Oncogenic Ras deregulates cell-substrate interactions during mitotic rounding and respreading to alter cell division orientation.

Oncogenic Ras has been shown to change the way cancer cells divide by increasing the forces generated during mitotic rounding. In this way, RasV12 enables cancer cells to divide across a wider range of mechanical environments than normal cells. Here, we identify a further role for oncogenic Ras-ERK signaling in division by showing that RasV12 expression alters the shape, division orientation, and respreading dynamics of cells as they exit mitosis. Many of these effects appear to result from the impact of RasV12 signaling on actomyosin contractility, because RasV12 induces the severing of retraction fibers that normally guide spindle positioning and provide a memory of the interphase cell shape. In support of this idea, the RasV12 phenotype is reversed by inhibition of actomyosin contractility and can be mimicked by the loss of cell-substrate adhesion during mitosis. Finally, we show that RasV12 activation also perturbs division orientation in cells cultured in 2D epithelial monolayers and 3D spheroids. Thus, the induction of oncogenic Ras-ERK signaling leads to rapid changes in division orientation that, along with the effects of RasV12 on cell growth and cell-cycle progression, are likely to disrupt epithelial tissue organization and contribute to cancer dissemination.

Modified metacognitive therapy for anorexia nervosa: An open trial in an outpatient setting.

OBJECTIVE: Current evidence indicates treatment for adults with anorexia nervosa (AN) requires improvement given recovery rates are low to moderate, and relapse rates are high. Metacognitive therapy (MCT) is an effective treatment for anxiety and depressive disorders. This study evaluates if MCT can be successfully modified to treat AN in a naturalistic clinical setting. METHOD: Twenty-four patients with AN participated in an open trial of modified metacognitive therapy (MCT-AN). Twelve of the 24 patients (50%) completed treatment. MCT was modified to include components specific to eating disorders. The MCT-AN was delivered by clinical psychologists who had undertaken training in MCT in a specialist outpatient service. Group and single participant data analyses were undertaken on those who completed treatment. RESULTS: As well as statistically significant differences from pre- to posttreatment in the group data there were also clinically significant improvements at the individual patient level for eating disorder and depressive symptoms, as well as weight. The mean number of therapy sessions was 18. DISCUSSION: These findings indicate that MCT-AN may be a promising intervention in the treatment of AN, warranting further investigation. PUBLIC SIGNIFICANCE: Treatment for anorexia nervosa in adults requires improvement. Research indicates that Metacognitive therapy (MCT) is an effective treatment for anxiety and depression and may be applicable to the eating disorders. This small open trial suggests that MCT can be modified successfully to treat patients with anorexia nervosa (AN). The results are preliminary and require further research to provide more evidence on the effectiveness of this treatment for AN.

Psychosocial and financial impacts for carers of those with eating disorders in New Zealand.

BACKGROUND: Eating disorders (ED) can have profound effects on family members and carers. These impacts can be experienced across multiple domains and may contribute to the maintenance of ED symptoms. In the absence of any New Zealand studies quantifying this, and given country-specific differences in access to care and treatment, this study explores the psychosocial and economic impacts on those caring for someone with an ED in New Zealand. METHODS: Carers (N = 121) of those who had, or still had, a self-reported ED (82.6% anorexia nervosa) completed an online survey open between December 2016 and October 2020, adapted to the New Zealand context. Questions addressed ED recency and recovery status of the individual cared for, treatment access, and the financial and psychosocial impact on the carer. Data analysis included descriptive statistics, with financial cost data converted to the equivalent of 2020 New Zealand dollars. RESULTS: Most (88.6%) recruited carers reported still caring for someone with ED symptoms of varying severity. A majority reported difficulty accessing treatment for the person they cared for, with a sizable minority (45%) paying for private treatment, despite few having private insurance. Carer losses typically included reduced income and productivity, travel costs, and other miscellaneous costs. Carers reported significant psychosocial impacts across a range of dimensions including family life, interpersonal relationships, and their own personal well-being. CONCLUSIONS: Carers in New Zealand report impacts which are far reaching and longstanding, covering their own personal and interpersonal well-being and that of those around them. While most of those they care for get access to public (free) treatment at some time or another, the wider financial and economic impacts on carers are significant, and likely to take years to recoup. Though not unique to EDs, interventions and supports for carers are much needed in New Zealand, alongside more comprehensive research methodology to further determine positive and other impacts of EDs over the long course of the caregiving role. HIGHLIGHTS: A majority reported difficulty accessing treatment for the person they cared for 45% paid for private treatment, despite few having private insurance Carers reported reduced income and productivity, travel costs, and other costs. Carers reported significant psychosocial impacts on family life, interpersonal relationships, and their own personal well-being. Carers provide a pivotal role in supporting treatment and recovery in their family member with the These findings will be relevant for funders and service providers in developing further approaches to address barriers and gaps in service provision to reduce impacts on carers, and as a result, those with eating disorders.

