Community-based models have become increasingly prominent in prevention, and have special relevance for suicide prevention in circumpolar Indigenous communities. It follows that outcomes from circumpolar suicide prevention programs might be more completely understood at the community level. We present here a methodology for analysis at this level. This paper seeks to understand a cultural prevention program for rural Yup'ik youth in Alaska targeting suicide and co-occurring alcohol abuse as a community development process through changes at the community level.
Quasi-experimental design with assessment at pre- and post-intervention or at 4 time points. The community development process for this project began in October 2004. The first program baseline assessment began in November 2006, prior to prevention activities with youth and parents, and the post-intervention assessment concluded in March 2008.
Five key informants pre- and post-intervention completed a community readiness assessment, which is a structured procedure assessing a community's awareness of suicide as an issue and its organizational readiness for prevention programming. Forty-three adult caregivers or sponsors of youth in the prevention program completed an assessment of behaviours that contributed to community protective factors from youth suicide and alcohol abuse at 4 time points before, during and after the intervention. The 54 youth who participated in the prevention program completed an assessment of community protective factors, also at 4 time points before, during and after the intervention. The community protective factors from suicide that were assessed included safety, enforcement of alcohol prohibitions, role models, support and opportunities for youth.
Community readiness for the prevention efforts increased to new developmental stages of readiness post-intervention, and a trend in the data suggested community protective factors increased in the amount of protective behaviours performed by adults (slope estimate=0.0162, 95% CI-0.0028–0.0351, d=.55) and in the perceptions of youth (slope estimate=0.0148, 95% CI-0.0004–0.0291, d=.45), in a dose response relationship to the number of prevention program sessions attended by adults and youth.
Using data from a feasibility study, this paper demonstrates the feasibility and potential utility of methodological approaches that use community-level variables beyond individual level outcomes in circumpolar suicide prevention research.
Suicide; suicide prevention; Alaska Native; community readiness assessment; community-based participatory research
Concerns in some settings regarding the accuracy and ethics of employing direct questions about alcohol use suggest need for alternative assessment approaches with youth. Umyuangcaryaraq is a Yup’ik Alaska Native word meaning “Reflecting.” The Reflective Processes Scale is a youth measure tapping awareness and thinking over potential negative consequences of alcohol misuse as a protective factor that includes cultural elements often shared by many other Alaska Native and American Indian cultures. A bifactor model of the scale items with three content factors provided excellent fit to observed data. Item response theory analysis suggested a binary response format as optimal. Evidence of convergent and discriminant validity is presented.
American Indian and Alaska Native; adolescents; alcohol; alcohol expectancies
Community-based participatory research (CBPR) with American Indian and Alaska Native communities creates distinct interventions, complicating cross-setting comparisons. In this study, coding CBPR intervention activities from three communities for protective factors and latent class analysis identified five patterns of exposure to protective factors: Internal, External, Limits on alcohol, Community and family, and Low probabilities of all protective factors. Patterns differed significantly by community and youth age. Standardizing protective factors by the functions an intervention serves instead of its form or components can assist in refining CBPR interventions and evaluating effects in culturally distinct settings.
The Fenna-Matthews-Olson antenna protein from the green bacterium Pelodictyon phaeum mediates the energy transfer from a peripheral antenna complex to the membrane-bound reaction center. The three-dimensional structure of this protein has been previously modeled using X-ray diffraction to a resolution limit of 2.0 Å, with Rwork and Rfree values of 16.6% and 19.9% respectively (Larson et al. (2011) Photosyn Res 107: 139–150). This model shows the protein as consisting of β-sheets surrounding several bacteriochlorophyll cofactors. While most of the model clearly matches the electron density maps, in this paper we re-examine the electron density for a specific feature, namely the eighth bacteriochlorophyll a cofactor. This electron density is now interpreted as arising primarily from the end of an otherwise disordered polyethylene glycol molecule. Additional electron density is present but the density is weak and cannot be unambiguously assigned. The new model has Rwork and Rfree values of 16.2% and 19.0%, respectively.
