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Oxidative stress has long been implicated in Parkinson's disease (PD) pathogenesis, although the sources and regulation of reactive oxygen species (ROS) production are poorly defined. Pathogenic mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are associated with increased kinase activity and a greater risk of PD. The substrates and downstream consequences of elevated LRRK2 kinase activity are still being elucidated, but overexpression of mutant LRRK2 has been associated with oxidative stress, and antioxidants reportedly mitigate LRRK2 toxicity. Here, using CRISPR-Cas9 gene-edited HEK293 cells, RAW264.7 macrophages, rat primary ventral midbrain cultures, and PD patient-derived lymphoblastoid cells, we found that elevated LRRK2 kinase activity was associated with increased ROS production and lipid peroxidation and that this was blocked by inhibitors of either LRRK2 kinase or NADPH oxidase 2 (NOX2). Oxidative stress induced by the pesticide rotenone was ameliorated by LRRK2 kinase inhibition and was absent in cells devoid of LRRK2. In a rat model of PD induced by rotenone, a LRRK2 kinase inhibitor prevented the lipid peroxidation and NOX2 activation normally seen in nigral dopaminergic neurons in this model. Mechanistically, LRRK2 kinase activity was shown to regulate phosphorylation of serine-345 in the p47 phox subunit of NOX2. This, in turn, led to translocation of p47 phox from the cytosol to the membrane-associated gp91 phox (NOX2) subunit, activation of the NOX2 enzyme complex, and production of ROS. Thus, LRRK2 kinase activity may drive cellular ROS production in PD through the regulation of NOX2 activity.

Background: Children with Down syndrome present with behavioural and emotional difficulties, including noncompliance, rule-breaking, emotion dysregulation and delays in executive functioning. Few behavioural interventions have been designed specifically for children with Down syndrome. The Research Units in Behavioral Intervention (RUBI) Parent Training for Disruptive Behaviors is a structured empirically supported parent training programme developed for caregivers of children with autism. This feasibility trial explored the feasibility and acceptability of an abbreviated RUBI intervention with caregivers of children with Down syndrome and identified promising outcome measures to target in future larger clinical trials., Method: A double-blind randomised feasibility pilot clinical trial allocated participants to a behavioural intervention (BEH) or educational (EDU) group. BEH and EDU consisted of five individual sessions over the course of 5 to 8 weeks. Measures were administered to 20 caregivers and their youth with Down syndrome at three time points., Results: Both BEH and EDU were rated as feasible with high parental adherence and acceptable with high treatment satisfaction. Both BEH and EDU demonstrated decreased externalising behaviours, irritability and hyperactivity and improved behavioural regulation in executive functioning over time. No impact was noted on caregiver functioning., Conclusion: The feasibility trial has strong findings regarding feasibility and satisfaction and has promising findings regarding the selection of measures for future trials testing an adapted RUBI programme and an education programme to reduce behavioural challenges in children with Down syndrome. Larger scale clinical trials are needed to confirm promising findings of these feasible treatments., (© 2024 The Author(s). Journal of Intellectual Disability Research published by MENCAP and John Wiley & Sons Ltd.)

Idiopathic Parkinson's disease (PD) is epidemiologically linked with exposure to toxicants such as pesticides and solvents, which comprise a wide array of chemicals that pollute our environment. While most are structurally distinct, a common cellular target for their toxicity is mitochondrial dysfunction, a key pathological trigger involved in the selective vulnerability of dopaminergic neurons. We and others have shown that environmental mitochondrial toxicants such as the pesticides rotenone and paraquat, and the organic solvent trichloroethylene (TCE) appear to be influenced by the protein LRRK2, a genetic risk factor for PD. As LRRK2 mediates vesicular trafficking and influences endolysosomal function, we postulated that LRRK2 kinase activity may inhibit the autophagic removal of toxicant damaged mitochondria, resulting in elevated oxidative stress. Conversely, we suspected that inhibition of LRRK2, which has been shown to be protective against dopaminergic neurodegeneration caused by mitochondrial toxicants, would reduce the intracellular production of reactive oxygen species (ROS) and prevent mitochondrial toxicity from inducing cell death. To do this, we tested in vitro if genetic or pharmacologic inhibition of LRRK2 (MLi2) protected against ROS caused by four toxicants associated with PD risk - rotenone, paraquat, TCE, and tetrachloroethylene (PERC). In parallel, we assessed if LRRK2 inhibition with MLi2 could protect against TCE-induced toxicity in vivo, in a follow up study from our observation that TCE elevated LRRK2 kinase activity in the nigrostriatal tract of rats prior to dopaminergic neurodegeneration. We found that LRRK2 inhibition blocked toxicant-induced ROS and promoted mitophagy in vitro, and protected against dopaminergic neurodegeneration, neuroinflammation, and mitochondrial damage caused by TCE in vivo. We also found that cells with the LRRK2 G2019S mutation displayed exacerbated levels of toxicant induced ROS, but this was ameliorated by LRRK2 inhibition with MLi2. Collectively, these data support a role for LRRK2 in toxicant-induced mitochondrial dysfunction linked to PD risk through oxidative stress and the autophagic removal of damaged mitochondria., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

Mutations in leucine-rich repeat kinase 2 (LRRK2) that increase its kinase activity are strongly linked to genetic forms of Parkinson's disease (PD). However, the regulation of endogenous wild-type (WT) LRRK2 kinase activity remains poorly understood, despite its frequent elevation in idiopathic PD (iPD) patients. Various stressors such as mitochondrial dysfunction, lysosomal dyshomeostasis, or vesicle trafficking deficits can activate WT LRRK2 kinase, but the specific molecular mechanisms are not fully understood. We found that the production of 4-hydroxynonenal (4-HNE), a lipid hydroperoxidation end-product, is a common biochemical response to these diverse stimuli. 4-HNE forms post-translational adducts with Cys2024 and Cys2025 in the kinase activation loop of WT LRRK2, significantly increasing its kinase activity. Additionally, we discovered that the 4-HNE responsible for regulating LRRK2 is generated by the action of 15-lipoxygenase (15-LO), making 15-LO an upstream regulator of the pathogenic hyperactivation of LRRK2 kinase activity. Pharmacological inhibition or genetic ablation of 15-LO prevents 4-HNE post-translational modification of LRRK2 kinase and its subsequent pathogenic hyperactivation. Therefore, 15-LO inhibitors, or methods to lower 4-HNE levels, or the targeting of Cys2024/2025 could provide new therapeutic strategies to modulate LRRK2 kinase activity and treat PD.