Caring for a person with any significant health or disability condition can have a harmful effect on the carer's own wellbeing. This study estimates this burden of caring for people with either ongoing ED symptoms or who have recovered, in New Zealand. The study recruited 121 carers, of whom almost all (97.5%) were parents, with most caregivers reporting impacts as this related to caring for someone with anorexia nervosa (82.6%).Overall, the results found widespread impacts on carers, particularly impacts on multiple relationships in their life, especially family relationships. These impacts extended to reporting harmful effects on the relationship with the person with the ED. Carers are also burdened by difficulties accessing treatment and wider economic impacts such as loss of income and productivity at work. As New Zealand's health system is relatively unique and this is the first quantitative study in New Zealand exploring ED carer impacts, larger studies are needed to capture the full extent of this in order to better meet these needs both at a health service and government level.

Life is different now - impacts of eating disorders on Carers in New Zealand: a qualitative study.

BACKGROUND: Eating disorders are widely recognized as serious illnesses, with complex psychological and physiological comorbidities. Affected individuals face a protracted and challenging treatment journey which, particularly for children and adolescents, requires significant input from family members as carers. This study investigated the perspectives and experiences of those caring for family members with an eating disorder diagnosis. METHOD: Participants were carers of affected individuals with an eating disorder, and were recruited from an online survey, subsequently consenting to a semi-structured qualitative interview. Inductive thematic analysis was undertaken to identify themes. RESULTS: Most participants in the sample were parents of affected individuals. Significant and ongoing psychological and emotional impacts were identified across the sample. The diagnosis, treatment journey and overall impact of the carer role created a situation captured by the over-arching theme 'life is different now'. Impacts profoundly influenced relationships and were felt across all aspects of life by carers, affected individuals and other family members. Heightened worry and vigilance experienced by carers continued beyond improvement or recovery. CONCLUSIONS: This research highlighted challenging and often exhausting impacts felt by carers due to their pivotal role in eating disorder treatment. Inadequate support for most carers in this sample has clear implications for families as well as service and funding providers. Further research should more fully investigate carer experience with different eating disorders to explore the type of support necessary to build capacity and resilience to reduce carer burden.

Eating disorders (EDs) are serious illnesses with a challenging treatment journey that often requires significant input from family members as carers. This study investigated perspectives and experiences of those caring for family members with an ED diagnosis.Carers reported significant impacts on many facets of their lives. The findings revealed levels of emotional and psychological stress both during and beyond the treatment journey. For carers 'normal' life was profoundly impacted by the complicated and often unanticipated role of treatment provider, and navigating the maze of treatment while trying to understand the diagnosis itself. For those with other children, the impacts were compounded by the distress also witnessed in those children. Relationships were significantly affected by the demands of treatment and the serious consequences of the disorder, as well as misunderstandings and stigma related to EDs.A 'new normal' state of constant worry and ongoing vigilance defined life post-diagnosis. The results indicate the impacts of an ED diagnosis in a loved one, and the demands of caring for that person and assuming the role of treatment provider were life-changing. For these carers, 'life is different now'.

Eating disorders in New Zealand: Implications for Maori and health service delivery.

