RESUMO
ABSTRACT Planting material with superior resistance to Armillaria root disease was identified in a field trial established to investigate variation in Armillaria infection among different Pinus radiata nursery stock types. At stand age 6.4 years, total infection incidence, mortality, and degree of root collar girdling by Armillaria spp. were all significantly lower among trees derived from both rooted stool bed cuttings (physiological age 1 to 3 years) and rooted field cuttings (physiological age 3 to 6 years) than among those grown from seedlings. Cutting types did not differ significantly from one another. No significant differences were found between stock types in stem diameter, but trees from stool bed cuttings were significantly taller than seedling trees. Whether these differences remain detectable later in the rotation, initial results suggest that it may be advantageous to plant robust stock, of either cuttings or seedlings, on Armillaria-infested sites. The incidence of infection in living, green-crowned trees was unevenly distributed across the trial site, and was greater nearer to trees killed by Armillaria spp. than further away (significant within a radius of 10 m). By mapping visible Armillaria-caused mortality prior to thinning, it may be possible to delineate areas with a higher incidence of concealed chronic infection, thus defining infested sites for postharvest treatment.
RESUMO
Integrated self-transmissible elements called conjugative transposons have been found in many different bacteria, but little is known about how they excise from the chromosome to form the circular intermediate, which is then transferred by conjugation. We have now identified a gene, exc, which is required for the excision of the Bacteroides conjugative transposon, CTnDOT. The int gene of CTnDOT is a member of the lambda integrase family of recombinases, a family that also contains the integrase of the Gram-positive conjugative transposon Tn916. The exc gene was located 15 kbp from the int gene, which is located at one end of the 65 kbp element. The exc gene, together with the regulatory genes, rteA, rteB and rteC, were necessary to excise a miniature form of CTnDOT that contained only the ends of the element and the int gene. Another open reading frame (ORF) in the same operon and upstream of exc, orf3, was not essential for excision and had no significant amino acid sequence similarity to any proteins in the databases. The deduced amino acid sequence of the CTnDOT Exc protein has significant similarity to topoisomerases. A small ORF (orf2) that could encode a small, basic protein comparable with lambda and Tn916 excision proteins (Xis) was located immediately downstream of the CTnDOT int gene. Although Xis proteins are required for excision of lambda and Tn916, orf2 had no effect on excision of the element. Excision of the CTnDOT mini-element was not affected by the site in which it was integrated, another difference from Tn916. Our results demonstrate that the Bacteroides CTnDOT excision system is tightly regulated and appears to be different from that of any other known integrated transmissible element, including those of some Bacteroides mobilizable transposons that are mobilized by CTnDOT.
Assuntos
Bacteroides/genética , Conjugação Genética/genética , Elementos de DNA Transponíveis/genética , DNA Bacteriano/metabolismo , Genes Bacterianos/genética , Recombinação Genética/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Clonagem Molecular , DNA Topoisomerases Tipo I/química , DNA Bacteriano/genética , DNA Circular/genética , DNA Circular/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
The bacteriophage lambda excisionase (Xis) is a sequence-specific DNA binding protein required for excisive recombination. Xis binds cooperatively to two DNA sites arranged as direct repeats on the phage DNA. Efficient excision is achieved through a cooperative interaction between Xis and the host-encoded factor for inversion stimulation as well as a cooperative interaction between Xis and integrase. The secondary structure of the Xis protein was predicted to contain a typical amphipathic helix that spans residues 18 to 28. Several mutants, defective in promoting excision in vivo, were isolated with mutations at positions encoding polar amino acids in the putative helix (T. E. Numrych, R. I. Gumport, and J. F. Gardner, EMBO J. 11:3797-3806, 1992). We substituted alanines for the polar amino acids in this region. Mutant proteins with substitutions for polar amino acids in the amino-terminal region of the putative helix exhibited decreased excision in vivo and were defective in DNA binding. In addition, an alanine substitution at glutamic acid 40 also resulted in altered DNA binding. This indicates that the hydrophilic face of the alpha-helix and the region containing glutamic acid 40 may form the DNA binding surfaces of the Xis protein.