Energy transfer; light harvesting complex; three dimensional structure; green bacteria
In yeast (Saccharomyces cerevisiae) and animals, the sulfhydryl oxidase Erv1 functions with Mia40 in the import and oxidative folding of numerous cysteine-rich proteins in the mitochondrial intermembrane space (IMS). Erv1 is also required for Fe-S cluster assembly in the cytosol, which uses at least one mitochondrially derived precursor. Here, we characterize an essential Erv1 orthologue from the protist Trypanosoma brucei (TbERV1), which naturally lacks a Mia40 homolog. We report kinetic parameters for physiologically relevant oxidants cytochrome c and O2, unexpectedly find O2 and cytochrome c are reduced simultaneously, and demonstrate that efficient reduction of O2 by TbERV1 is not dependent upon a simple O2 channel defined by conserved histidine and tyrosine residues. Massive mitochondrial swelling following TbERV1 RNA interference (RNAi) provides evidence that trypanosome Erv1 functions in IMS protein import despite the natural absence of the key player in the yeast and animal import pathways, Mia40. This suggests significant evolutionary divergence from a recently established paradigm in mitochondrial cell biology. Phylogenomic profiling of genes also points to a conserved role for TbERV1 in cytosolic Fe-S cluster assembly. Conversely, loss of genes implicated in precursor delivery for cytosolic Fe-S assembly in Entamoeba, Trichomonas, and Giardia suggests fundamental differences in intracellular trafficking pathways for activated iron or sulfur species in anaerobic versus aerobic eukaryotes.
This qualitative study examines the resources that Vietnamese refugee parents use in raising their adolescent youth in exile and how they, and their adolescents, regard their experiences of different parenting styles. The study is based on 55 semi-structured interviews and several focus groups performed with a small sample of Vietnamese refugee parents and their adolescent children. Three main themes from the interviews were identified: the role of the extended family and siblings in bringing up children; language acquisition and cultural continuity and, finally, religion and social support. Our findings suggest extended kin are involved in the raising of adolescent children, providing additional family ties and support. Parents regarded Vietnamese language acquisition by their youth as facilitating both communication with extended kin and cultural transmission. Several parents stressed the importance of religious community to socialising and creating a sense of belonging for their youth. Vietnamese refugee parents seek a balance between Vietnamese values and their close extended family social networks, and the opportunities in Norway to develop autonomy in pursuit of educational and economic goals. Together these parenting practices constituted a mobilization of resources in support of their youth. These findings may have important implications for future research on resiliency and the role of these strategies as protective factors mediating mental health outcomes. They may also have implications for treatment, in terms of the types of resources treatment can access and for prevention strategies that maximize key cultural resources for Vietnamese refugee youth.
Vietnamese; Refugees; Parenting; Well-being; Resilience; Qualitative method
Radon-222 is a naturally occurring radioactive gas that is responsible for approximately half of the human annual background radiation exposure globally. Chronic exposure to radon and its decay products is estimated to be the second leading cause of lung cancer behind smoking, and links to other forms of neoplasms have been postulated. Ionizing radiation emitted during the radioactive decay of radon and its progeny can induce a variety of cytogenetic effects that can be biologically damaging and result in an increased risk of carcinogenesis. Suggested effects produced as a result of alpha particle exposure from radon include mutations, chromosome aberrations, generation of reactive oxygen species, modification of the cell cycle, up or down regulation of cytokines and the increased production of proteins associated with cell-cycle regulation and carcinogenesis. A number of potential biomarkers of exposure, including translocations at codon 249 of TP53 in addition to HPRT mutations, have been suggested although, in conclusion, the evidence for such hotspots is insufficient. There is also substantial evidence of bystander effects, which may provide complications when calculating risk estimates as a result of exposure, particularly at low doses where cellular responses often appear to deviate from the linear, no-threshold hypothesis. At low doses, effects may also be dependent on cellular conditions as opposed to dose. The cellular and molecular carcinogenic effects of radon exposure have been observed to be both numerous and complex and the elevated chronic exposure of man may therefore pose a significant public health risk that may extend beyond the association with lung carcinogenesis.