This study evaluated the appropriateness of scoring the Behavior Rating Inventory of Executive Function-Preschool (BRIEF-P) using age-equivalent scores generated from multiple measures of cognition and language among school-age children with Down syndrome (DS). Subscale T scores for 95 children with DS were contrasted using standard scoring on the Behavior Rating Inventory of Executive Function-Second edition (BRIEF-2; based on chronological age) to alternate scoring using the BRIEF-P (based on age-equivalent) for patterns of subscale intercorrelations, differences in mean scores, and agreement on findings from clinical cut-off scores. Results with children with DS suggested using (1) the BRIEF-P for children ages 2-5 years old, (2) the BRIEF-2 with chronological-age scoring or the BRIEF-P with age-equivalent scoring (with some caveats) for research on children ages 5-10 years old, and (3) the BRIEF-2 for children ages 11 and older., (©AAIDD.)

Background: Inhibitory control measures have been commonly used when assessing individuals with Down syndrome. However, minimal attention has been devoted to evaluating the appropriateness of specific assessments for use in this population, potentially leading to erroneous conclusions. This study aimed to examine the psychometric properties of measures of inhibitory control among youth with Down syndrome. We sought to examine the feasibility, presence of floor or practice effects, test-retest reliability, convergent validity and correlations with broader developmental domains of a set of inhibitory control tasks., Methods: A sample of 97 youth with Down syndrome aged 6 to 17 years old participated in verbal and visuospatial tasks of inhibitory control including the Cat/dog Stroop, Neuropsychological Assessment Second Edition (NEPSY-II) Statue, National Institutes of Health (NIH) Toolbox Cognition Battery (TCB) Flanker, Leiter-3 Attention Sustained, and the Test of Attentional Performance for Children (KiTAP) Go/No-go and Distractibility subtests. Youth also completed standardised assessments of cognition and language, and caregivers completed rating scales. Psychometric properties on the tasks of inhibitory control were evaluated against a priori criteria., Results: Apart from demonstrating negligible practice effects, adequate psychometric properties were not observed for any inhibitory control measure within the current sample's age range. One task with low working memory demands (NEPSY-II Statue) generally had better psychometric properties than the other tasks assessed. Subgroups of participants with an IQ greater than 30 and age more than 8 years were shown to be more likely to be able to complete the inhibition tasks., Conclusions: Findings suggest better feasibility for analogue tasks rather than computerised assessments of inhibitory control. Given the weak psychometrics of several common measures, future studies are required to evaluate other inhibitory control measures, specifically those with reduced working memory demands for youth with Down syndrome. Recommendations for use of the inhibitory control tasks among youth with Down syndrome are provided., (© 2023 The Authors. Journal of Intellectual Disability Research published by MENCAP and International Association of the Scientific Study of Intellectual and Developmental Disabilities and John Wiley & Sons Ltd.)

Objectives: Obstructive sleep apnea (OSA) is highly prevalent among individuals with Down syndrome (DS), and the nonphysiological consequences of OSA require examination to inform treatment planning. This study aimed to investigate the association between OSA and aspects of language, executive functioning, behavioral, social abilities, and sleep problems in youth with DS aged 6 to 17 years., Methods: Multivariate analysis of covariance was used to compare 3 groups adjusted for age, participants with DS with untreated OSA (n = 28), participants with DS without OSA (n = 38), and participants with DS with treated OSA (n = 34). To be eligible for the study, participants had to have an estimated mental age of 3 years. No children were excluded based on estimated mental age., Results: After adjusting for age, participants with untreated OSA showed a common pattern of lower estimated marginal mean scores than those with treated OSA and those with no OSA in expressive and receptive vocabulary and higher estimated marginal mean scores with executive functions, everyday memory, attention, internalizing and externalizing behavior, social behavior, and sleep problems. However, only the group differences for executive function (emotional regulation) and internalizing behavior were statistically significant., Conclusion: Study findings corroborate and extend prior findings related to OSA and clinical outcomes for youth with DS. The study highlights the importance of OSA treatment in youth with DS and provides clinical recommendations for this population. Additional studies are necessary to control the effects of health and demographic variables., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Reliable and valid cognitive outcome measures, including examiner-administered and computer-facilitated assessments of processing speed and reaction time, are necessary for future clinical trials that include individuals with Down syndrome (DS). The current study evaluated the score distributions and psychometric properties of four examiner-administered and three computerized processing speed and reaction time measures. Participants included 97 individuals with DS, aged 6 to 17 (M = 12.6, SD = 3.3). Two examiner-administered measures (Differential Ability Scales-II Rapid Naming and Cat/dog Stroop Congruent) met most predetermined psychometric criteria. Other assessments demonstrated good test-retest reliability and had negligible practice effects but lacked adequate feasibility. Recommendations for using processing speed and reaction time assessments in research and suggestions for modifications of measures are discussed.

Mitochondrial dysfunction and oxidative stress are strongly implicated in Parkinson's disease (PD) pathogenesis and there is evidence that mitochondrially-generated superoxide can activate NADPH oxidase 2 (NOX2). Although NOX2 has been examined in the context of PD, most attention has focused on glial NOX2, and the role of neuronal NOX2 in PD remains to be defined. Additionally, pharmacological NOX2 inhibitors have typically lacked specificity. Here we devised and validated a proximity ligation assay for NOX2 activity and demonstrated that in human PD and two animal models thereof, both neuronal and microglial NOX2 are highly active in substantia nigra under chronic conditions. However, in acute and sub-acute PD models, we observed neuronal, but not microglial NOX2 activation, suggesting that neuronal NOX2 may play a primary role in the early stages of the disease. Aberrant NOX2 activity is responsible for the formation of oxidative stress-related post-translational modifications of α-synuclein, and impaired mitochondrial protein import in vitro in primary ventral midbrain neuronal cultures and in vivo in nigrostriatal neurons in rats. In a rat model, administration of a brain-penetrant, highly specific NOX2 inhibitor prevented NOX2 activation in nigrostriatal neurons and its downstream effects in vivo, such as activation of leucine-rich repeat kinase 2 (LRRK2). We conclude that NOX2 is an important enzyme that contributes to progressive oxidative damage which in turn can lead to α-synuclein accumulation, mitochondrial protein import impairment, and LRRK2 activation. In this context, NOX2 inhibitors hold potential as a disease-modifying therapy in PD., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

This study evaluates the psychometric properties of a verbal fluency task for potential use as an outcome measure in future clinical trials involving children with Down syndrome. Eighty-five participants attempted a modified version of the Neuropsychological Assessment of Children, Second Edition Word Generation Task at two time points. In the full sample, the measure fell below a priori reliability and feasibility criteria, though feasibility of the semantic trials were higher than feasibility of the phonemic trials. Performance on the measure correlated with chronological age and IQ scores, and no sex-related effects were found. Additional analyses suggested that the semantic verbal fluency trials might be appropriate for children with Down syndrome 10 years of age and older., (©AAIDD.)