OBJECTIVE: Lifetime prevalence rates in Te Rau Hinengaro (The New Zealand Mental Health Survey) suggest eating disorders are at least as common in the Maori population as the non-Maori population, yet little is known at a population level about those accessing specialist mental health treatment for eating disorders in New Zealand. The aim of this study was to describe the population undergoing specialist mental health treatment for eating disorders and compare Maori and non-Maori clinical characteristics and service use. METHOD: This study uses the Programme for the Integration of Mental Health Data data set, managed by the New Zealand Ministry of Health to describe the characteristics of people with eating disorders and their use of specialist mental health services from 2009 to 2016. RESULTS: There were 3,835 individuals with a diagnosed eating disorder who had contact with specialist mental health services in this time period, 7% of whom were Maori. Within the cohort, Maori had a higher prevalence for a bulimia nervosa diagnosis, fewer diagnosed with anorexia nervosa, and a higher prevalence of other psychiatric comorbidity than non-Maori. DISCUSSION: There is discrepancy between the proportion of service users accessing specialist mental health services who are Maori and the assessed crude prevalence of eating disorders for Maori in national estimates. Once Maori are in specialist services; however, their use of services is comparable to non-Maori. Further research is needed to highlight the experiences of those Maori with eating disorders and address barriers to accessing services for Maori with eating disorders.

Heart rate, energy expenditure, and affective responses from children participating in trampoline park sessions compared with traditional extra-curricular sports clubs.

BACKGROUND: Lack of physical activity (PA) is becoming an issue in younger populations. Trampoline parks are newly popular environments for PA yet research on their use is scarce. Thus the present study compared heart rate, energy expenditure, and affective responses in children participating in trampoline park sessions compared with extracurricular sports clubs. METHODS: Children (aged 6-11 years; N.=16 females, N.=10 males) participated in 3 trampoline park sessions and 3 extracurricular sports club sessions lasting ~45 minutes over 3 weeks. Heart rate, energy expenditure, and affective responses through the circumplex model, were measured. RESULTS: Both conditions elicited moderate-vigorous PA. Average heart rate (mean difference [95%CIs]=27.6 fc [23.5 to 31.8]), peak heart rate (mean difference [95%CIs]=24.2 fc [20.8 to 27.6]) and energy expenditure (mean difference [95%CIs]=3.2 kcals.min-1 [2.7 to 3.6]) were all significantly higher for the trampoline sessions. Affective responses for both conditions elicited feelings of "excitement." CONCLUSIONS: Both extracurricular sports clubs and trampoline park activities provide moderate-vigorous PA, though the latter may result in higher heart rate and energy expenditure responses. Both however produce similar positive affective responses. As such, both could be valuable options for PA opportunities for children.

Everyday executive function impairments predict comorbid psychopathology in autism spectrum and attention deficit hyperactivity disorders.

OBJECTIVE: Autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD) both have psychiatric comorbidities and distinctive profiles of executive dysfunction. Although there is evidence that executive function (EF) plays a role in the expression of specific behaviors and psychiatric symptoms, it is not known whether specific EF deficits in ASD and ADHD may be pathways to comorbidities in the disorders. This study examines whether parent reported problems with flexibility in ASD and inhibition in ADHD mediate the disorders' associations with anxiety/depression and oppositional/aggressive behavior, respectively. METHOD: Parent report data from the Behavior Rating Inventory of Executive Function (BRIEF) and the Child Behavior Checklist (CBCL) were obtained for 125 children (70 ASD, 55 ADHD Hyperactive/Impulsive or Combined type) as part of a neuropsychological assessment. Diagnostic status, BRIEF Shift (shifting/flexibility) and Inhibit (behavioral inhibition) scale scores, and CBCL Anxious/Depressed (anxiety/depression) and Aggressive Behavior (oppositionality/aggression) scale scores were analyzed with a path analysis to investigate the relation of flexibility and inhibition to comorbid symptoms in children with ASD and ADHD. RESULTS: In a path model with good fit ASD predicted greater inflexibility which predicted greater anxiety/depression, while ADHD predicted greater disinhibition that predicted greater aggression, consistent with our mediational hypotheses. Unexpectedly, the greater inflexibility associated with ASD also predicted greater aggression. CONCLUSIONS: Findings support the importance of everyday EF problems in ASD and ADHD as predictors of comorbid psychopathology and as crucial intervention targets for potential prevention and mitigation of comorbid symptoms.