Assuntos
Bacteriófago lambda/enzimologia , Bacteriófago lambda/genética , DNA Nucleotidiltransferases/genética , DNA Nucleotidiltransferases/metabolismo , Proteínas Virais , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , Sítios de Ligação , DNA Nucleotidiltransferases/química , DNA Viral/química , DNA Viral/genética , DNA Viral/metabolismo , Escherichia coli/genética , Escherichia coli/virologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sequências Repetitivas de Ácido NucleicoRESUMO
OBJECTIVE: The aim of this study was to evaluate the association of age, race, and comorbid illness with procedures and complications in hospitalized patients with invasive carcinoma of the cervix in a statewide population-based database over a 3-year period. METHODS: Hospitalizations were classified into homogeneous subgroups based on a diagnosis of invasive cervical cancer. Cancer-related complications and comorbid diseases were evaluated. chi(2) and t tests determined differences in means or proportions. Linear regression techniques were applied to build models for hospitalization charges and lengths of stay (LOS). RESULTS: There were 1009 admissions. The mean age was 49.5, with a median age of 46 (21-100, SD 15.4). Of the total, 606/1009 (60%) were white, 354/1009 (35%) were African-American (AA), and 5% were "other" races. AAs were more likely to have Medicaid or be uninsured (44% vs 23%, P = 0. 001) and were more likely to be admitted for an emergency (unadjusted odds ratio (OR) = 1.6; 1.2-2.2), to have a comorbid illness (P = 0.001), to be admitted for a cancer-related complication (P = 0.036), to be admitted for a transfusion (P = 0. 01), and to be admitted for radiation therapy rather than surgery (P = 0.001). The following were associated with LOS and higher hospital costs: emergency admissions for complications of cancer, comorbid illness, and older age. CONCLUSIONS: Racial differences exist in patterns of admission, type of therapy, and severity of illness; however, there were no differences in charges or LOS for similar procedures. The large percentage of African-Americans uninsured or insured by government-supported programs indicates the potential impact of public policy on the care of these patients. Socioeconomic status rather than phenotypic appearance may be a more important determinant of outcome.
Assuntos
Hospitalização/estatística & dados numéricos , Neoplasias do Colo do Útero/terapia , Adulto , Negro ou Afro-Americano/estatística & dados numéricos , Fatores Etários , Comorbidade , Bases de Dados Factuais , Feminino , Acessibilidade aos Serviços de Saúde , Preços Hospitalares , Custos Hospitalares , Hospitalização/economia , Humanos , Seguro Saúde , Tempo de Internação , Maryland/epidemiologia , Pessoa de Meia-Idade , Análise Multivariada , Metástase Neoplásica , Grupos Raciais , Fatores Socioeconômicos , Resultado do Tratamento , Neoplasias do Colo do Útero/economia , Neoplasias do Colo do Útero/epidemiologia , População Branca/estatística & dados numéricosRESUMO
Integration host factor (IHF) is a protein that binds to the H' site of bacteriophage lambda with sequence specificity. Genetic experiments implicated amino acid residue Glu(44) of the beta-subunit of IHF in discrimination against substitution of A for T at position 44 of the TTR submotif of the binding site (Lee, E. C., Hales, L. M., Gumport, R. I., Gardner, J. F. (1992) EMBO J., 11, 305-313). We have extended this observation by generating all possible single-base substitutions at positions 43, 44, and 45 of the H' site. IHF failed to bind these H' site substitution mutants in vivo. The K(d)(app) value for each H' site substitution, except for H'45A mutant, was reduced >2000-fold relative to the wild-type site. Substitution of amino acid beta-Glu(44) with alanine prevented IHF from discriminating against the H'44A variant but not the other H' site substitution mutants. Further analysis with other substitutions at position beta44 demonstrated that both oxygens of the wild-type glutamic acid are necessary for discrimination of AT at position 44. Because the beta-Glu(44) residue does not contact the DNA, this residue probably enforces binding specificity indirectly through interaction with amino acids that themselves contact the DNA.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas de Bactérias/química , Pareamento de Bases , Ácido Glutâmico , Fatores Hospedeiros de Integração , Mutagênese Sítio-Dirigida , Subunidades Proteicas , Relação Estrutura-AtividadeRESUMO
Bacteroides conjugative transposons (CTns) are thought to transfer by first excising themselves from the chromosome to form a nonreplicating circle, which is then transferred by conjugation to a recipient. Earlier studies showed that transfer of most Bacteroides CTns is stimulated by tetracycline, but it was not known which step in transfer is regulated. We have cloned and sequenced both ends of the Bacteroides CTn, CTnDOT, and have used this information to examine excision and integration events. A segment of DNA that contains the joined ends of CTnDOT and an adjacent open reading frame (ORF), intDOT, was necessary and sufficient for integration into the Bacteroides chromosome. Integration of this miniature form of the CTn was not regulated by tetracycline. Excision of CTnDOT and formation of the circular intermediate were detected by PCR, using primers designed from the end sequences. Sequence analysis of the PCR products revealed that excision and integration involve a 5-bp coupling sequence-type mechanism possibly similar to that used by CTn Tn916, a CTn found originally in enterococci. PCR analysis also demonstrated that excision is a tetracycline-regulated step in transfer. The integrated minielement containing intDOT and the ends of CTnDOT did not excise, nor did a larger minielement that also contained an ORF located immediately downstream of intDOT designated orf2. Thus, excision involves other genes besides intDOT and orf2. Both intDOT and orf2 were disrupted by single-crossover insertions. Analysis of the disruption mutants showed that intDOT was essential for excision but orf2 was not. Despite its proximity to the integrase gene, orf2 appears not to be essential for excision.