radon; carcinogenesis; cytogenetics; DNA damage; alpha particles; bystander effect; chromosome aberrations; micronuclei; linear; no-threshold; hormesis
Self-mastery refers to problem-focused coping facilitated through personal agency. Communal mastery describes problem solving through an interwoven social network. This study investigates an adaptation of self- and communal mastery measures for youth. Given the important distinction between family and peers in the lives of youth, these adaptation efforts produced Mastery-Family and Mastery-Friends subscales, along with a Mastery-Self subscale. We tested these measures for psychometric properties and internal structure with 284 12 to 18-year-old predominately Yup’ik Eskimo Alaska Native adolescents from rural, remote communities — a non-Western culturally distinct group hypothesized to display higher levels of collectivism and communal mastery. Results demonstrate a subset of items adapted for youth function satisfactorily, a three-response alternative format provided meaningful information, and the subscale’s underlying structure is best described through three distinct first-order factors organized under one higher order mastery factor.
American Indian and Alaska Native; mastery; self-efficacy; communal mastery; coping
The Relationship dimension of the Family Environment Scale (FES; Moos & Moos, 1994), which consists of the Cohesion, Expressiveness, and Conflict subscales, measures a person's perception of the quality of their family relationship functioning. This study investigates an adaptation of the Relationship dimension of the FES for Alaska Native youth. We tested the adapted measure, the Brief Family Relationship Scale (BFRS), for psychometric properties and internal structure with 284 12 to18 year-old predominately Yup'ik Eskimo Alaska Native adolescents from rural, remote communities. This non-Western cultural group is hypothesized to display higher levels of collectivism traditionally organized around an extended kinship family structure. Results demonstrate a subset of the adapted items function satisfactorily, a three-response alternative format provided meaningful information, and the subscale's underlying structure is best described through three distinct first-order factors, organized under one higher order factor. Convergent and discriminant validity of the BFRS was assessed through correlational analysis.
Alaska Native; cohesion; expressiveness; conflict; family relationships; family climate
Orofacial clefts are common birth defects with strong evidence for both genetic and environmental causal factors. Candidate-gene studies combined with exposures known to influence the outcome provide a highly targeted approach to detecting GxE interactions. We developed a new statistical approach that combines the case-control and offspring-parent triad designs into a “hybrid design” to search for GxE interactions among 334 autosomal cleft candidate genes and maternal first-trimester exposure to smoking, alcohol, coffee, folic acid supplements, dietary folate, and vitamin A. The study population comprised 425 case-parent triads of isolated clefts and 562 control-parent triads derived from a nationwide study of orofacial clefts in Norway (1996-2001). A full maximum-likelihood model was used in combination with a Wald test statistic to screen for statistically significant GxE interaction between strata of exposed and unexposed mothers. In addition, we performed pathway-based analyses on 28 detoxification genes and 21 genes involved in folic acid metabolism. With the possible exception of the T-box 4 gene (TBX4) and dietary folate interaction in isolated CPO, there was little evidence overall of GxE interaction in our data. This study is the largest to date aimed at detecting interactions between orofacial clefts candidate genes and well-established risk exposures.
Birth defects; orofacial cleft; cleft lip; cleft palate; genetic epidemiology
We describe important elements in the process of engagement with tribal communities in research with children and youth and their families. We believe it helpful to understand the research relationship with tribal communities through the lens of kinship relations. This calls for re-examination of the nature of research and researcher, with important implications for the research process, design and organization, recovery from errors, and dissemination of results. Implications include a re-examination of some of our canons of research methods and research ethics, along with a willingness to address new challenges, to share control of the research process, and to be open to new conceptual perspectives, including alternative research strategies. Its repercussions hold promise for a deepening of the research relationship, and the role of researcher in the community.
American Indian; Alaska Native; community based participatory research; tribal participatory research; youth; children
The nuclear family is often the point of departure in much of the existing acculturation research on refugee youth and children of refugees. The influence of other extended family members appears to receive less attention in understanding acculturation processes and intergenerational perspectives. This qualitative study explores the influence of extended family members upon a small sample of Vietnamese refugee parents and their adolescents while they undergo acculturation through their long-term resettlement process in Norway. With repeated interviews over a time span of 3 years, we identified situations and processes in family life in which extended kin become particularly activated and influential. Vietnamese refugee families in Norway keep close contact with extended kin even in the face of geographical distance to kin remaining in Vietnam, or globally dispersed. Aunts, uncles, and cousins are experienced as significant persons in the lives of many adolescents. Additionally, birth order of parents can often influence relationship dynamics among siblings and siblings children. Extended kin surfaced as especially important and influential at critical stages and crisis situations in family life. Extended family, and in particular, parental siblings play important roles in the acculturation experience and family functioning of Vietnamese refugee families in Norway. This has important implications for the study of Vietnamese and other refugee and immigrant families in acculturation research.
