DNA double-strand break repair: a theoretical framework and its application

DNA double-strand breaks (DSBs) are formed as a result of genotoxic insults, such as exogenous ionizing radiation, and are among the most serious types of DNA damage. One of the earliest molecular responses following DSB formation is the phosphorylation of the histone H2AX, giving rise to γH2AX. Many copies of γH2AX are generated at DSBs and can be detected in vitro as foci using well-established immuno-histochemical methods. It has previously been shown that anti-γH2AX antibodies, modified by the addition of the cell-penetrating peptide TAT and a fluorescent or radionuclide label, can be used to visualize and quantify DSBs in vivo. Moreover, when labelled with a high amount of the short-range, Auger electron-emitting radioisotope, ¹¹¹In, the amount of DNA damage within a cell can be increased, leading to cell death. In this report, we develop a mathematical model that describes how molecular processes at individual sites of DNA damage give rise to quantifiable foci. Equations that describe stochastic mean behaviours at individual DSB sites are derived and parametrized using population-scale, time-series measurements from two different cancer cell lines. The model is used to examine two case studies in which the introduction of an antibody (anti-γH2AX-TAT) that targets a key component in the DSB repair pathway influences system behaviour. We investigate: (i) how the interaction between anti-γH2AX-TAT and γH2AX effects the kinetics of H2AX phosphorylation and DSB repair and (ii) model behaviour when the anti-γH2AX antibody is labelled with Auger electron-emitting ¹¹¹In and can thus instigate additional DNA damage. This work supports the conclusion that DSB kinetics are largely unaffected by the introduction of the anti-γH2AX antibody, a result that has been validated experimentally, and hence the hypothesis that the use of anti-γH2AX antibody to quantify DSBs does not violate the image tracer principle. Moreover, it provides a novel model of DNA damage accumulation in the presence of Auger electron-emitting ¹¹¹In that is supported qualitatively by the available experimental data.     

Genetic and other determinants of AMP deaminase activity in healthy adult skeletal muscle

AMPD1 genotype, relative fiber type composition, training status, and gender were evaluated as contributing factors to the reported variation in AMP deaminase enzyme activity in healthy skeletal muscle. Multifactorial correlative analyses demonstrate that AMPD1 genotype has the greatest effect on enzyme activity. An AMPD1 mutant allele frequency of 13.7 and a 1.7% incidence of enzyme deficiency was found across 175 healthy subjects. Homozygotes for the AMPD1 normal allele have high enzyme activities, and heterozygotes display intermediate activities. When examined according to genotype, other factors were found to affect variability as follows: AMP deaminase activity in homozygotes for the normal allele exhibits a negative correlation with the relative percentage of type I fibers and training status. Conversely, residual AMP deaminase activity in homozygotes for the mutant allele displays a positive correlation with the relative percentage of type I fibers. Opposing correlations in different homozygous AMPD1 genotypes are likely due to relative fiber-type differences in the expression of AMPD1 and AMPD3 isoforms. Gender also contributes to variation in total skeletal muscle AMP deaminase activity, with normal homozygous and heterozygous women showing only 85-88% of the levels observed in genotype-matched men.     

ExoY, an adenylate cyclase secreted by the Pseudomonas aeruginosa type III system

The exoenzyme S regulon is a set of coordinately regulated virulence genes of Pseudomonas aeruginosa. Proteins encoded by the regulon include a type III secretion and translocation apparatus, regulators of gene expression, and effector proteins. The effector proteins include two enzymes with ADP-ribosyltransferase activity (ExoS and ExoT) and an acute cytotoxin (ExoU). In this study, we identified ExoY as a fourth effector protein of the regulon. ExoY is homologous to the extracellular adenylate cyclases of Bordetella pertussis (CyaA) and Bacillus anthracis (EF). The homology among the three adenylate cyclases is limited to two short regions, one of which possesses an ATP-binding motif. In assays for adenylate cyclase activity, recombinant ExoY (rExoY) catalyzed the formation of cAMP with a specific activity similar to the basal activity of CyaA. In contrast to CyaA and EF, rExoY activity was not stimulated or activated by calmodulin. A 500-fold stimulation of activity was detected following the addition of a cytosolic extract from Chinese hamster ovary (CHO) cells. These results indicate that a eukaryotic factor, distinct from calmodulin, enhances rExoY catalysis. Site-directed mutagenesis of residues within the putative active site of ExoY abolished adenylate cyclase activity. Infection of CHO cells with ExoY-producing strains of P. aeruginosa resulted in the intracellular accumulation of cAMP. cAMP accumulation within CHO cells depended on an intact type III translocation apparatus, demonstrating that ExoY is directly translocated into the eukaryotic cytosol.     