Parents of 30 school-age children with Down syndrome participated in a small-scale randomized clinical trial of a behavioral sleep treatment designed specifically for children with Down syndrome. The aim was to improve child sleep, child daytime behavior problems, caregiver sleep, and caregiver stress. The intervention spanned 5-8 weeks, and assessments occurred pre-treatment, immediately post-treatment, and three months post-treatment using a double-blinded design. Both the active treatment and a treatment-as-usual attention-controlled comparison group showed improvements in actigraphy and parent-report measures of child sleep, parent-reported child internalizing behaviors, and actigraphy measures of parent-sleep. The behavioral sleep treatment did not yield significantly different outcomes than a treatment-as-usual approach supplemented with non-sleep-specific behavioral or education sessions. Possible interpretations of study findings are discussed., (©AAIDD.)

Background: Improving short-term memory (STM) performance for individuals with Down syndrome (DS) has been a target of recent clinical trials. Validation of STM outcome measures is essential for research rigor in trials among children and adolescents with DS., Aims: The current study investigated the psychometric properties of four direct STM assessments and one everyday memory parent form., Methods and Procedures: Measures were administered to a sample of 74 youth with DS at two visits, two weeks apart. Overall cognitive abilities were also assessed., Outcomes and Results: The OMQ-PF had good feasibility and distribution of scores, but floor effects were prominent for direct measures. Test-retest reliability was poor to moderate for all measures and practice effects were problematic for the NEPSY-II List Memory and DAS-II Recall of Objects subtests. Commonalities in responses were observed, including primacy/recency effects, and some STM scores were correlated with overall cognitive abilities., Conclusions and Implications: The OMQ-PF met most study criteria, but no direct measure met sufficient criteria to be strongly recommended for future clinical trials. Because higher cognitive abilities were related to assessment completion, STM measures may require adaptation for use in broader samples of youth with DS across all levels of cognitive ability., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Expressive language delays and executive functioning challenges are common in youth with Down syndrome (DS). Verbal fluency is one method to investigate these constructs. We examined semantic verbal fluency responses to determine patterns in response generation and the psychometric properties of coded cluster formations. Participants were 97 children and adolescents with DS ranging in age from 6 to 19 years old. The semantic verbal fluency task was administered at two time points, two weeks apart. Heterogeneity in performance was observed for responses when coded either with conventional or contextual classifications. Overall, the number of switches in conventional classifications was greater than contextual classifications. This implies that participants did not use traditional (conventional) categories to organize their semantic verbal fluency responses, but may have been using contextual strategies. However, the number of switches and cluster size variables had poor to moderate test-retest reliability, which indicated that participants did not stay consistent with their performance over the two-week testing interval, regardless of the strategies used. Therefore, conventional and contextual clusters and switches as a measure of executive control may not be appropriate for all individuals with DS and additional attention is warranted to determine the utility of response coding in this population.

Missense mutations in leucine rich-repeat kinase 2 (LRRK2) cause forms of familial Parkinson's disease and have been linked to 'idiopathic' Parkinson's disease. Assessment of LRRK2 kinase activity has been very challenging due to its size, complex structure, and relatively low abundance. A standard in the field to assess LRRK2 kinase activity is to measure the level of substrate phosphorylation (pThr73-Rab10) or autophosphorylation of serine 1292 ( i.e ., phosphoserine 1292; pS1292). The levels of pS1292 have typically been assessed by western blotting, which limits cellular and anatomical resolution. Here, we describe the method for a novel proximity ligation assay (PLA) that can detect endogenous LRRK2 kinase activity (PLA LRRK2) in situ at cellular and subcellular resolutions. PLA is a fluorescence- or chromogen-based assay that can be used to either (1) detect protein-protein interactions or (2) detect and amplify post-translational modifications on proteins. We used PLA for in situ detection and amplification of LRRK2 autophosphorylation levels at serine 1292. Our findings demonstrate that PLA LRRK2 is a highly sensitive and specific assay that can be used for assessing kinase activity in cultured cells and postmortem tissues., Competing Interests: Competing interestsThe authors declare they have no competing interests., (Copyright © 2021 The Authors; exclusive licensee Bio-protocol LLC.)

Sleep problems have a bi-directional impact on the daytime performance of children, parental well-being, and overall family functioning in the general population. Children with Down syndrome (DS) are at a high risk of sleep problems, yet the relationship between sleep problems, adaptive functioning, and family stress in children with DS is not well documented. We examined the relationship between sleep (i.e., duration and quality) and child and parent/family functioning. Sixty-six children with DS wore an actigraph for a week to assess their sleep duration and sleep efficiency. Their parents completed ratings on child sleep duration and parasomnias, child adaptive functioning, parental depression and sleep, and family stress. The parents' reports of their children's sleep duration were associated with parental depressive symptoms. The parents' reports of their children's restless sleep behaviors were associated with poorer performances in child-compliant/calm behaviors, worse parental sleep, and negative parental feelings and sibling relationships. The findings from actigraph measures of the children's sleep demonstrated that greater sleep efficiency was associated with greater child adaptive functioning and fewer parental depressive symptoms. The study findings provide preliminary evidence that sleep problems are related to child adaptive functioning, parental functioning, and family stress in children with DS.

The current study evaluates the concurrent relationship between parent ratings of executive functioning and maladaptive behavior among children and adolescents with Down syndrome and then repeats this evaluation using teacher reports. Parents and teachers of 63 school-age children with Down syndrome rated the child's executive functioning (Behavior Rating Inventory of Executive Function) and behaviors (Achenbach Child Behavior Checklist). For parent and teacher ratings, elevated behavior dysregulation predicted higher levels of rule-breaking, aggressive, and externalizing behavior. For teacher ratings, elevated behavior dysregulation also predicted higher levels of inattention problems. Among both parent and teacher ratings, greater metacognitive difficulties predicted challenges with attention. Understanding the relationship between these constructs has important implications for targets of intervention and developing preventative strategies., (©AAIDD.)