Assuntos
Bacteroides/genética , Conjugação Genética , Elementos de DNA Transponíveis , Sequência de Aminoácidos , Clonagem Molecular , DNA Bacteriano/metabolismo , DNA Circular/genética , Genes Bacterianos , Integrases/genética , Dados de Sequência Molecular , Recombinação Genética , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
The integrase (Int) proteins encoded by bacteriophages HK022 and lambda catalyse similar site-specific integration and excision reactions between specific DNA regions known as attachment (att) sites. However, the Int proteins of HK022 and lambda are unable to catalyse recombination between non-cognate att sites. The att sites of both phages contain weak binding sites for Int, known as 'core-type' sites. Negatively acting nucleotide determinants associated with specific core sites (lambda B', HK022 B', HK022 C) are responsible for the barrier to non-cognate recombination. In this study, we used challenge phages to demonstrate that the lambda and HK022 Ints cannot bind to core sites containing non-cognate specificity determinants in vivo. We isolated mutants of the HK022 Int, which bind the lambda B' core site. Two mutants, D99N and D99A, have changed a residue in the core-binding (CB) domain, which may be directly contacting the core site DNA. We suggest that binding to the lambda B' site was accomplished by removing the negatively charged aspartate residue, which normally participates in a conflicting interaction with the G4 nucleotide of the lambda B' site. We showed that, although our mutants retain the ability to recombine their cognate att sites, they are unable to recombine lambda att sites.
Assuntos
Integrases/genética , Integrases/metabolismo , Salmonella typhimurium/virologia , Siphoviridae/enzimologia , Proteínas do Core Viral/metabolismo , Sequência de Aminoácidos , Sítios de Ligação Microbiológicos , Bacteriófago lambda/enzimologia , Bacteriófago lambda/metabolismo , Escherichia coli/virologia , Integrases/química , Dados de Sequência Molecular , Mutação , Plasmídeos/genética , Estrutura Secundária de Proteína , Recombinação Genética , Siphoviridae/genéticaRESUMO
CONTEXT: Recent reports on the use of psychotropic medications for preschool-aged children with behavioral and emotional disorders warrant further examination of trends in the type and extent of drug therapy and sociodemographic correlates. OBJECTIVES: To determine the prevalence of psychotropic medication use in preschool-aged youths and to show utilization trends across a 5-year span. DESIGN: Ambulatory care prescription records from 2 state Medicaid programs and a salaried group-model health maintenance organization (HMO) were used to perform a population-based analysis of three 1-year cross-sectional data sets (for the years 1991, 1993, and 1995). SETTING AND PARTICIPANTS: From 1991 to 1995, the number of enrollees aged 2 through 4 years in a Midwestern state Medicaid (MWM) program ranged from 146,369 to 158,060; in a mid-Atlantic state Medicaid (MAM) program, from 34,842 to 54,237; and in an HMO setting in the Northwest, from 19,107 to 19,322. MAIN OUTCOME MEASURES: Total, age-specific, and gender-specific utilization prevalences per 1000 enrollees for 3 major psychotropic drug classes (stimulants, antidepressants, and neuroleptics) and 2 leading psychotherapeutic medications (methylphenidate and clonidine); rates of increased use of these drugs from 1991 to 1995, compared across the 3 sites. RESULTS: The 1995 rank order of total prevalence in preschoolers (per 1000) in the MWM program was: stimulants (12.3), 90% of which represents methylphenidate (11.1); antidepressants (3.2); clonidine (2.3); and neuroleptics (0.9). A similar rank order was observed for the MAM program, while the HMO had nearly 3 times more clonidine than antidepressant use (1.9 vs 0.7). Sizable increases in prevalence were noted between 1991 and 1995 across the 3 sites for clonidine, stimulants, and antidepressants, while neuroleptic use increased only slightly. Methylphenidate prevalence in 2- through 4-year-olds increased at each site: MWM, 3-fold; MAM, 1.7-fold; and HMO, 3.1-fold. Decreases occurred in the relative proportions of previously dominant psychotherapeutic agents in the stimulant and antidepressant classes, while increases occurred for newer, less established agents. CONCLUSIONS: In all 3 data sources, psychotropic medications prescribed for preschoolers increased dramatically between 1991 and 1995. The predominance of medications with off-label (unlabeled) indications calls for prospective community-based, multidimensional outcome studies.