Acculturation; Extended Family; Vietnamese; Refugees; Qualitative Methods; Mental Health
The native people of Alaska have experienced historical trauma and rapid changes in culture and lifestyle patterns. As a consequence, these populations shoulder a disproportionately high burden of psychological stress. The Yup'ik Experiences of Stress and Coping project originated from rural Yup'ik communities’ concerns about stress and its effects on health. It aimed to understand the stressful experiences that affect Yup'ik communities, to identify coping strategies used to deal with these stressors and to inform culturally responsive interventions.
Here, we examine the process of moving from research (gaining understanding) to disseminating project findings to translation into intervention priorities. We highlight the importance of community participation and discuss challenges encountered, strategies to address these challenges and ethical considerations for responsible intervention research with indigenous communities that reflect their unique historical and current socio-cultural realities.
Community-wide presentations and discussions of research findings on stress and coping were followed by smaller Community Planning Group meetings. During these meetings, community members contextualized project findings and discussed implications for interventions. This process placed priority on community expertise in interpreting findings and translating results and community priorities into grant applications focused on intervention development and evaluation.
Challenges included translation between English and Yup'ik, funding limitations and uncertainties, and the long timelines involved in moving from formative research to intervention in the face of urgent and evolving community needs. The lack of congruence between institutional and community worldviews in the intervention research enterprise highlights the need for “principled cultural sensitivity”.
Cultural sensitivity requires sharing results that have practical value, communicating openly, planning for sustainability and incorporating indigenous knowledge and expertise through a community-guided process. Our research findings will inform continued work within our partnership as we co-develop culturally based strategies for multilevel community interventions to address stress.
Alaska Native; stress; coping; reporting research results; community-based participatory research (CBPR); research ethics
Smad3/Akt/mTOR/S6K/S6RP signaling plays a critical role in follistatin-mediated muscle growth that operates independently of myostatin-driven mechanisms.
Follistatin is essential for skeletal muscle development and growth, but the intracellular signaling networks that regulate follistatin-mediated effects are not well defined. We show here that the administration of an adeno-associated viral vector expressing follistatin-288aa (rAAV6:Fst-288) markedly increased muscle mass and force-producing capacity concomitant with increased protein synthesis and mammalian target of rapamycin (mTOR) activation. These effects were attenuated by inhibition of mTOR or deletion of S6K1/2. Furthermore, we identify Smad3 as the critical intracellular link that mediates the effects of follistatin on mTOR signaling. Expression of constitutively active Smad3 not only markedly prevented skeletal muscle growth induced by follistatin but also potently suppressed follistatin-induced Akt/mTOR/S6K signaling. Importantly, the regulation of Smad3- and mTOR-dependent events by follistatin occurred independently of overexpression or knockout of myostatin, a key repressor of muscle development that can regulate Smad3 and mTOR signaling and that is itself inhibited by follistatin. These findings identify a critical role of Smad3/Akt/mTOR/S6K/S6RP signaling in follistatin-mediated muscle growth that operates independently of myostatin-driven mechanisms.
To review the existing epidemiological literature on suicide and alcohol related disorders and their social determinants in the U.S. Arctic, as it relates to U.S. government research and evaluation efforts, and to offer recommendations to boost research capacity in the U.S. Arctic and collaborations across the circumpolar arctic as part of global health initiatives. Study design: Synthetic literature review.
Published literature, federal and state reports on suicide and alcohol-related disorders, federal databases on research and program evaluation in the U.S Arctic were reviewed, with a focus on epidemiological trends over the past 50 years.