Individuals with Down syndrome (DS) are often described as socially engaged; however, challenges with social cognition, expressive language, and social interaction are also common in DS and are prospective outcomes of interest for clinical trials. The current study evaluates the psychometric properties of standardized measurements of social cognition and social behavior for potential use as outcome measures for children and adolescents with DS. Seventy-three youth ages 6 to 17 years old (M = 12.67, SD = 3.16) with DS were assessed on social cognition subtests of a neuropsychological assessment at two time points. Caregivers also completed a parent-report measure of social behavior. Measures were evaluated for feasibility, test-retest reliability, practice effects, convergent validity, and associations with broader developmental domains (i.e., age, cognition, and language). All social cognition and behavior measures met criteria for a portion of the psychometric indices evaluated, yet feasibility limitations were identified for the Developmental Neuropsychological Assessment, Second Edition (NEPSY-II) Affect Recognition subtest, and the NEPSY-II Theory of Mind subtest had problematic floor effects for percentile ranks. The Social Responsiveness Scale, Second Edition (SRS-2; T-scores) had high feasibility, moderate to excellent test-retest reliability, and no practice effects, suggesting this measure could be appropriate for use in clinical trials involving youth with DS.

The current study evaluates the psychometric properties of the Behavior Rating Inventory of Executive Function (BRIEF) with children with Down syndrome. Caregivers of 84 children with Down syndrome rated their child's behavior with the BRIEF. Teacher ratings were obtained for 57 children. About 40% of children with Down syndrome were reported by parents, and 70% by teachers, to exhibit clinically significant challenges with executive functioning. Distribution of scores was normal, internal consistency for subscales was questionable to primarily excellent, and inter-rater reliability was poor to good. Normative data conversions controlled for age, IQ, and gender differences, with some exceptions. The study findings suggest that the BRIEF and its subscales generally performed in a psychometrically sound manner among children with Down syndrome.

Background: Behavioural problems are common among children with Down syndrome (DS). Tools to detect and evaluate maladaptive behaviours have been developed for typically developing children and have been evaluated for use among children with intellectual and developmental disabilities. However, these measures have not been evaluated for use specifically in children with DS. This psychometric evaluation is important given that some clinically observed behaviours are not addressed in currently available rating scales. The current study evaluates the psychometric properties of the Child Behavior Checklist (CBCL), a commonly used screening tool developed for typically developing children and commonly used with children with intellectual and developmental disabilities., Methods: The study investigated the psychometric properties of the CBCL among school-aged children with DS, including an assessment of the rate of detecting behaviour problems, concerns with distribution, internal consistency, inter-rater reliability and convergent and discriminant validity with the Aberrant Behavior Checklist and Nisonger Child Behavior Rating Form. Caregivers of 88 children with DS aged 6-18 years rated their child's behaviour with the CBCL, Aberrant Behavior Checklist and Nisonger Child Behavior Rating Form. Teachers completed the Teacher Report Form., Results: About one-third of children with DS were reported to exhibit behaviours of clinical concern on the total score of the CBCL. Internal consistency for CBCL sub-scales was poor to excellent, and inter-rater reliability was generally acceptable. The sub-scales of the CBCL performed best when evaluating convergent validity, with variable discriminant validity. Normative data conversions controlled for age and gender differences in this sample., Conclusions: The study findings suggest that, among children with DS, some CBCL sub-scales generally performed in a psychometrically sound and theoretically appropriate manner in relation to other measures of behaviour. Caution is warranted when interpreting specific sub-scales (Anxious/Depressed, Somatic Complaints and Thought Problems). The CBCL can continue to be used as a screening measure when evaluating behavioural concerns among children with DS, acknowledging poor discriminant validity and the possibility that key behaviour concerns in DS may not be captured by the CBCL screen., (© 2018 MENCAP and International Association of the Scientific Study of Intellectual and Developmental Disabilities and John Wiley & Sons Ltd.)

Missense mutations in leucine-rich repeat kinase 2 (LRRK2) cause familial Parkinson's disease (PD). However, a potential role of wild-type LRRK2 in idiopathic PD (iPD) remains unclear. Here, we developed proximity ligation assays to assess Ser1292 phosphorylation of LRRK2 and, separately, the dissociation of 14-3-3 proteins from LRRK2. Using these proximity ligation assays, we show that wild-type LRRK2 kinase activity was selectively enhanced in substantia nigra dopamine neurons in postmortem brain tissue from patients with iPD and in two different rat models of the disease. We show that this occurred through an oxidative mechanism, resulting in phosphorylation of the LRRK2 substrate Rab10 and other downstream consequences including abnormalities in mitochondrial protein import and lysosomal function. Our study suggests that, independent of mutations, wild-type LRRK2 plays a role in iPD. LRRK2 kinase inhibitors may therefore be useful for treating patients with iPD who do not carry LRRK2 mutations., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background: Sleep problems have an impact on executive functioning in the general population. While children with Down syndrome (DS) are at high risk for sleep problems, the impact of these sleep problems on executive functioning in school-age children with DS is less well documented. Our study examined the relationship between parent-reported and actigraphy-measured sleep duration and sleep quality with parent and teacher reports and neuropsychology assessments of executive functioning among school-age children with DS., Method: Thirty school-age children with DS wore an actigraph watch for a week at home at night. Their parent completed ratings of the child's sleep during that same week. Children completed a neuropsychology assessment of their inhibitory control, ability to shift and working memory. Their parents and teachers completed rating scales to assess these same constructs of executive functioning., Results: Parent reports of restless sleep behaviours on the Children's Sleep Habits Questionnaire (CSHQ), but not actigraph-measured sleep period or efficiency, were predictive of parent reports of concerns with inhibitory control, shifting and working memory, and of teacher reports of inhibitory control. No measure of sleep was predictive of executive functioning as measured by the neuropsychology assessment., Conclusion: The study findings corroborate the preliminary literature that parent-reported sleep problems are related to executive functioning in school-age children with DS, particularly in the area of inhibitory control across home and school. These findings have implications for understanding contributing factors to academic performance and school behaviour in school-age children with DS., (© 2018 MENCAP and International Association of the Scientific Study of Intellectual and Developmental Disabilities and John Wiley & Sons Ltd.)

