RESUMO
We identified a subset of impulsive, aggressive children as having symptoms that met criteria for Intermittent Explosive Disorder (IED) using the Interview Module for Intermittent Explosive Disorder (M-IED). The M-IED was administered to 34 children and adolescents between the ages of 10 and 17. These data provide initial evidence for the M-IED as a useful instrument in the diagnosis of IED in adolescents. The M-IED displayed a high level of inter-rater reliability and adequate test-retest reliability. Construct validity was supported by the fact that the subjects with IED symptomatology had significantly more lifetime aggression, oppositionality, inattention and hyperactivity/impulsivity compared to community controls. In addition, the subjects with IED symptomatology had a significantly greater number of episodes of lifetime physical aggression and documented episodes of aggression while in residential treatment compared to psychiatric controls. The subjects with IED symptomatology had a greater number of positive screening questions for DSM-IV diagnoses using the Swanson, Nolan and Pelham questionnaire (SNAP-IV), particularly those related to IED and posttraumatic stress disorder than psychiatric controls.
Assuntos
Agressão , Transtornos Disruptivos, de Controle do Impulso e da Conduta/diagnóstico , Escalas de Graduação Psiquiátrica/normas , Adolescente , Estudos de Casos e Controles , Criança , Transtornos Disruptivos, de Controle do Impulso e da Conduta/classificação , Transtornos Disruptivos, de Controle do Impulso e da Conduta/psicologia , Feminino , Humanos , Masculino , Projetos Piloto , Valor Preditivo dos Testes , Psicometria , Reprodutibilidade dos TestesRESUMO
The role of several regulatory elements in environmental modulation of mucoidy in Pseudomonas aeruginosa was studied. Transcriptional activation of algD, necessary for the mucoid phenotype, was found to depend on FUS, the newly identified far-upstream sites of the algD promoter. The FUS were delimited to a region spanning nucleotides -432 to -332 relative to the algD mRNA start site. Insertional inactivation of algR in PAO568 abolished the algD promoter response to nitrogen availability and greatly diminished but did not completely eliminate reactivity to changes in salt concentration. Insertional inactivation of rpoN (ntrA) in PAO568 did not affect algR and algD transcription.
Assuntos
Dioxigenases , Genes Bacterianos , Regiões Promotoras Genéticas , Pseudomonas aeruginosa/genética , Sequência de Bases , Catecol 2,3-Dioxigenase , DNA Bacteriano/genética , Dados de Sequência Molecular , Mutagênese Insercional , Oxigenases/metabolismo , Pseudomonas aeruginosa/fisiologia , RNA Mensageiro/genética , Transcrição GênicaRESUMO
A novel procaryotic transcriptional regulatory element, AlgP, with a histone H1-like carboxy-terminal domain was identified in Pseudomonas aeruginosa. AlgP is required for transcription of the key biosynthetic gene algD, which is necessary for production of the exopolysaccharide alginate causing mucoidy in P. aeruginosa. Mucoidy is a critical virulence determinant of P. aeruginosa invariably associated with the respiratory infections causing high mortality in cystic fibrosis. Here we show that AlgP and histones H1 both have repeated units of the Lys-Pro-Ala-Ala motif (KPAA) and its variations within their long (over 100 amino acids) carboxy-terminal domains. This region of histone H1 tails has been shown to bind to the linker DNA in eucaryotic chromatin fibers. A synthetic 50-mer peptide consisting of repeats from the AlgP carboxy-terminal domain was found to bind DNA in a mobility shift DNA-binding assay. AlgP is encoded by a gene that contains multiple direct repeats organized as tandem, head-to-tail, 12-base-pair (bp) units overlapping with six highly conserved 75-bp units. The repetitive structure of the algP gene appears to participate in the processes underlying the metastable character of mucoidy in P. aeruginosa. Relatively large DNA rearrangements spanning the region with tandem direct repeats encoding the carboxy-terminal histone H1-like structure of AlgP were detected in several strains upon conversion from the mucoid to the nonmucoid phenotype. The frequency of the detectable algP rearrangements associated with the transition into the nonmucoid state varied from strain to strain and ranged from 0 to 50%. The nonmucoid derivatives with the clearly rearranged chromosomal copy of algP were complemented to mucoidy with plasmids containing algP from P. aeruginosa PAO. When a random collection of mucoid strains, isolated from different cystic fibrosis patients, was analyzed by using polymerase chain reaction, an additional level of strain-dependent sequence variation in algP was observed. Variations in the number of the 12-bp repeats were found; however, they did not appear to influence the mucoid status of the strains examined. Thus, the repeated region of algP appears to be a hot spot for DNA rearrangements and strain-dependent variability.