Suicide and alcohol-related disorders play a significant role in health disparities t in the U.S. Arctic, with evidence of a disturbing prevalence trend over the past 50 years. Important variations exist in suicide rates across different regions of Alaska with different majority populations of Alaska Native cultural groups, and in selected key instances, within these regions, with immense implications for guiding effective prevention efforts. Consequences of alcohol abuse are severe and particularly significant in their impact upon Alaska Native people. Health related conditions associated with alcohol abuse are among leading causes of mortality.
Recommendations to boost research capacity in behavioural health in the U.S. Arctic are offered; specifically on strategies and methods of inquiry and analysis, distinctions between populations and communities in rural circumpolar contexts, future epidemiological and implementation research.
Arctic; Alaska; Alaska Native; suicide; alcohol; U.S. Arctic research and research infrastructure
Epidemiological studies suggest that chronic exposure to inorganic arsenic is associated with cancer of the skin, urinary bladder and lung as well as the kidney and liver. Previous experimental studies have demonstrated increased incidence of liver, lung, ovary, and uterine tumors in mice exposed to 85 ppm (∼8 mg/kg) inorganic arsenic during gestation. To further characterize age susceptibility to arsenic carcinogenesis we administered 85 ppm inorganic arsenic in drinking water to C3H mice during gestation, prior to pubescence and post-pubescence to compare proliferative lesion and tumor outcomes over a one-year exposure period. Inorganic arsenic significantly increased the incidence of hyperplasia in urinary bladder (48%) and oviduct (36%) in female mice exposed prior to pubescence (beginning on postnatal day 21 and extending through one year) compared to control mice (19 and 5%, respectively). Arsenic also increased the incidence of hyperplasia in urinary bladder (28%) of female mice continuously exposed to arsenic (beginning on gestation day 8 and extending though one year) compared to gestation only exposed mice (0%). In contrast, inorganic arsenic significantly decreased the incidence of tumors in liver (0%) and adrenal glands (0%) of male mice continuously exposed from gestation through one year, as compared to levels in control (30 and 65%, respectively) and gestation only (33 and 55%, respectively) exposed mice. Together, these results suggest that continuous inorganic arsenic exposure at 85 ppm from gestation through one year increases the incidence and severity of urogenital proliferative lesions in female mice and decreases the incidence of liver and adrenal tumors in male mice. The paradoxical nature of these effects may be related to altered lipid metabolism, the effective dose in each target organ, and/or the shorter one-year observational period.
arsenic; carcinogenesis; life-stage; age susceptibility; urinary bladder; C3H mice
Research with Native Americans has identified connectedness as a culturally-based protective factor against substance abuse and suicide. Connectedness refers to the interrelated welfare of the individual, one’s family, one’s community, and the natural environment. We developed an 18-item quantitative assessment of awareness of connectedness and tested it with 284 Alaska Native youth. Evaluation with confirmatory factor analysis and item response theory identified a 12-item subset that functions satisfactorily in a second-order, four-factor model. The proposed Awareness of Connectedness Scale displays good convergent and discriminant validity and correlates positively with hypothesized protective factors such as reasons for living and communal mastery. The measure has utility in the study of culture-specific protective factors and as an outcomes measure for behavioral health programs with Native American youth.
American Indian and Alaska Native youth; assessment; indigenous psychology
This report describes how multiple community constituents came together to work with university researchers on developing a shared agenda for studying young indigenous people in five international circumpolar communities. The paper focuses on the set up and process of an initial face-to-face methodological planning workshop involving youth and adult community members and academics. Members of Yup'ik, Inupiat, Eveny, Inuit and Sámi communities from Siberia to Norway participated in the workshop and engaged in negotiations to arrive at shared research interests. This was essential since the ultimate goal of the research is translational and transformative, spurring social action in communities. Describing the beginning stage of this project and the underlying participatory methodology offers reader insight into the how the approach engaged community members with varying degrees of sustained interest and practical success. It, therefore, articulates a methodological approach for those interested in doing community-based participatory research in international contexts.
youth; resilience; indigenous; circumpolar; international; community-based participatory research
Formation of a functional nervous system requires neurons to migrate to the correct place within the developing brain. Tangentially migrating neurons are guided by a leading process which extends towards the target and is followed by the cell body. How environmental cues are coupled to specific cytoskeletal changes to produce and guide leading process growth is unknown. One such cytoskeletal modulator is drebrin, an actin-binding protein known to induce protrusions in many cell types and be important for regulating neuronal morphology.