Background: There is a need for rigorous measures of sleep in children with Down syndrome as sleep is a substantial problem in this population and there are barriers to obtaining the gold standard polysomnography (PSG). PSG is cost-prohibitive when measuring treatment effects in some clinical trials, and children with Down syndrome may not cooperate with undergoing a PSG. Minimal information is available on the validity of alternative methods of assessing sleep in children with Down syndrome, such as actigraphy and parent ratings. Our study examined the concurrent and convergent validity of different measures of sleep, including PSG, actigraphy and parent reports of sleep among children with Down syndrome., Method: A clinic (n = 27) and a community (n = 47) sample of children with Down syndrome were examined. In clinic, children with Down syndrome wore an actigraph watch during a routine PSG. In the community, children with Down syndrome wore an actigraph watch for a week at home at night as part of a larger study on sleep and behaviour. Their parent completed ratings of the child's sleep during that same week., Results: Actigraph watches demonstrated convergent validity with PSG when measuring a child with Down syndrome's total amount of sleep time, total wake time after sleep onset and sleep period efficiency. In contrast, actigraph watches demonstrated poor correlations with parent reports of sleep, and with PSG when measuring the total time in bed and total wake episodes. Actigraphy, PSG and parent ratings of sleep demonstrated poor concurrent validity with clinical diagnosis of obstructive sleep apnoea., Conclusion: Our current data suggest that actigraph watches demonstrate convergent validity and are sensitive to measuring certain sleep constructs (duration, efficiency) in children with Down syndrome. However, parent reports, such as the Children's Sleep Habits Questionnaire, may be measuring other sleep constructs. These findings highlight the importance of selecting measures of sleep related to target concerns., (© 2018 MENCAP and International Association of the Scientific Study of Intellectual and Developmental Disabilities and John Wiley & Sons Ltd.)

Background: In the general population, sleep problems have an impact on daytime performance. Despite sleep problems being common among children with Down syndrome, the impact of sleep problems on daytime behaviours in school-age children with Down syndrome is an understudied topic. Our study examined the relationship between parent-reported and actigraphy-measured sleep duration and sleep quality with parent and teacher reports of daytime behaviour problems among school-age children with Down syndrome., Method: Thirty school-age children with Down syndrome wore an actigraph watch for a week at home at night. Their parent completed ratings of the child's sleep during that same week. Their parent and teacher completed a battery of measures to assess daytime behaviour., Results: Parent reports of restless sleep behaviours on the Children's Sleep Habits Questionnaire, but not actigraph-measured sleep efficiency, was predictive of parent and teacher behavioural concerns on the Nisonger Child Behaviour Rating Form and the Vanderbilt ADHD Rating Scales. Actigraph-measured sleep period and parent-reported sleep duration on the Children's Sleep Habits Questionnaire was predictive of daytime parent-reported inattention. Actigraph-measured sleep period was predictive of parent-reported hyperactivity/impulsivity., Conclusion: The study findings suggest that sleep problems have complex relationships to both parent-reported and teacher-reported daytime behaviour concerns in children with Down syndrome. These findings have implications for understanding the factors impacting behavioural concerns and their treatment in school-age children with Down syndrome., (© 2017 MENCAP and International Association of the Scientific Study of Intellectual and Developmental Disabilities and John Wiley & Sons Ltd.)

Background: Behavioural sleep disturbances are common among children with Down syndrome (DS). However, tools used to detect and evaluate behavioural sleep disturbances were developed for typically developing children and have not been evaluated for use among children with DS. The current study evaluates the psychometric properties of three measures of behavioural sleep disturbances that are currently being used with children with intellectual and developmental disabilities, including children with DS., Method: Caregivers of 30 children with DS rated their child's sleep with the Behavioral Evaluation of Disorders of Sleep (BEDS), Children's Sleep Habits Questionnaire (CSHQ) and Sleep Disturbances Scale for Children (SDSC). Caregivers also provided information on sleep diagnoses and completed a 7-night sleep and behaviour diary., Results: The study investigated the rate of detecting sleep problems, internal consistency, and convergent and concurrent validity of the BEDS, CSHQ and SDSC. Children with DS were reported to exhibit behavioural sleep disturbances at different rates depending on the measure used; 0% BEDS, 79.3% CSHQ and 17.2% SDSC. Internal consistency was comparable for all three measures for their total scores. However, when evaluating the internal consistency of subscale scores, those on the CSHQ and SDSC performed more strongly. The subscales of the CSHQ performed best when evaluating convergent and concurrent validity, with the SDSC subscales performing moderately well., Conclusion: The study findings suggest that, among children with DS, the CSHQ and its subscales performed in a psychometrically sound and theoretically appropriate manner in relation to other measures of sleep, medical history of sleep problems, and daily reports of sleep and associated behaviours. The SDSC performed moderately well. When evaluating behavioural sleep disturbances among children with DS, the CSHQ is recommended based on its stronger psychometric properties., (© 2016 MENCAP and International Association of the Scientific Study of Intellectual and Developmental Disabilities and John Wiley & Sons Ltd.)

Objective: To characterize practice patterns regarding sleep evaluation and intervention among children with Down syndrome (DS)., Method: Data were obtained from electronic health records from 2009 to 2013 for a retrospective cohort of 954 children with DS, aged 5 to 21 years during the time sampled. International Classification of Diseases, Ninth Revision, diagnoses were used to identify children with obstructive sleep apnea and/or behavioral sleep disturbances. Primary outcomes were confirmed by participation in an overnight diagnostic polysomnography (PSG) and/or documented provision of specified sleep interventions including positive airway pressure, otolaryngology (ENT) surgery, sleep medication, and behavioral sleep therapy., Results: Overall, 47.7% of children with DS had undergone PSG, 39.1% had diagnosed sleep problems, and of those diagnosed with sleep problems, 81.2% had received sleep intervention. Consistent with best practice clinical care, sleep treatments matched the diagnosed sleep problems. Age, gender, and race, but not body mass index (BMI), were associated with PSG completion rate and occurrence rates for ENT surgery and sleep medication usage. BMI was associated with obstructive sleep apnea., Conclusion: Despite high rates of reported sleep problems in children with DS, less than half underwent PSG. Children diagnosed with sleep problems received treatment consistent with their sleep diagnosis. However, age and gender were associated with differential rates of treatment delivery that was incongruous with prevalence rates for diagnosed sleep problems. These findings underscore the importance of screening for sleep problems in children with DS, and referring for and providing appropriate targeted sleep interventions., Competing Interests: Dr Esbensen has served as a consultant to Roche Products. Dr Beebe is currently receiving a grant (R01 HL120879) from NIH. No other conflicts of interests were declared by the authors.