Assuntos
Proteínas de Ligação a DNA/genética , Genes Bacterianos , Genes Reguladores , Histonas/genética , Pseudomonas aeruginosa/genética , Sequências Repetitivas de Ácido Nucleico , Sequência de Aminoácidos , Sequência de Bases , DNA Bacteriano/genética , Proteínas de Ligação a DNA/metabolismo , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , Pseudomonas aeruginosa/fisiologia , RNA Bacteriano/genética , Mapeamento por Restrição , Homologia de Sequência do Ácido NucleicoRESUMO
The complete nucleotide sequence of a 3.2-kilobase-pair chromosomal region containing the algP and algQ genes was determined. The algQ gene encodes an acidic 18-kilodalton polypeptide required for transcriptional activation of the algD gene. The algD gene product catalyzes a critical step in alginate biosynthesis, and its overproduction is necessary for the emergence of mucoid Pseudomonas aeruginosa during chronic infections in cystic fibrosis. A novel genetic element, algP, was identified immediately downstream of algQ. This gene appears to act synergistically with algQ. Unlike a biosynthetic gene, algD, and another regulatory gene, algR, which undergo transcriptional activation in mucoid cells, both algP and algQ are equally transcribed in mucoid and nonmucoid isogenic strains of P. aeruginosa. The promoter regions of algP and algQ were mapped by using S1 nuclease protection analysis. The algQ promoter was also analyzed and showed activity in an in vitro transcriptional runoff assay with major RNA polymerase species from P. aeruginosa and Escherichia coli. The putative algQ and algP promoter sequences, unlike algD and algR, resemble sigma 70-utilized promoters from E. coli and appeared constitutively transcribed at a low level in P. aeruginosa. The algP gene has an unusual DNA sequence, with multiple direct repeats organized in six highly conserved, tandemly arranged, 75-base-pair (bp) units. At a lower level, this sequence had 45 degenerate repeats of 12 bp overlapping with the 75-bp repeats and extending beyond the region of 75-bp repeats. The algP repeats appeared important for the function of the algQ-algP regulatory region in maintaining mucoidy.
Assuntos
Alginatos/metabolismo , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Família Multigênica , Pseudomonas aeruginosa/genética , Transcrição Gênica , Sequência de Bases , Sondas de DNA , Genes Reguladores , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação , Sondas de Oligonucleotídeos , Plasmídeos , Pseudomonas aeruginosa/fisiologia , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Homologia de Sequência do Ácido NucleicoRESUMO
Increased levels of alginate biosynthesis cause mucoidy in Pseudomonas aeruginosa, a virulence factor of particular importance in cystic fibrosis. The algR gene product, which controls transcription of a key alginate biosynthetic gene, algD, is homologous to the activator members of the two-component, environmentally responsive systems (NtrC, OmpR, PhoB, ArcA, etc). In this report, we show that mutations in the muc loci, (muc-2, muc-22, and muc-23, in the standard genetic P. aeruginosa strain PAO, as well as a mapped muc allele in an isolate from a cystic fibrosis patient) affect transcription of algD and algR. This influence was strongly dependent on environmental factors. Regulation by nitrogen was observed in all strains examined, but the absolute transcriptional levels, determining the mucoid or nonmucoid status, were strain (muc allele)-dependent. Increased concentrations of NaCl in the medium, an osmolyte which is elevated in cystic fibrosis lung secretions, resulted in an increased algD transcription and mucoid phenotype in a muc-2 strain; the same conditions, however, produced a nonmucoid phenotype in the muc-23 background and abolished algD transcription. Mutations in the muc loci may cause mucoidy by deregulating the normal response of the alginate system to environmental stimuli.