Assuntos
Controle Comportamental , Transtornos do Comportamento Infantil/tratamento farmacológico , Padrões de Prática Médica/tendências , Psicotrópicos/uso terapêutico , Mudança Social , Transtorno do Deficit de Atenção com Hiperatividade/tratamento farmacológico , Estimulantes do Sistema Nervoso Central/uso terapêutico , Pré-Escolar , Uso de Medicamentos/tendências , Feminino , Sistemas Pré-Pagos de Saúde , Humanos , Masculino , Medicaid , Metilfenidato/uso terapêutico , Prevalência , Estados Unidos/epidemiologiaRESUMO
The purpose of this study was to determine whether patients with end stage renal disease treated with hemodialysis were correlated in dialysis adequacy within facilities. This was a retrospective analysis of dialysis adequacy based on urea reduction ratio (URR) values from 6969 patients dialyzed at 154 facilities. The within-center correlation was quantified using the between-center variation and the parameter p that was derived using ANOVA tables and mixed effects models. The variation in center means for URR was wider than expected for independent observations (52.9-76.1 versus 60.7-73.8, respectively). Furthermore, there was a significant within-center correlation in URR values across all facilities (p = 0.136, P<0.0001), which persisted after adjusting for patient specific covariates, facility characteristics, and state. In conclusion, there was a substantial within-center correlation in dialysis adequacy that reflected important center effects on the outcome of ESRD patients.
Assuntos
Nitrogênio da Ureia Sanguínea , Unidades Hospitalares de Hemodiálise/normas , Falência Renal Crônica/terapia , Avaliação de Resultados em Cuidados de Saúde , Feminino , Humanos , Masculino , Mid-Atlantic Region , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
This study evaluated the association of race, comorbid illness, insurance status, and other prognostic factors on treatment and survival of patients with invasive carcinoma of the cervix, using as its methodology a retrospective study of patients with invasive carcinoma of the cervix treated from 1991 to 1998 at the University of Maryland. Of 161 such patients, 153 met the study criteria. Of these 153 women, 83 (54%) were African Americans and 70 (46%) were white. The mean (+/- SE) age of African American women was 52 +/- 1.8 years and that of white women was 50 +/- 1.8 years. African American women were more likely to present with stages II-IV disease (P = 0.01) and, as a consequence, underwent radiation therapy rather than surgery (P = 0.04). The survival of African American women with stage I-II disease was significantly lower than for white women. In the final regression model, stage III-IV disease (hazard ratio 3.2, 95% (CI 1.7, 6.1) African American race, (hazard ratio 1.9; 95% CI 1.0, 3.3) and comorbid illnesses (hazard ratio 2.3; 95% CI 1.3-4.0) were associated with poor survival. Adverse outcomes of African American women with cervical cancer persist after adjustment for stage of disease and other factors and are particularly apparent in stage I-II disease. The role of comorbid illness is deserving of further study.