α-Synuclein accumulation and mitochondrial dysfunction have both been strongly implicated in the pathogenesis of Parkinson's disease (PD), and the two appear to be related. Mitochondrial dysfunction leads to accumulation and oligomerization of α-synuclein, and increased levels of α-synuclein cause mitochondrial impairment, but the basis for this bidirectional interaction remains obscure. We now report that certain posttranslationally modified species of α-synuclein bind with high affinity to the TOM20 (translocase of the outer membrane 20) presequence receptor of the mitochondrial protein import machinery. This binding prevented the interaction of TOM20 with its co-receptor, TOM22, and impaired mitochondrial protein import. Consequently, there were deficient mitochondrial respiration, enhanced production of reactive oxygen species, and loss of mitochondrial membrane potential. Examination of postmortem brain tissue from PD patients revealed an aberrant α-synuclein-TOM20 interaction in nigrostriatal dopaminergic neurons that was associated with loss of imported mitochondrial proteins, thereby confirming this pathogenic process in the human disease. Modest knockdown of endogenous α-synuclein was sufficient to maintain mitochondrial protein import in an in vivo model of PD. Furthermore, in in vitro systems, overexpression of TOM20 or a mitochondrial targeting signal peptide had beneficial effects and preserved mitochondrial protein import. This study characterizes a pathogenic mechanism in PD, identifies toxic species of wild-type α-synuclein, and reveals potential new therapeutic strategies for neuroprotection., Competing Interests: The authors declare that they have no competing financial interests., (Copyright © 2016, American Association for the Advancement of Science.)

More than 80 years after iron accumulation was initially described in the substantia nigra (SN) of Parkinson's disease (PD) patients, the mechanisms responsible for this phenomenon are still unknown. Similarly, how iron is delivered to its major recipients in the cell - mitochondria and the respiratory complexes - has yet to be elucidated. Here, we report a novel transferrin/transferrin receptor 2 (Tf/TfR2)-mediated iron transport pathway in mitochondria of SN dopamine neurons. We found that TfR2 has a previously uncharacterized mitochondrial targeting sequence that is sufficient to import the protein into these organelles. Importantly, the Tf/TfR2 pathway can deliver Tf bound iron to mitochondria and to the respiratory complex I as well. The pathway is redox-sensitive and oxidation of Tf thiols to disulfides induces release from Tf of highly reactive ferrous iron, which contributes to free radical production. In the rotenone model of PD, Tf accumulates in dopamine neurons, with much of it accumulating in the mitochondria. This is associated with iron deposition in SN, similar to what occurs in PD. In the human SN, TfR2 is also found in mitochondria of dopamine neurons, and in PD there is a dramatic increase of oxidized Tf in SN. Thus, we have discovered a novel mitochondrial iron transport system that goes awry in PD, and which may provide a new target for therapeutic intervention.

Animal and human studies of addiction indicate that the D2 dopamine receptor (DRD2) plays a critical role in the mechanism of drug reward. D2 receptor density in the brains of alcoholics has been shown to be reduced relative to controls. Previous studies of DRD2 in association with alcohol dependence using variation in the TaqI A locus were highly controversial. Recently, a synonymous mutation, C957T, in the coding region of the human DRD2 gene has been identified which appears to have functional effects including alteration in receptor availability. In order to determine if susceptibility to alcohol dependence (AD) within multiplex alcohol dependence families would be altered by the C957T in the coding region of the D2 gene, within-family association was studied in members of Caucasian multiplex alcohol dependence families. Members of control families with no personal alcohol or substance dependence history were included for case/control comparisons. Analyses performed to detect within-family association showed evidence favoring an association for the C957T polymorphism (P = 0.038). Linkage analyses of polymorphisms in this region showed that only the C957T locus remained of interest (P = 0.015). Evidence for the C957T T allele having a role in AD susceptibility at the population level using a case/control comparison was statistically marginal (P = 0.062), but was consistent with the family data results. These results support a role for DRD2 as a susceptibility gene for alcohol dependence within multiplex families at high risk for developing alcohol dependence., ((c) 2007 Wiley-Liss, Inc.)

Purpose: To develop a mouse behavioral assay that can assess differential nocioceptive sensitivity to light (photophobia)., Materials and Methods: Normal C57BL/6J mice and congenic albino mice, C57BL/6J-Tyr c-2J/j, were habituated to a light/dark box testing chamber and then tested for a preference for the dark versus the light compartment in response to increasing brightness of the light compartment., Results: We found a statistically significant difference between the normal and the albino mice (N = 5/strain) in their preference for the dark compartment when the ambient condition in the light compartment was 1,000 lux, whereas at 0 lux, both groups of animals exhibited no preference for either compartment., Conclusions: The approach described here presents the first mouse behavioral assay for assessing aversion/avoidance behavior in response to light that appears to be comparable to human photophobia. This approach can be used to test other causes of sustained photophobia in mouse models, as well as to assess the efficacy of drugs for the relief of photophobia.

Objective: The genes encoding the gamma-aminobutyric acid(A) (GABA(A)) receptor have been the focus of several recent studies investigating the genetic etiology of alcohol dependence. Analyses of multiplex families found a particular gene, GABRA2, to be highly associated with alcohol dependence, using within-family association tests and other methods. Results were confirmed in three case-control studies. The objective of this study was to investigate the GABRA2 gene in another collection of multiplex families., Method: Analyses were based on phenotypic and genotypic data available for 330 individuals from 65 bigenerational pedigrees with a total of 232 alcohol-dependent subjects. A proband pair of same-sex siblings meeting Diagnostic and Statistical Manual of Mental Disorders, Third Edition, criteria for alcohol dependence was required for entry of a family into the study. One member of the proband pair was identified while in treatment for alcohol dependence. Linkage and association of GABRA2 and alcohol dependence were evaluated using SIBPAL (a nonparametric linkage package) and both the Pedigree Disequilibrium Test and the Family-Based Association Test, respectively., Results: We find no evidence of a relationship between GABRA2 and alcohol dependence. Linkage analyses exhibited no linkage using affected/affected, affected/unaffected, and unaffected/unaffected sib pairs (all p's < .13). There was no evidence of a within-family association (all p's > .39)., Conclusions: Comorbidity may explain why our results differ from those in the literature. The presence of primary drug dependence and/or other psychiatric disorders is minimal in our pedigrees, although several of the other previously published multiplex family analyses exhibit a greater degree of comorbidity.