Assuntos
Desidrogenases de Carboidrato/genética , Fibrose Cística/microbiologia , Glicosaminoglicanos/biossíntese , Polissacarídeos Bacterianos/biossíntese , Pseudomonas aeruginosa/genética , Fatores de Transcrição/genética , Alginatos/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Mutação , Nitrogênio/metabolismo , Regiões Promotoras Genéticas , Pseudomonas aeruginosa/enzimologiaRESUMO
A new alginate regulatory gene, algQ, was identified in a chromosomal region which, when tandemly amplified, induces mucoidy in Pseudomonas aeruginosa. The algQ gene was found closely linked to the previously identified algR gene. Both algQ and algR were required for transcription of the key alginate biosynthetic gene, algD. In addition, expression of the algR gene was studied. The algR promoter was mapped by S1 nuclease and reverse transcription and found to be activated in mucoid cells. However, even in nonmucoid cells, transcription of algR was detectable at an approximately 50-fold-lower level, as opposed to the algD promoter, which was silent in the nonmucoid background. Transcription of both promoters was studied by using algR- and algD-specific oligonucleotides and total cellular RNA from fresh cystic fibrosis isolates of mucoid P. aeruginosa and their nonmucoid revertants. Identical patterns of activity were found in all strains: in mucoid cells, both algR and algD were activated. This finding indicated that common mechanisms were involved in the regulation of alginate gene expression. However, when the algR gene was cloned behind the tac promoter on a broad-host-range-controlled expression vector, induction of transcription with isopropropyl-beta-D-thiogalactopyranoside (IPTG) caused the appearance of a nonmucoid phenotype in previously mucoid cells. This effect was transient, since removal of the inducer (IPTG) made cells mucoid again. Since the algR gene product is homologous to transcriptional regulators from a class of environmentally responsive systems (known to have a second, sensory component), the algQ gene could be a candidate for the sensory component of the alginate system.
Assuntos
Alginatos/genética , Genes Bacterianos , Genes Reguladores , Pseudomonas aeruginosa/patogenicidade , Transcrição Gênica , Sequência de Bases , Deleção Cromossômica , Fibrose Cística/genética , Fibrose Cística/microbiologia , Endonucleases , Humanos , Dados de Sequência Molecular , Mapeamento de Nucleotídeos , Regiões Promotoras Genéticas , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiologia , Endonucleases Específicas para DNA e RNA de Cadeia Simples , VirulênciaRESUMO
The Pseudomonas aeruginosa capsule, composed of polysaccharide alginate, is an important Pseudomonas virulence factor encountered primarily in cystic fibrosis. The regulatory algR gene positively controls transcription of a key alginate biosynthetic gene, algD. The algR gene was subcloned and sequenced by creating a set of nested deletions in M13 bacteriophage. DNA sequence analysis of algR revealed the homology of its gene product with a recently recognized class of environmentally responsive bacterial regulatory genes, including ompR, phoB, sfrA, ntrC, spoOA, dctD, and virG; these transcriptional activators control cellular reactions to osmotic pressure, phosphate limitations, or specific chemical compounds present in the medium or released from wounded host tissue. These findings indicate that novel conditions in lungs affected by cystic fibrosis may be participating in the control of mucoidy.
Assuntos
Alginatos/metabolismo , Genes Bacterianos , Genes Reguladores , Pseudomonas aeruginosa/genética , Transativadores , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Clonagem Molecular , Dados de Sequência Molecular , Pseudomonas aeruginosa/patogenicidade , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica , VirulênciaRESUMO
A set of bacteriophage and plasmid vectors containing xylE as a reporter gene was constructed for the analysis of promoters functional in Escherichia coli and in other Gram-negative bacteria. Two M13 bacteriophage derivatives, M13mVDX18 and M13mMK010, were designed for rapid cloning, screening and sequencing of DNA fragments promoting transcription in E. coli. To demonstrate their utility, total cellular DNA from a variety of bacterial species including Pseudomonas aeruginosa strain PAO was shotgun cloned in M13 vectors and clones displaying promoter activity in E. coli were isolated. These randomly cloned promoters from P. aeruginosa, Borrelia burgdorferi, Streptococcus pneumoniae and other bacterial species were sequenced without a need for further subcloning manipulation. The promoter activity of P. aeruginosa clones was verified by subcloning inserts on a broad-host-range promoter probe vector pVDX18 and assaying the xylE transcription from these promoters in P. aeruginosa. The pVDX18 vector was also used for initial characterization of the algD promoter controlling mucoidy in P. aeruginosa. The activities of the wild-type and deletion clones of the algD promoter were compared. Results indicated that the region containing direct and inverted repeats at -55 to -110 bp upstream of the mRNA 5' end was important for the activation of the algD transcription in mucoid P. aeruginosa infecting cystic fibrosis patients.