Using the migration of oculomotor neurons as a model, we have shown that drebrin is necessary for the generation and guidance of the leading process. In the absence of drebrin, leading processes are not formed and cells fail to migrate although axon growth and pathfinding appear grossly unaffected. Conversely, when levels of drebrin are elevated the leading processes turn away from their target and as a result the motor neuron cell bodies move along abnormal paths within the brain. The aberrant trajectories were highly reproducible suggesting that drebrin is required to interpret specific guidance cues. The axons and growth cones of these neurons display morphological changes, particularly increased branching and filopodial number but despite this they extend along normal developmental pathways.
Collectively these results show that drebrin is initially necessary for the formation of a leading process and subsequently for this to respond to navigational signals and grow in the correct direction. Furthermore, we have shown that the actions of drebrin can be segregated within individual motor neurons to direct their migration independently of axon guidance.
OMN, oculomotor nucleus; PCN, precerebellar nuclei; YFP, yellow fluorescent protein; Drebrin; Actin-binding; Migration; Leading process; Oculomotor
To report on a participatory research process in southwest Alaska focusing on youth involvement as a means to facilitate health promotion. We propose youth-guided community-based participatory research (CBPR) as way to involve young people in health promotion and prevention strategizing as part of translational science practice at the community-level.
We utilized a CBPR approach that allowed youth to contribute at all stages.
Implementation of the CBPR approach involved the advancement of three key strategies including: (a) the local steering committee made up of youth, tribal leaders, and elders, (b) youth-researcher partnerships, and (c) youth action-groups to translate findings.
The addition of a local youth-action and translation group to the CBPR process in the southwest Alaska site represents an innovative strategy for disseminating findings to youth from a research project that focuses on youth resilience and wellbeing. This strategy drew from two community-based action activities: (a) being useful by helping elders and (b) being proud of our village.
In our study, youth informed the research process at every stage, but most significantly youth guided the translation and application of the research findings at the community level. Findings from the research project were translated by youth into serviceable action in the community where they live. The research created an experience for youth to spend time engaged in activities that, from their perspectives, are important and contribute to their wellbeing and healthy living. Youth-guided CBPR meant involving youth in the process of not only understanding the research process but living through it as well.
Alaska Native; Yup'ik Eskimo; youth-guided; community-based participatory research; youth prevention; translational science
Proto-oncogene activation caused by retroviral vector integration can cause malignancies in gene therapy trials. This has led investigators to search for less genotoxic vectors with minimal enhancer activity and a decreased risk of influencing neighboring chromosomal gene expression after integration. We previously showed that foamy virus vectors expressing the canine CD18 gene from an internal murine stem cell virus promoter could cure canine leukocyte adhesion deficiency. Here we have repeated these studies using a foamy virus vector expressing canine CD18 from a phosphoglycerate kinase gene promoter. In vitro analysis showed that this vector did not contain an enhancer that activated neighboring genes, and it expressed CD18 efficiently in canine neutrophils and CD34+ cells. However, dogs that received hematopoietic stem cells transduced with the PGK-CD18 vector continued to suffer from leukocyte adhesion deficiency, and sometimes died prematurely of the disease. These studies show that the phosphoglycerate kinase promoter cannot effectively replace the murine stem cell virus promoter in CD18-expressing foamy virus vectors, and they suggest that vectors containing a strong promoter/enhancer may be necessary for the treatment of human leukocyte adhesion deficiency.
Foamy virus; retrovirus; gene therapy; immunodeficiency; hematopoietic stem cell; canine
Background: Heme attachment to cytochrome c is a catalyzed post-translational modification.
Results: We identify a ternary complex of the cytochrome c biogenesis protein CcmE, heme, and a cytochrome, and demonstrate its functional significance.
Conclusion: The complex is a trapped catalytic intermediate at the point of heme transfer from the cytochrome biogenesis apparatus to the cytochrome.