Alcoholism is currently one of the most serious public health problems in the US. Lifetime prevalence rates are relatively high with one in five men and one in 12 women meeting criteria for this condition. Identification of genetic loci conferring an increased susceptibility to developing alcohol dependence could strengthen prevention efforts by informing individuals of their risk before abusive drinking ensues. Families identified through a double proband methodology have provided an exceptional opportunity for gene-finding because of the increased recurrence risks seen in these sibships. A total of 360 markers for 22 autosomes were spaced at an average distance of 9.4 cM and genotyping performed for 330 members of these multiplex families. Extensive clinical data, personality variation, and event-related potential characteristics were available for reducing heterogeneity and detecting robust linkage signals. Multipoint linkage analysis using different analytic strategies give strong support for loci on chromosomes 1, 2, 6, 7, 10, 12, 14, 16, and 17., (Copyright 2004 Wiley-Liss, Inc.)

Reactive oxygen species (ROS) such as superoxide, peroxide and hydroxyl radicals are generated during normal cellular metabolism and are increased in acute injury and in many chronic disease states. When their production is inadequately regulated, ROS accumulate and irreversibly damage cell components, causing impaired cellular function and death. Antioxidant enzymes such as superoxide dismutase (SOD) play a vital role in minimizing ROS levels and ROS-mediated damage. The cytosolic form of Cu/Zn-SOD appears specialized to remove superoxide produced as a result of injury. 'Knockout' mice with targeted deletion of Sod1, the gene that codes for Cu/Zn-SOD, develop normally but show enhanced susceptibility to central nervous system injury. Since loud noise is injurious to the cochlea and is associated with elevated cochlear ROS, we hypothesized that Sod1 knockout mice would be more susceptible to noise-induced permanent threshold shifts (PTS) than wild-type and heterozygous control mice. Fifty-nine mice (15 knockout, 29 heterozygous and 15 wild type for Sod1) were exposed to broad-band noise (4.0-45.0 kHz) at 110 dB SPL for 1 h. Hearing sensitivity was evaluated at 5, 10, 20 and 40 kHz using auditory brainstem responses before exposure and 1, 14 and 28 days afterward. Cu/Zn-SOD deficiency led to minor (0-7 dB) threshold elevations prior to noise exposure, and about 10 dB of additional noise-induced PTS at all test frequencies, compared to controls. The distribution of thresholds at 10 and 20 kHz at 28 days following exposure contained three modes, each showing an effect of Cu/Zn-SOD deficiency. Thus another factor, possibly an additional unlinked gene, may account for the majority of the observed PTS. Our results indicate that genes involved in ROS regulation can impact the vulnerability of the cochlea to noise-induced hearing loss.

The role of oxidative damage in neurodegenerative disease was investigated in mice lacking cytoplasmic Cu/Zn superoxide dismutase (SOD), created by deletion of the SOD1 gene (SOD1(-/-)). SOD1(-/-) mice developed a chronic peripheral hindlimb axonopathy. Mild denervation of muscle was detected at 2 months, and behavioral and physiological motor deficits were present at 5-7 months of age. Ventral root axons were shrunken but were normal in number. The somatosensory system in SOD1(-/-) mice was mildly affected. SOD1(-/-) mice expressing Cu/Zn SOD only in brain and spinal cord were generated using transgenic mice expressing mouse SOD1 driven by the neuron-specific synapsin promoter. Neuron-specific expression of Cu/Zn SOD in SOD1(-/-) mice rescued motor neurons from the neuropathy. Therefore, Cu/Zn SOD is not required for normal motor neuron survival, but is necessary for the maintenance of normal neuromuscular junctions by hindlimb motor neurons.

To investigate the roles of CuZn superoxide dismutase (CuZnSOD) and Mn superoxide dismutase (MnSOD) in oxygen radical-mediated cytotoxicity and to distinguish the actions of these two enzymes, fetal fibroblasts were derived from mouse fetuses that are either deficient in CuZnSOD (Sod1-/+ and -/-) or MnSOD (Sod2-1+ and -/-) for in vitro studies. Whereas the phenotype of the Sod1 mutant animals did not differ from that of their normal littermates, the growth of Sod1-/- fetal fibroblasts was only 25% of that of the -/+ and +/+ cells. On the other hand, although almost all homozygous Sod2 mutant animals (-/-) died within 10 days after birth, cultivation of Sod2-/- fetal fibroblasts was possible and their growth was about 60% that of -/+ and +/+ cells. When cultured cells were subjected to treatment with paraquat to assess their ability to grow in the presence of high levels of superoxide radicals, Sod1-/- cells were 80 times more sensitive and Sod2-/- cells were 12 times more sensitive to paraquat than wild-type cells. In addition, whereas the loss of 50% CuZnSOD rendered Sod1-/+ cells almost twice more sensitive to paraquat than +/+ cells, loss of 50% MnSOD had no effect on paraquat sensitivity. Our results suggest that CuZnSOD-deficient cells are more sensitive to oxygen toxicity than are MnSOD-deficient cells, that paraquat causes free radical-induced damage in both the mitochondria and cytoplasm, and that SOD compartmentalized in the cytosol cannot compensate for the loss of SOD in the mitochondria and vice versa.

Apoptotic neuronal cell death has recently been associated with the development of infarction after cerebral ischemia. In a variety of studies, CuZn-superoxide dismutase (CuZn-SOD) has been shown to protect the brain from ischemic injury. A possible role for CuZn-SOD-related modulation of neuronal viability is suggested by the finding that CuZn-SOD inhibits apoptotic neuronal cell death in response to some forms of cellular damage. We evaluated this possibility in the model of transient focal cerebral ischemia in mice bearing a disruption of the CuZn-SOD gene (Sod1). Homozygous mutant (Sod1 -/-) mice had no detectable CuZn-SOD activity, and heterozygous mutants (Sod1 +/-) showed a 50% decrease compared with wild-type mice. Sod1 -/- mice showed a high level of blood-brain barrier disruption soon after 1 hr of middle cerebral artery occlusion and 100% mortality at 24 hr after ischemia. Sod1 +/- mice showed 30% mortality at 24 hr after ischemia, and neurological deficits were exacerbated compared with wild-type controls. The Sod1 +/- animals also had increased infarct volume and brain swelling, accompanied by increased apoptotic neuronal cell death as indicated by the in situ nick-end labeling technique to detect DNA fragmentation and morphological criteria. These results suggest that oxygen-free radicals, especially superoxide anions, are an important factor for the development of infarction by brain edema formation and apoptotic neuronal cell death after focal cerebral ischemia and reperfusion.