Characterizing the factor structure of parent reported executive function in autism spectrum disorders: the impact of cognitive inflexibility.

Parents of children with autism spectrum disorders (ASD) consistently report executive functioning (EF) deficits. This study investigates the factor structure of the Behavior Rating Inventory of Executive Function (BRIEF) as reported by parents of children with ASD and typically developing children (TDC). BRIEFs for 411 children with ASD and 467 TDC were examined. Confirmatory factor analysis of a nine-factor model met thresholds for goodness-of-fit in TDC, but not in the ASD sample. We found globally elevated EF problems in the ASD sample, especially on the Shift scale. These findings confirm that children with ASD exhibit significant EF deficits. Further investigation is needed to understand the pervasive nature of cognitive inflexibility in children with ASD.

Cognitive-behavioral therapy for bulimia nervosa and atypical bulimic nervosa: effectiveness in clinical settings.

OBJECTIVE: The efficacy of cognitive-behavioral therapy (CBT) for bulimic disorders has been established in research trials. This study examined whether that efficacy can be translated into effectiveness in routine clinical practice. METHOD: Seventy-eight adult women with bulimic disorders (bulimia nervosa and atypical bulimia nervosa) undertook individual CBT, with few exclusion criteria and a treatment protocol based on evidence-based approaches, utilizing individualized formulations. Patients completed measures of eating behaviors, eating attitudes, and depression pre- and post-treatment. Eight patients dropped out. The mean number of sessions attended was 19.2. RESULTS: No pretreatment features predicted drop-out. Treatment outcome was similar whether using treatment completer or intent to treat analyses. Approximately 50% of patients were in remission by the end of treatment. There were significant improvements in mood, eating attitudes, and eating behaviors. Reductions in bingeing and vomiting were comparable to efficacy trials. DISCUSSION: The improvements in this "real-world" trial of CBT for adults with bulimic disorders mirrored those from large, funded research trials, though the conclusions that can be reached are inevitably limited by the nature of the trial (e.g., lack of control group and therapy validation).

Impairments in real-world executive function increase from childhood to adolescence in autism spectrum disorders.

OBJECTIVE: Although several studies have investigated developmental trajectories of executive functioning (EF) in individuals with autism spectrum disorders (ASD) using lab-based tasks, no study to date has directly measured how EF skills in everyday settings vary at different ages. The current study seeks to extend prior work by evaluating age-related differences in parent-reported EF problems during childhood and adolescence in a large cross-sectional cohort of children with ASD. METHOD: Children (N = 185) with an ASD without intellectual disability participated in the study. Participants were divided into four groups based on age (5-7, 8-10, 11-13, and 14-18-year-olds). The four age groups did not differ in IQ, sex ratio, or autism symptoms. RESULTS: There were significant age effects (i.e., worsening scores with increasing age) in three of G. A. Gioia, P. K. Isquith, S. Guy, and L. Kenworthy's (2000) BRIEF: Behavior Rating Inventory of Executive Function, Odessa, FL, Psychological Assessment Resources scale scores: Initiate (p = .007), working memory (p = .003), and organization of materials (p = .023). In addition, analysis of the BRIEF scale profile revealed that, although multiple scales were elevated, the shift scale showed the greatest problems in both the youngest and oldest age cohorts. CONCLUSIONS: Older children with ASD show greater EF problems compared with the normative sample than younger children with ASD. Specifically, there is a widening divergence from the normative sample in metacognitive executive abilities in children with ASD as they age. This, in combination with significant, albeit more stable, impairments in flexibility, has implications for the challenges faced by high-functioning individuals with ASD as they attempt to enter mainstream work and social environments.

Persistence, perseveration and perfectionism in the eating disorders.

