Biomedical subjects

A R Clarke

Publications and source records attributed to A R Clarke.

At least 37 records · Page 2Linked to original sources

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↗

PubMed2001

From efficacy to effectiveness: managing organisational change to improve health services for young people with deliberate self harm behaviour.

Repeat Deliberate Self Harm is a recognised risk factor for completed suicide and therefore reduction by effective health service response represents a valid contribution to suicide prevention. However, only a small fraction of people with deliberate self harm presentations to general health settings actually reach specialist mental health follow-up appointments. Therefore, even if responses at that point are known to be effective they do not make a significant contribution to reducing repeat self-harm overall. We describe health system organisational change strategies to improve health service engagement for the target group, and present data demonstrating the effectiveness of these strategies.

Adolescent↗

PubMed2001

Electroencephalogram differences in two subtypes of attention-deficit/hyperactivity disorder.

This study investigated EEG differences between children with two subtypes of Attention-Deficit/Hyperactivity Disorder (ADHD) and normal control subjects. EEG was recorded during an eyes-closed resting condition and Fourier transformed to provide absolute and relative power estimates for the delta, theta, alpha, and beta bands, and the mean frequency for each band was calculated. Ratio coefficients were also calculated between frequency bands. Mean group differences were found in the theta, alpha, and beta bands between all three groups. Similarly, differences were found between all three groups for the theta/alpha and theta/beta ratios and for the mean frequency of the total EEG. These results support a model of ADHD resulting from a developmental deviation rather than a maturational lag in the central nervous system. Differences between the clinical groups in frontal activity suggest that different neuroanatomical systems are involved in the different subtypes of ADHD.

Adolescent↗

PubMed2000

A novel role for IRF-1 as a suppressor of apoptosis.

The tumour suppressor IRF-1 is a transcription factor involved in the induction of apoptosis in several in vitro systems. Post-lactational involution of the mammary gland is characterized by extensive apoptosis of the epithelial cells. We have previously shown that signal transducer and activator of transcription (Stat) 3 drives apoptosis and involution in the mouse mammary gland. Since one of the downstream targets of the Stat signalling pathway is IRF-1, we have used IRF-1 knockout mice to address the potential role of this transcription factor in involution. Surprisingly, in the absence of IRF-1 significantly higher numbers of apoptotic cells were found in involuting glands at 48 h compared to control glands. In addition, the alveolar structure in IRF-1 null mammary glands had collapsed whereas in control glands the alveoli remained intact and distended. However, by 72 h control and null glands were morphologically similar suggesting that IRF-1 suppresses apoptosis only during the early, reversible, stage of involution. This suggests a survival role for IRF-1 in mammary epithelia and demonstrates a novel role for IRF-1 in vivo--suppression of premature epithelial apoptosis during mammary gland involution.

Animals↗

PubMed2000

Progressive stabilization of intermediate and transition states in protein folding reactions by introducing surface hydrophobic residues.

It can be argued from the principle of solvent exclusion that the introduction of hydrophobic residues onto the surface of a protein will not destabilize the folded state because the nonpolar side chain will be at least as exposed in the unfolded state as it is when the protein chain is folded. A comparison of the folding pathway of wild type and 11 site-directed mutants of CD2.d1 shows this to be true. In fact, owing to partial burial of nonpolar groups as folding proceeds, we find that the rapidly formed intermediate state and, to a greater extent, the transition state are generally stabilized by hydrophobic surface mutations. This effect is slightly moderated in the folded state presumably by the perturbation of van der Waals' contacts and/or local electrostatic interactions that have a greater influence in this fully compact structure. The fact that in all but one case we find that stabilization of the rapidly collapsed intermediate is accompanied by a faster acquisition of the folded state refutes the argument that I states are generally "off pathway" conformations or ensembles that lead to the inhibition of otherwise more rapid folding trajectories.

Amino Acid Substitution↗

PubMed2000

P53 null mice: damaging the hypothesis?