Point mutations occurring within the Cu/Zn superoxide dismutase (SOD1) gene have been implicated in the etiology of some cases of familial amyotrophic lateral sclerosis (FALS). In order to better understand the functional consequences of these mutations, we have introduced FALS mutations into the mouse SOD1 gene and studied the expression of the mutant templates in stably transformed cell lines. Pulse-chase analyses of lysates derived from cell lines stably expressing the Cu/Zn SOD isoforms indicate that the FALS mutant Cu/Zn SOD proteins are turned over more rapidly than wild-type SOD. Protease inhibitors specific for the major intracellular proteolytic activities were used to characterize the degradative pathways involved in the turnover of mutant Cu/Zn SOD. Inhibition of the chymotrypsin-like activity of the proteasome (also known as multicatalytic proteinase or ubiquitin, ATP-dependent proteinase) by a synthetic dipeptide aldehyde led to a significant increase in levels of the mutant Cu/Zn SOD implicating this proteolytic pathway in the turnover of the FALS mutant SOD proteins.

The discovery that some cases of familial amyotrophic lateral sclerosis (FALS) are associated with mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) has focused much attention on the function of SOD1 as related to motor neuron survival. Here we describe the creation and characterization of mice completely deficient for this enzyme. These animals develop normally and show no overt motor deficits by 6 months in age. Histological examination of the spinal cord reveals no signs of pathology in animals 4 months in age. However Cu/Zn SOD-deficient mice exhibit marked vulnerability to motor neuron loss after axonal injury. These results indicate that Cu/Zn SOD is not necessary for normal motor neuron development and function but is required under physiologically stressful conditions following injury.

We have been studying a transformed derivative of a mouse fibroblast line (3T3DM) that stably maintains double minute chromosomes (DMs). In this report we describe a comprehensive analysis of the structure of the DMs within this cell line, utilizing a combination of long-range mapping via pulsed-field gel electrophoresis, screening of DM-enriched genomic libraries, and DM sizing using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Our data indicate that the minute particles in these cells exist as a homogeneous population of circular molecules, roughly 4 Mb in size, upon which three genes are amplified. One of these is the mdm2 oncogene, which has also been found to be amplified in a number of human sarcomas. Further, we present evidence that these three genes are arranged as two identical inverted repeat units linked by spacer regions of heterogeneous size. This work has led to the first model for the structure of an entire double minute particle containing an amplified oncogene; this model provides clues to later events occurring in the gene amplification process in tumor cells.

Evidence is provided that isoproterenol, 4-methylcatechol and serum induce NGF by three separate mechanisms. Isoproterenol and 4-methylcatechol induced NGF and NGF mRNA in mouse fibroblast L929 cells in either the presence or absence of serum. Propranolol prevented NGF induction by isoproterenol, but not by 4-methylcatechol or serum. All possible combinations of these inducers resulted in additive increases in the levels of NGF and NGF mRNA.

To gain insight into the normal controls mediating expression of the c-Ki-ras protooncogene, we have identified DNA sequence elements within its promoter that are essential for transcriptional activity. Transient expression assays using the bacterial chloramphenicol acetyltransferase gene were used initially to localize regions directing primary promoter function. Stepwise deletion of 5' promoter sequences resulted in a gradual decrease in the ability to drive transcription of the reporter gene, suggesting that this promoter is composed of multiple cis-acting elements. Gel mobility-shift and DNase protection studies involving a 166-base-pair DNA fragment allowed the identification of protein-binding sites corresponding to these multiple regulatory elements. One element demonstrating particular transcriptional influence exists as a homopurine/homopyrimidine-rich region that in vitro exhibits S1 nuclease sensitivity and binds at least one nuclear protein. Data from competition binding experiments suggest that this nuclear factor may be influential in the regulation of other essential growth-control genes as well.

We isolated and characterized the 5' region of the mouse c-Ki-ras gene, including a 5' untranslated exon (exon 0). These studies used genetic material from Y1 mouse adrenocortical tumor cells in which the c-Ki-ras gene is amplified and overexpressed. Our data demonstrate that transcription initiates at multiple sites, predicting size heterogeneity at the 5' ends of the c-Ki-ras mRNAs. Using transient expression assays, we identified a genomic fragment within the 5' region which exhibits bidirectional promoter activity.

Enhanced expression of the cKi-ras proto-oncogene in a bone marrow-derived mouse cell line, 416B, has been shown to be associated with the integration of Friend viral DNA into the cellular gene. Here we report the results of experiments designed to clarify the molecular mechanism responsible for the cKi-ras overexpression. Based on primer extension analyses and DNA sequencing of cKi-ras cDNA clones, we have obtained evidence that the 416B cells contain viral-host chimaeric transcripts that initiate within the 3' long terminal repeat (LTR) of the integrated provirus. Processing of the transcripts from the rearranged cKi-ras gene includes an unexpected splicing event associated with the fortuitous creation of a cryptic donor splice site at the junction between the proviral and cellular DNA sequences. These data demonstrate that enhanced cKi-ras expression in the 416B cells results from a retroviral promoter insertion mechanism of transcriptional activation.

Fusion genes containing segments of the promoter region of the human LDL receptor gene and the coding sequence of the bacterial enzyme, chloramphenicol acetyltransferase (CAT), were introduced into JEG-3 human choriocarcinoma cells. Constructs containing 177 base pairs of 5'-flanking DNA (pLDLR-CAT 234) or 6500 base pairs (pLDLR-CAT 6500) promoted CAT activity in transient expression assays. Although both pLDLR-CAT 234 and pLDLR-CAT 6500 contain sequences related to the recently reported consensus sequence for cyclic AMP responsiveness, treatment of the transfected JEG-3 cells with 8-bromo-cAMP did not increase CAT activity. The cyclic AMP analog did, however, stimulate steroidogenesis and hCG secretion and increase CAT activity in cells transfected with p18X2SV1CAT, which contains two copies of an 18 base pair sequence corresponding to the cAMP-responsive element of the human alpha chorionic gonadotropin gene.