BACKGROUND: Perseveration, persistence and perfectionism are traits that have been suggested to be relevant to the eating disorders. This study explored the levels and correlates of these three traits in the eating disorders and control groups. METHOD: A measure of these three elements (the Persistence, Perseveration and Perfectionism Questionnaire - PPPQ-22) was administered to 99 women with eating disorders, 25 women with other psychiatric disorders, and 91 non-clinical women. Differences in PPPQ-22 scores across groups were measured, as were the associations between PPPQ-22 scores and eating attitudes. RESULTS: The eating disordered groups showed lower levels of persistence (the drive towards goal achievement) than the non-clinical group, but did not show higher levels of perseveration (the following of rules, without considering whether goals are achieved). Both women with eating disorders and non-clinical controls showed correlations between eating disorder symptoms and perseveration. CONCLUSIONS: The current study, using a relatively new measure, suggests that low levels of persistence, rather than high levels of perseveration, may be implicated in the eating disorders. It was less clear that perfectionism per se was a useful construct in understanding eating pathology. If confirmed by future research, persistence should be considered in treatment of these complex and challenging conditions.

Enzyme mediated silicon-oxygen bond formation; the use of Rhizopus oryzae lipase, lysozyme and phytase under mild conditions.

The potential for expanding the variety of enzymic methods for siloxane bond formation is explored. Three enzymes, Rhizopus oryzae lipase (ROL), lysozyme and phytase are reported to catalyse the condensation of the model compound, trimethylsilanol, formed in situ from trimethylethoxysilane, to produce hexamethyldisiloxane in aqueous media at 25 °C and pH 7. Thermal denaturation and reactant inhibition experiments were conducted to better understand the catalytic role of these enzyme candidates. It was found that enzyme activities were significantly reduced following thermal treatment, suggesting a potential key-role of the enzyme active sites in the catalysis. Similarly, residue-specific modification of the key-amino acids believed to participate in the ROL catalysis also had a significant effect on the silicon bio-catalysis, indicating that the catalytic triad of the lipase may be involved during the enzyme-mediated formation of the silicon-oxygen bond. E. coli phytase was found to be particularly effective at catalysing the condensation of trimethylsilanol in a predominantly organic medium consisting of 95% acetonitrile and 5% water. Whereas the use of enzymes in silicon chemistry is still very much a developing and frontier activity, the results presented herein give some grounds for optimism that the variety of enzyme mediated reactions will continue to increase and may one day become a routine element in the portfolio of the synthetic silicon chemist.

The biochemical basis for stereochemical control in polyketide biosynthesis.

One of the most striking features of complex polyketides is the presence of numerous methyl- and hydroxyl-bearing stereogenic centers. To investigate the biochemical basis for the control of polyketide stereochemistry and to establish the timing and mechanism of the epimerization at methyl-bearing centers, a series of incubations was carried out using reconstituted components from a variety of modular polyketide synthases. In all cases the stereochemistry of the product was directly correlated with the intrinsic stereospecificity of the ketoreductase domain, independent of the particular chain elongation domains that were used, thereby establishing that methyl group epimerization, when it does occur, takes place after ketosynthase-catalyzed chain elongation. The finding that there were only minor differences in the rates of product formation observed for parallel incubations using an epimerizing ketoreductase domain and the nonepimerizing ketoreductase domain supports the proposal that the epimerization is catalyzed by the ketoreductase domain itself.

Emotional awareness among eating-disordered patients: the role of narcissistic traits.

The narcissistic defences and a lack of emotional awareness (alexithymia) are both salient features of eating disorder pathology, as well as being linked to each other. As each of these characteristics impacts independently on treatment, it is important to understand how they interact within an eating-disordered population. The present study assessed the associations between the three core elements of alexithymia and the core and defensive elements of narcissism in this clinical group. Seventy eating-disordered patients completed standardised measures of alexithymia and narcissism, and multiple regression analyses were conducted in order to examine the relationship between these variables. Core narcissism (e.g. grandiosity, entitlement) was associated with difficulties in describing feelings to others, whereas the narcissistic defences were associated with difficulties in identifying feelings and distinguishing them from somatic experiences. These patterns of association suggest that different aspects of alexithymia are associated with different aspects of narcissism. Clinical suggestions are made for how these characteristics might require modifications of standard treatment approaches for the eating disorders.

