The development of language and communication may play an important role in the emergence of behavioral problems in young children, but they are rarely included in predictive models of behavioral development. In this study, cross-sectional relationships between language, attention, and behavior problems were examined using parent report, videotaped observations, and performance measures in a sample of 116 severely and profoundly deaf and 69 normally hearing children ages 1.5 to 5 years. Secondary analyses were performed on data collected as part of the Childhood Development After Cochlear Implantation Study, funded by the National Institutes of Health. Hearing-impaired children showed more language, attention, and behavioral difficulties, and spent less time communicating with their parents than normally hearing children. Structural equation modeling indicated there were significant relationships between language, attention, and child behavior problems. Language was associated with behavior problems both directly and indirectly through effects on attention. Amount of parent–child communication was not related to behavior problems.
Proteins can form reversible mixed disulfides with glutathione (GSH). It has been hypothesized that protein glutathionylation may represent a mechanism of redox regulation, in a fashion similar to that mediated by protein phosphorylation. We investigated whether GSH has a signaling role in the response of HL60 cells to hydrogen peroxide (H 2O2), in addition to its obvious antioxidant role. We identified early changes in gene expression induced at different times by H 2O2 treatment, under conditions that increase protein glutathionylation and minimal toxicity. We then investigated the effect of prior GSH depletion by buthionine sulfoximine and diethylmaleate on this response. The analysis revealed 2,016 genes regulated by H 2O2. Of these, 215 genes showed GSHdependent expression changes, classifiable into four clusters displaying down-or up-regulation by H 2O2, either potentiated or inhibited by GSH depletion. The modulation of 20 selected genes was validated by real-time RT-PCR. The biological process categories overrepresented in the largest cluster (genes whose upregulation was inhibited by GSH depletion) were NF-B activation, transcription, and DNA methylation. This cluster also included several cytokine and chemokine ligands and receptors, the redox regulator thioredoxin interacting protein, and the histone deacetylase sirtuin. The cluster of genes whose up-regulation was potentiated by GSH depletion included two HSPs (HSP40 and HSP70) and the AP-1 transcription factor components Fos and FosB. This work demonstrates that GSH, in addition to its antioxidant and protective function against oxidative stress, has a specific signaling role in redox regulation.chemokines ͉ cytokines ͉ hydrogen peroxide ͉ oxidative stress G lutathione (GSH) protects the cell from damage induced by high levels of reactive oxygen species (ROS), defined as oxidative stress (1). Its main antioxidant activity consists of the detoxification of peroxides, partly with the aid of various GSH peroxidases. Its role as a major thiol antioxidant is demonstrated by the fact that various conditions of oxidative stress are exacerbated by GSH depletion, for instance by inhibitors of its synthesis, and ameliorated by the addition of GSH or its precursors, including N-acetylcysteine (NAC). Experimentally, thiol antioxidants, including GSH and NAC, have been used as tools to investigate the role of ROS in biological systems.In addition to scavenging ROS, GSH can form mixed disulfides with proteins, a phenomenon known as protein glutathionylation (2-4). This can occur by various reactions, either by thiol͞disulfide exchange between protein cysteines and oxidized GSH (GSH disulfide, GSSG), by direct oxidation, or through the NO-mediated formation of S-nitrosothiols (3, 4).The effect of protein glutathionylation is generally considered deleterious in the framework of oxidative stress, because it is one of the many forms of thiol oxidation induced by ROS. However, according to the more recent concept of redox regulation, several protein cysteines can be d...