Population parameters, sample statistics, and individual observations: A lesson in the law of small numbers.

Comments on the original article "Predoctoral internship training in Canada--I: Internship settings and supervisory issues," by J. L. Howes, T. M. Vallis, A. Wilson, M. Ross, and H. Louisy (see record 1996-06731-004). It has come to our attention that the data presented for Manitoba are misleading. It is stated (p. 175) that the average stipend in Manitoba decreased from 1992-93 to 1996-97. However, this is a statistical artifact due to the addition of a second program which paid less than the existing program. Rather than a reduction, the program participating in the 1992-93 survey actually increased its funding by $1,746 (from $28,671 to $30,417). A second program opened between '92-3 and '96-7 (with a stipend of $19,000). As well, on page 174 of our paper Manitoba's stipend is listed as $27,671 when it in fact was $28,671. We regret any confusion that reporting provincial averages may have caused. To clarify the funding issue, we reanalyzed our data on a program basis, not provincial basis (i.e., programs are the unit of measurement). We included only those 30 funded programs operating in 1992-93 in this re-analysis, the results of which are presented here. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Therapist competence ratings in relation to clinical outcome in cognitive therapy of depression.

This study reports on the relationship of therapist competence to the outcome of cognitive-behavioral treatment in the National Institute of Mental Health Treatment of Depression Collaborative Research Program. Outpatients suffering from major depressive disorder were treated by cognitive-behavioral therapists at each of 3 U.S. sites using a format of 20 sessions in 16 weeks. Findings provide some support for the relationship of therapist competence (as measured by the Cognitive Therapy Scale) to reduction of depressive symptomatology when controlling for therapist adherence and facilitative conditions. The results are, however, not as strong or consistent as expected. The component of competence that was most highly related to outcome is a factor that reflects the therapist's ability to structure the treatment. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Predoctoral internship training in Canada - I: Internship settings and supervisory issues.

Presents the results of our 1992 survey of internship directors and clinical supervisors in Canadian predoctoral internship training programs belonging to the Canadian Council of Professional Psychology Programs. 25 internship directors and 58 clinical supervisors responded to the survey. On average, individual programs offered 2.28 funded positions, at an average stipend of $20,269 per annum. Within the internship programs, a wide range of training opportunities were offered to interns, but limited opportunities existed for training in geriatric psychology, and in custody, competence, and forensic assessments. Over the 3 yrs surveyed there was a high degree of stability in terms of the number of applicants, number of interns interviewed, and acceptance rates. The majority of the clinical supervisors were well trained clinicians, but had received limited formal training in supervision. The supervisors were flexible in providing supervision in a wide range of training experiences, but with some limitations. Individual sessions were the most frequently employed form of supervision. The majority of supervisors reported no problems in supervision. The most frequently reported problems in supervision were related to interns' personal concerns and stress, and inadequate pre-internship training. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Inhibition of receptor-mediated endocytosis by the amphiphysin SH3 domain