Emotional awareness and core beliefs among women with eating disorders.

Patients with eating disorders have been shown to experience the emotional components of alexithymia-difficulties in identifying and describing emotions. In keeping with cognitive theories, which stress the role of schema-level beliefs in understanding emotions, this study examined the core beliefs that are associated with this difficulty in women with eating disorders. Seventy eating-disordered women completed standardised measures of core beliefs and alexithymia. There were no differences in alexithymia between diagnostic groups, so the women were treated as a single, transdiagnostic group. Multiple regression analyses showed specific patterns of association between the core beliefs and the emotional elements of alexithymia. Difficulties in identifying emotions were associated with entitlement beliefs, while difficulties in describing emotions were associated with both abandonment and emotional inhibition beliefs. These findings suggest that it may be necessary to work with core beliefs in order to reduce levels of alexithymia, prior to addressing the emotions that drive and maintain pathological eating behaviours.

Cognitive content and process in eating-disordered patients with obsessive-compulsive features.

OBJECTIVE: This study examined the schema-level cognitive content and processes that might explain the presence of compulsive behaviours among patients with eating disorders. METHOD: Each of 62 eating-disordered women completed standardised measures of schema-level core beliefs and cognitive processes, and was interviewed for diagnosis and obsessive compulsive features. RESULTS: Compulsive behaviours were associated with four core beliefs--'mistrust/abuse', 'defectiveness/shame', 'dependence/incompetence' and 'subjugation'. Higher levels of avoidance of emotional arousal were also associated with compulsivity. DISCUSSION: Schema-level beliefs and processes are associated with the presence of compulsivity. The key cognitions might impact via the activation of negative affect, meaning that the compulsive behaviours serve the function of emotion regulation. Such cognitions are likely to be a necessary target in CBT for those presenting with compulsive behaviours in the eating disorders.

Bacterial flavodoxins support nitric oxide production by Bacillus subtilis nitric-oxide synthase.

Unlike animal nitric-oxide synthases (NOSs), the bacterial NOS enzymes have no attached flavoprotein domain to reduce their heme and so must rely on unknown bacterial proteins for electrons. We tested the ability of two Bacillus subtilis flavodoxins (YkuN and YkuP) to support catalysis by purified B. subtilis NOS (bsNOS). When an NADPH-utilizing bacterial flavodoxin reductase (FLDR) was added to reduce YkuP or YkuN, both supported NO synthesis from either L-arginine or N-hydroxyarginine and supported a linear nitrite accumulation over a 30-min reaction period. Rates of nitrite production were directly dependent on the ratio of YkuN or YkuP to bsNOS. However, the V/Km value for YkuN (5.2 x 10(5)) was about 20 times greater than that of YkuP (2.6 x 10(4)), indicating YkuN is more efficient in supporting bsNOS catalysis. YkuN that was either photo-reduced or prereduced by FLDR transferred an electron to the bsNOS ferric heme at rates similar to those measured for heme reduction in the animal NOSs. YkuN supported a similar NO synthesis activity by a different bacterial NOS (Deinococcus radiodurans) but not by any of the three mammalian NOS oxygenase domains nor by an insect NOS oxygenase domain. Our results establish YkuN as a kinetically competent redox partner for bsNOS and suggest that FLDR/flavodoxin proteins could function physiologically to support catalysis by bacterial NOSs.

Trauma and multi-impulsivity in the eating disorders.

BACKGROUND: Multiple impulsive behaviours are common in the eating disorders, and multi-impulsive patients appear to do more poorly in treatment. However, comparatively little is known about the origins of multi-impulsivity in such cases. This study addresses the links between reported childhood trauma and multi-impulsivity in the eating disorders, examining whether specific types of trauma are predictive of specific impulsive behaviours in this population. METHOD: The sample consisted of 102 individuals who met strict criteria for an eating disorder, and who were interviewed regarding trauma history and comorbid impulsive behaviours. RESULTS: Any reported history of childhood trauma was associated with a higher number of impulsive behaviours and with the presence of multi-impulsivity. Childhood sexual abuse was particularly important, and was associated with self-cutting, alcohol abuse, and substance abuse (amphetamines, cocaine, cannabis and 'other substances', including ketamine and benzodiazepines). DISCUSSION: These findings indicate the importance of considering the psychological consequences of trauma during both assessment and treatment of the eating disorders. In particular, eating-disordered women who report a history of childhood sexual abuse should be examined for a pattern of comorbid impulsive behaviours.