Significance: An insight into biosynthesis of heme proteins.
c-Type cytochromes are widespread proteins, fundamental for respiration or photosynthesis in most cells. They contain heme covalently bound to protein in a highly conserved, highly stereospecific post-translational modification. In many bacteria, mitochondria, and archaea this heme attachment is catalyzed by the cytochrome c maturation (Ccm) proteins. Here we identify and characterize a covalent, ternary complex between the heme chaperone CcmE, heme, and cytochrome c. Formation of the complex from holo-CcmE occurs in vivo and in vitro and involves the specific heme-binding residues of both CcmE and apocytochrome c. The enhancement and attenuation of the amounts of this complex correlates completely with known consequences of mutations in genes for other Ccm proteins. We propose the complex is a trapped catalytic intermediate in the cytochrome c biogenesis process, at the point of heme transfer from CcmE to the cytochrome, the key step in the maturation pathway.
Cytochrome c; Cytochromes; Energy Metabolism; Enzymes; Heme; CcmE; Cytochrome c Biogenesis; Post-translational Modification
Generalized anxiety is thought to result, in part, from impairments in contingency awareness during conditioning to cues that predict aversive or fearful outcomes. Dopamine neurons of the ventral midbrain exhibit heterogeneous responses to aversive stimuli that are thought to provide a critical modulatory signal to facilitate orienting to environmental changes and assignment of motivational value to unexpected events. Here, we describe a mouse model in which activation of dopamine neurons in response to an aversive stimulus is attenuated by conditional genetic inactivation of functional N–methyl–D–aspartate–type glutamate receptors (NMDARs) on dopamine neurons. We discovered that altering the magnitude of excitatory responses by dopamine neurons in response to an aversive stimulus is associated with impaired conditioning to a cue that predicts an aversive outcome. Impaired conditioning by these mice is associated with development of a persistent, generalized anxiety–like phenotype. These data are consistent with a role for dopamine in facilitating contingency awareness that is critical for the prevention of generalized anxiety.
The ferredoxin-dependent nitrite reductase from the green alga Chlamydomonas reinhardtii has been cloned, expressed in Escherichia coli as a His-tagged recombinant protein, and purified to homogeneity. The spectra, kinetic properties and substrate-binding parameters of the C. reinhardtii enzyme are quite similar to those of the ferredoxin-dependent spinach chloroplast nitrite reductase. Computer modeling, based on the published structure of spinach nitrite reductase, predicts that the structure of C. reinhardtii nitrite reductase will be similar to that of the spinach enzyme. Chemical modification studies and the ionic-strength dependence of the enzyme’s ability to interact with ferredoxin are consistent with the involvement of arginine and lysine residues on C. reinhardtii nitrite reductase in electrostatically-stabilized binding to ferredoxin. The C. reinhardtii enzyme has been used to demonstrate that hydroxylamine can serve as an electron-accepting substrate for the enzyme and that the product of hydroxylamine reduction is ammonia, providing the first experimental evidence for the hypothesis that hydroxylamine, bound to the enzyme, can serve as a late intermediate during the reduction of nitrite to ammonia catalyzed by the enzyme.
Nitrite reductase; NII1; PETF; ferredoxin; hydroxylamine reduction; tertiary structure
A growing body of research supports the development of recombinant adeno-associated viral vectors (rAAV) for delivery of gene expression cassettes to striated musculature as a method of treating severe neuromuscular conditions. However, it is unclear whether delivery protocols that achieve extensive gene transfer in mice can be adapted to produce similarly extensive gene transfer in larger mammals and ultimately patients. Consequently, we sought to investigate methodological modifications that would facilitate rAAV-mediated gene transfer to the striated musculature of canines. A simple procedure incorporating acute a) occlusion of limb blood flow, b) exsanguination via compression bandage, and c) vector “dwell” time of < 20 minutes, markedly enhanced the transduction of limb muscles, compared with a simple bolus limb infusion of vector. A complementary method whereby vector was infused into the jugular vein led to efficient transduction of cardiomyocytes and to a lesser degree the diaphragm. Together these methods can be used to achieve transgene expression in heart, diaphragm, and limb muscles of juvenile dogs using rAAV6 vectors. These results establish that rAAV-mediated gene delivery is a viable approach to achieving systemic transduction of striated musculature in mammals approaching the dimensions of newborn humans.