provide estimates for total power, absolute and relative power in the delta, theta, alpha and beta bands, and for theta/alpha and theta/beta ratios. RESULTS: Absolute delta activity decreased with age. Relative delta and theta decreased and alpha and beta increased with increasing age. The theta/alpha and theta/beta ratios decreased with increasing age. All of these indicated a developmental reduction in slow wave activity. Maturational differences were found in the rates of change between the midline and the two hemispheres. In the absolute delta and the theta/beta ratio, the midline and the two hemispheres became more equipotential with age. In the beta band, power increased at a greater rate than in the two hemispheres. Sex differences were found, with males having less theta and more alpha than females. CONCLUSIONS: These results indicated that maturation occurs earlier at the midline than in the two hemispheres. Females were also found to have a developmental lag in the EEG compared with males.

Age Factors↗

PubMed2001

EEG-defined subtypes of children with attention-deficit/hyperactivity disorder.

OBJECTIVES: This study investigated the presence of EEG clusters within a sample of children with the combined type of attention-deficit/hyperactivity disorder (ADHD). METHODS: Subjects consisted of 184 boys with ADHD and 40 age-matched controls. EEG was recorded from 21 sites during an eyes-closed resting condition and Fourier transformed to provide estimates for total power, and relative power in the delta, theta, alpha and beta bands, and for the theta/beta ratio. Factor analysis was used to group sites into 3 regions, covering frontal, central and posterior regions. These data were subjected to cluster analysis. RESULTS: Three distinct EEG clusters of children with ADHD were found. These were characterized by (a) increased slow wave activity and deficiencies of fast wave, (b) increased high amplitude theta with deficiencies of beta activity, and (c) an excess beta group. CONCLUSIONS: These results indicate that children with ADHD do not constitute a homogenous group in EEG profile terms. This has important implications for studies of the utility of EEG in the diagnosis of ADHD. Efforts aimed at using EEG as a tool to discriminate ADHD children from normals must recognize the variability within the ADHD population if such a tool is to be valid and reliable in clinical practice.

Attention Deficit Disorder with Hyperactivity↗