Adenosine-to-inosine RNA editing, a fundamental RNA modification, is regulated by adenosine deaminase (AD) domain containing proteins. Within the testis, RNA editing is catalyzed by ADARB1 and is regulated in a cell-type dependent manner. This study examined the role of two testis-specific AD domain proteins, ADAD1 and ADAD2, on testis RNA editing and male germ cell differentiation. ADAD1, previously shown to localize to round spermatids, and ADAD2 had distinct localization patterns with ADAD2 expressed predominantly in mid-to late-pachytene spermatocytes suggesting a role for both in meiotic and post-meiotic germ cell RNA editing. AD domain analysis showed the AD domain of both ADADs was likely catalytically inactive, similar to known negative regulators of RNA editing. To assess the impact of Adad mutation on male germ cell RNA editing, CRISPRinduced alleles of each were generated in mouse. Mutation of either Adad resulted in complete male sterility with Adad1 mutants displaying severe teratospermia and Adad2 mutant germ cells unable to progress beyond round spermatid. However, mutation of neither Adad1 nor Adad2 impacted RNA editing efficiency or site selection. Taken together, these results demonstrate ADAD1 and ADAD2 are essential regulators of male germ cell differentiation with molecular functions unrelated to A-to-I RNA editing. RNA editing is a class of post-transcriptional modification that enhances the complexity of the transcriptome 1. On a molecular level, RNA editing is the irreversible chemical modification of a nucleotide within an intact RNA. Two basic types of RNA editing are observed in mammals, adenosine to inosine and cytosine to uridine, of which adenosine to inosine (A-to-I) occurs much more frequently 2. A-to-I RNA editing may occur at one or more sites in a given target RNA and across the entire population of a target RNA or a fraction thereof. To date, A-to-I RNA editing has been observed in a diverse range of RNAs including mRNAs, small RNAs, and long noncoding RNAs 3,4. Functionally, inosine mimics the behavior of guanine and is read as such by the translational machinery 5 , thus A-to-I RNA editing events behave as A-to-G mutations on the RNA level. As a consequence, the outcome of A-to-I RNA editing varies widely based on the RNA target and the edited site or sites within the target. Reported impacts of RNA editing include altered protein coding potential 6 , splicing patterns 7 , and microRNA recognition (either from edits within miRNAs 8 themselves or their targets 2). The physiological relevance of RNA editing is clear as animals deficient for A-to-I RNA editing enzymes often show severe physiological defects 9-11. In mammals, RNA editing is catalyzed by two adenosine deaminase (AD) domain-containing proteins: Adenosine Deaminase, RNA-specific 1 and 2 (ADAR1 and ADAR2 in the human, and ADAR and ADARB1 in the mouse, respectively). Both enzymes contain at least one double-stranded RNA binding motif and an AD domain, which directly catalyzes the conversion of adenosine to inosine 5. ...
Transgenesis has been a mainstay of mouse genetics for over 30 yr, providing numerous models of human disease and critical genetic tools in widespread use today. Generated through the random integration of DNA fragments into the host genome, transgenesis can lead to insertional mutagenesis if a coding gene or an essential element is disrupted, and there is evidence that larger scale structural variation can accompany the integration. The insertion sites of only a tiny fraction of the thousands of transgenic lines in existence have been discovered and reported, due in part to limitations in the discovery tools. Targeted locus amplification (TLA) provides a robust and efficient means to identify both the insertion site and content of transgenes through deep sequencing of genomic loci linked to specific known transgene cassettes. Here, we report the first large-scale analysis of transgene insertion sites from 40 highly used transgenic mouse lines. We show that the transgenes disrupt the coding sequence of endogenous genes in half of the lines, frequently involving large deletions and/or structural variations at the insertion site. Furthermore, we identify a number of unexpected sequences in some of the transgenes, including undocumented cassettes and contaminating DNA fragments. We demonstrate that these transgene insertions can have phenotypic consequences, which could confound certain experiments, emphasizing the need for careful attention to control strategies. Together, these data show that transgenic alleles display a high rate of potentially confounding genetic events and highlight the need for careful characterization of each line to assure interpretable and reproducible experiments.
L-Asparaginase is important in the induction regimen for treating acute lymphoblastic leukemia. Cytotoxic complications are clinically significant problems lacking mechanistic insight. To reveal tissue-specific molecular responses to this drug, mice were administered asparaginase from either Escherichia coli (clinically used) or Wolinella succinogenes (novel, glutaminasefree form). Both enzymes abolished serum asparagine, but only the E. coli form reduced circulating glutamine. E. coli asparaginase reduced protein synthesis in liver and spleen but not pancreas via increased phosphorylation of the translation factor eIF2. In contrast, treatment with Wolinella caused no untoward changes in protein synthesis in any tissue examined. Treating mice deleted for the eIF2 kinase, GCN2, with the E. coli enzyme showed eIF2 phosphorylation to be GCN2-dependent, but only initially. Furthermore, although eIF2 phosphorylation was not increased in the pancreas or by Wolinella asparaginase, expression of the amino acid stress response genes, asparagine synthetase and CHOP/GADD153, increased as a result of both enzymes, even in tissues demonstrating no change in eIF2 phosphorylation. Finally, signaling downstream of the mammalian target of rapamycin kinase was repressed in liver and pancreas by E. coli but not Wolinella asparaginase. These data demonstrate that the nutrient stress response to asparaginase is tissuespecific and exacerbated by glutamine depletion. Importantly, increased expression of asparagine synthetase and CHOP does not require eIF2 phosphorylation, signifying alternate or auxiliary means of inducing gene expression under conditions of amino acid depletion in the whole animal.
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