BACKGROUND: Receptor-mediated endocytosis appears to require the GTP-binding protein dynamin, but the process by which dynamin is recruited to clathrin-coated pits remains unclear. Dynamin contains several proline-rich clusters that bind to Src homology 3 (SH3) domains, which are short modules found in many signalling proteins and which mediate protein-protein interactions. Amphiphysin, a protein that is highly expressed in the brain, interacts with dynamin in vitro, as do Grb2 and many other SH3 domain-containing proteins. In this study, we examined the role of amphiphysin in receptor-mediated endocytosis in vivo. RESULTS: To address the importance of the amphiphysin SH3 domain in dynamin recruitment, we used a transferrin and epidermal growth factor (EGF) uptake assay in COS-7 fibroblasts. Amphiphysin is present in these cells at a low level and indeed in other peripheral tissues. Confocal immunofluorescence revealed that cells transfected with the amphiphysin SH3 domain showed a potent blockade in receptor-mediated endocytosis. To test whether the cellular target of amphiphysin is dynamin, COS-7 cells were contransfected with both dynamin and the amphiphysin SH3 domain; here, transferrin uptake was efficiently rescued. Importantly, the SH3 domains of Grb2, phospholipase C gamma and spectrin all failed to exert any effect on endocytosis. The mechanism of amphiphysin action in recruiting dynamin was additionally tested in vitro: amphiphysin could associate with both dynamin and alpha-adaptin simultaneously, further supporting a role for amphiphysin in endocytosis. CONCLUSIONS: Our results suggest that the SH3 domain of amphiphysin recruits dynamin to coated pits in vivo, probably via plasma membrane adaptor complexes. We propose that amphiphysin is not only required for synaptic-vesicle endocytosis, but might also be a key player in dynamin recruitment in all cells undergoing receptor-mediated endocytosis.     

Crystal structure of the amphiphysin-2 SH3 domain and its role in the prevention of dynamin ring formation

The amphiphysins are brain-enriched proteins, implicated in clathrin-mediated endocytosis, that interact with dynamin through their SH3 domains. To elucidate the nature of this interaction, we have solved the crystal structure of the amphiphysin-2 (Amph2) SH3 domain to 2.2 A. The structure possesses several notable features, including an extensive patch of negative electrostatic potential covering a large portion of its dynamin binding site. This patch accounts for the specific requirement of amphiphysin for two arginines in the proline-rich binding motif to which it binds on dynamin. We demonstrate that the interaction of dynamin with amphiphysin SH3 domains, unlike that with SH3 domains of Grb2 or spectrin, prevents dynamin self-assembly into rings. Deletion of a unique insert in the n-Src loop of Amph2 SH3, a loop adjacent to the dynamin binding site, significantly reduces this effect. Conversely, replacing the n-Src loop of the N-terminal SH3 domain of Grb2 with that of Amph2 causes it to favour dynamin ring disassembly. Transferrin uptake assays show that shortening the n-Src loop of Amph2 SH3 reduces the ability of this domain to inhibit endocytosis in vivo. Our data suggest that amphiphysin SH3 domains are important regulators of the multimerization cycle of dynamin in endocytosis.     

Red wine quality: Correlations between colour,aroma and flavour and pigment and other parameters of young Beaujolais

In 15 young Beaujolais red wines from the 1974 and 1975 vintages statistically significant linear correlations were found between overall quality and the contents of total pigments, total anthocyanins, coloured anthocyanins and tasters' mean colour scores; and also between flavour and the contents of total pigments and total anthocyanins. In 1974 additional significant correlations were found between (a) overall quality and wine colour density; non-coloured anthocyanins and pH, (b) flavour and colour scores and coloured anthocyanins, (c) aroma and total pigments, total anthocyanins, coloured and non-coloured anthocyanins and pH and (d) the chemical parameters of pH and total pigments and total anthocyanins. In 1975 flavour was correlated also with non-coloured anthocyanins and pH. The results demonstrate the desirable effects of anthocyanins on wine flavour and quality, whereas the polymeric pigments formed on ageing of Beaujolais appear to be indifferent features of quality. Thus, the negative correlations found between quality factors and wine ?chemical age’? tend to support the opinion that most Beaujolais should be drunk young. Comparison of the results with data on Australian and Swiss red wines suggests that sulphur dioxide affects not only quality itself but also the nature of the quality correlation found. Simple colour measurement of acidified wine provides a useful indication of quality in young red wines of the same grape cultivar and age, having minimal sulphur dioxide content.     

The field of clinical psychology: Arriving at a definition.

Summarizes the development of the definition of the field of clinical psychology by the Executive and members of the Section on Clinical Psychology of the Canadian Psychological Association. The need for a definition and the process of drafting the definition are highlighted. Strategies for the use of the definition in advancing the field of clinical psychology are also addressed. The definition of clinical psychology is appended. (French abstract) (PsycINFO Database Record (c) 2010 APA, all rights reserved)