Attributional style in the eating disorders.

Previous research has shown that patients with eating disorders have a characteristic cognitive bias, making internal attributions when evaluating negative events. However, there is less clarity about their attributions for positive events. There are suggestions that this cognitive style might be influenced by depressed mood. This study examines attributional style in the eating disorders for positive and negative events, independent of covariant effects of depression. Twenty-five eating-disordered women and 26 nonclinical women each completed measures of attributional style, depressed mood, and eating pathology. They also completed a measure of verbal intelligence (to ensure comparability of groups). Women with an eating disorder had a greater tendency to attribute negative situations to the self when compared with nonclinical women, even when differences in depressed mood were controlled for. There were no comparable differences in positive attributional biases. Women with an eating disorder adopt a self-blaming style when evaluating negative events, and such self-blame is likely to contribute to the maintenance of an eating disorder. This suggests that therapy for the eating disorders should include an element that focuses on highlighting and re-evaluating such interpretations.

Expression and characterization of the two flavodoxin proteins of Bacillus subtilis, YkuN and YkuP: biophysical properties and interactions with cytochrome P450 BioI.

The two flavodoxins (YkuN and YkuP) from Bacillus subtilis have been cloned, overexpressed in Escherichia coli and purified. DNA sequencing, mass spectrometry, and flavin-binding properties showed that both YkuN and YkuP were typical short-chain flavodoxins (158 and 151 amino acids, respectively) and that an error in the published B. subtilis genome sequence had resulted in an altered reading frame and misassignment of YkuP as a long-chain flavodoxin. YkuN and YkuP were expressed in their blue (neutral semiquinone) forms and reoxidized to the quinone form during purification. Potentiometry confirmed the strong stabilization of the semiquinone form by both YkuN and YkuP (midpoint reduction potential for oxidized/semiquinone couple = -105 mV/-105 mV) with respect to the hydroquinone (midpoint reduction potential for semiquinone/hydroquinone couple = -382 mV/-377 mV). Apoflavodoxin forms were generated by trichloroacetic acid treatment. Circular dichroism studies indicated that flavin mononucleotide (FMN) binding led to considerable structural rearrangement for YkuP but not for YkuN. Both apoflavodoxins bound FMN but not riboflavin avidly, as expected for short-chain flavodoxins. Structural stability studies with the chaotrope guanidinium chloride revealed that there is moderate destabilization of secondary and tertiary structure on FMN removal from YkuN, but that YkuP apoflavodoxin has similar (or slightly higher) stability compared to the holoprotein. Differential scanning calorimetry reveals further differences in structural stability. YkuP has a lower melting temperature than YkuN, and its endotherm is composed of a single transition, while that for YkuN is biphasic. Optical and fluorimetric titrations with oxidized flavodoxins revealed strong affinity (K(d) values consistently <5 microM) for their potential redox partner P450 BioI, YkuN showing tighter binding. Stopped-flow reduction studies indicated that the maximal electron-transfer rate (k(red)) to fatty acid-bound P450 BioI occurs from YkuN and YkuP at approximately 2.5 s(-1), considerably faster than from E. coli flavodoxin. Steady-state turnover with YkuN or YkuP, fatty acid-bound P450 BioI, and E. coli NADPH-flavodoxin reductase indicated that both flavodoxins supported lipid hydroxylation by P450 BioI with turnover rates of up to approximately 100 min(-1) with lauric acid as substrate. Interprotein electron transfer is a likely rate-limiting step. YkuN and YkuP supported monohydroxylation of lauric acid and myristic acid, but secondary oxygenation of the primary product was observed with both palmitic acid and palmitoleic acid as substrates.