P53 is extremely well characterised as a tumour suppressor gene, and many activities have been attributed to it which are consistent with this function. However, despite being the subject of intense study it still remains unclear precisely which of these functions is crucial to its in vivo role as a tumour suppressor gene. This is particularly true of its role in the induction of apoptosis. The original observation of p53-dependent apoptosis gave rise to the following hypothesis: namely, that p53 deficiency leads to a persistence of DNA damaged cells which are the potential founders of malignancy. This review summarises the data for and against this hypothesis, with specific emphasis on data obtained from studies of the murine intestine. What emerges from these studies is a complex picture, where data can be obtained in support of this hypothesis, but there are many circumstances which exist where it is not supported. Taken together this collection of data suggests that the abrogation of p53-dependent apoptosis may indeed impact upon carcinogenesis and neoplastic progression, but that the simplistic notion of p53 as the single gatekeeper of this pathway is untenable.

Animals↗

PubMed2000

Domain rotations between open, closed and bullet-shaped forms of the thermosome, an archaeal chaperonin.

Three conformations of the thermosome, an archaeal group II chaperonin, have been determined by cryo-electron microscopy (EM). We describe an open form of the double-ring oligomer, a closed form and a bullet-shaped form with one ring open and the other closed. Domain movements have been deduced by docking atomic coordinates into the EM maps. The subunit apical domains, bearing the putative substrate binding sites, rotate about 30 degrees upwards and twist in the plane of the ring from the closed to the open conformation. The closed rings have their nucleotide binding pockets closed by the intermediate domains, but in the open rings, the pocket is accessible.

Archaea↗

PubMed2000

The major transition state in folding need not involve the immobilization of side chains.

During protein folding in which few, if any, definable kinetic intermediates are observable, the nature of the transition state is central to understanding the course of the reaction. Current experimental data does not distinguish the relative contributions of side chain immobilization and dehydration phenomena to the major rate-limiting transition state whereas this distinction is central to theoretical models that attempt to simulate the behavior of proteins during folding. Renaturation of the small proteinase inhibitor cystatin under oxidizing versus reducing conditions is the first experimental case in which these processes can be studied independently. Using this example, we show that sidechain immobilization occurs downstream of the major folding transition state. A consequence of this is the existence of states with disordered side chains, which are distinct from kinetic protein folding intermediates and which lie within the folded state free energy well.

Animals↗

PubMed2000

Effects of mutations on the thermodynamics of a protein folding reaction: implications for the mechanism of formation of the intermediate and transition states.

We have measured changes in heat capacity, entropy, and enthalpy for each step in the folding reaction of CD2.d1 and evaluated the effects of core mutations on these properties. All wild-type and mutant forms fold through a rapidly formed intermediate state that precedes the rate-limiting transition state. Mutations have a pronounced effect on the enthalpy of both the intermediate and folded states, but in all cases a compensatory change in entropy results in a small net free-energy change. While the enthalpy change in the folded state can be attributed to a loss of van der Waals interactions, it has already been shown that changes in the stability of the intermediate are dominated by changes in secondary structure propensity [Lorch et al. (1999) Biochemistry 38, 1377-1385]. It follows that the thermodynamic basis of beta-propensity is enthalpic in origin. The effects of mutations on the enthalpy and entropy of the transition state are smaller than on the ground states. This relative insensitivity to mutation is discussed in the light of theories concerning the nature of the rate-limiting barrier in folding reactions.

Animals↗

PubMed2000

Structural characterization of a methionine-rich, emulsifying protein from sunflower seed.

The 2 S seed storage protein, sunflower albumin 8, contains an unusually high proportion of hydrophobic residues including 16 methionines in a mature protein of 103 amino acids. A structural model, based on the known structure of a related protein, has been constructed as a four-helix bundle cross-linked by four disulphide bonds. This model structure is consistent with data from circular dichroism and nuclear magnetic resonance experiments. Analysis of the model's surface shows the presence of a large hydrophobic face that may be responsible for the highly stable emulsions this protein is known to form with oil/water mixtures.

2S Albumins, Plant↗