Assuntos
Negro ou Afro-Americano/estatística & dados numéricos , Carcinoma/mortalidade , Neoplasias do Colo do Útero/mortalidade , Carcinoma/etnologia , Carcinoma/patologia , Feminino , Acessibilidade aos Serviços de Saúde , Humanos , Maryland/epidemiologia , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Estudos Retrospectivos , Fatores Socioeconômicos , Neoplasias do Colo do Útero/etnologia , Neoplasias do Colo do Útero/patologia , População Branca/estatística & dados numéricosRESUMO
OBJECTIVES: (1) To describe temporal patterns of office visits for attention-deficit/hyperactivity disorder (ADHD) and stimulant treatment for 5- to 14-year-old US youths; (2) to compare youth visits for ADHD with and without melication according to patient demographics, physician specialty, reimbursement source, and comorbid diagnoses; and (3) to compare office visits for youths with ADHD in relation to common medication patterns (stimulants alone, stimulants with other psychotherapeutic medication, and nonstimulant psychotherapeutic medications alone). DESIGN: Survey based on a national probability sample of office-based physicians in the United States. SETTING: Physician offices. PARTICIPANTS: A systematically sampled group of office-based physicians. MAIN OUTCOME MEASURES: National estimates of office visits for ADHD and psychotherapeutic drug visits for ADHD for each year and for a combined 8-year period. RESULTS: Youth visits for ADHD as a percentage of total physician visits had a 90% increase, from 1.9% in 1989 to 3.6% in 1996. Stimulant therapy within ADHD youth visits rose from 62.6% in 1989 to 76.6% in 1996. While the majority of non-ADHD youth visits were conducted by primary care physicians, one third of ADHD youth visits were managed by psychiatry and neurology specialists. Health maintenance organization insurance was the reimbursement source for 17.9% of non-ADHD youth visits but only 11.7% of ADHD youth visits. Complex medication therapy was more likely to be prescribed by psychiatrists and less likely to be related to visits with health maintenance organization reimbursement. CONCLUSIONS: National survey estimates in the 1990s confirm the substantial increase in visits for youths diagnosed as having ADHD, with more than three quarters of these visits associated with psychotherapeutic medication treatment. Physician specialty and reimbursement source variables identify distinct patient populations with a gradient in psychotherapeutic medication patterns from single-drug standard (stimulant) therapy to complex multidrug treatment regimens for which evidence-based scientific information is lacking.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade/tratamento farmacológico , Estimulantes do Sistema Nervoso Central/uso terapêutico , Padrões de Prática Médica/estatística & dados numéricos , Psicotrópicos/uso terapêutico , Adolescente , Criança , Pré-Escolar , Quimioterapia Combinada , Feminino , Humanos , Reembolso de Seguro de Saúde , Masculino , Medicina , Visita a Consultório Médico/estatística & dados numéricos , Especialização , Fatores de Tempo , Estados UnidosRESUMO
BACKGROUND AND PURPOSE: In prior studies, age, race, job category, disability, and cortical functions such as praxis, language, and memory have been associated with vocational outcome, but the influence of stroke location on return to work has never been critically examined. METHODS: We examined the influence of stroke location on vocational outcome in patients with clinically confirmed acute ischemic stroke from the National Institute of Neurological Disorders and Stroke Stroke Data Bank. RESULTS: Of 143 patients working full time at the time of first ischemic stroke, 23 patients were dead and 120 were alive at 1 year. Employment status was known in 109 (mean age, 55 years; 51 [47%] were white, and 82 [75%] were male). Fifty-eight (53%) had returned to work; most (85%) worked full time. Younger age was positively associated with return to work (P<0.05). In an age-adjusted analysis, stroke severity as measured by the Barthel Index 7 to 10 days after stroke was negatively associated with return to work (P<0.001). Higher household income and absence of cortical neurological dysfunction 7 to 10 days after stroke were positively but less strongly associated with return to work (P<0.08). Stroke location, sex, and depression at time of stroke were not associated with vocational outcome. CONCLUSIONS: Our data suggest that stroke location may be less important than other more easily measured factors in predicting vocational outcome.
Assuntos
Emprego/estatística & dados numéricos , Acidente Vascular Cerebral/patologia , Fatores Etários , Idoso , Análise de Variância , Afasia/etiologia , Córtex Cerebral/patologia , Circulação Cerebrovascular , Feminino , Humanos , Infarto da Artéria Cerebral Anterior/complicações , Infarto da Artéria Cerebral Anterior/patologia , Infarto da Artéria Cerebral Anterior/fisiopatologia , Infarto da Artéria Cerebral Posterior/complicações , Infarto da Artéria Cerebral Posterior/patologia , Infarto da Artéria Cerebral Posterior/fisiopatologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Índice de Gravidade de Doença , Fatores Socioeconômicos , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/fisiopatologiaRESUMO
To analyze the role of amino acids in the steroid receptor DNA binding domain (DBD) recognition helix in binding of the receptor to the estrogen response element (ERE), we adapted the powerful P22 challenge phage selection system for use with a vertebrate protein. We used the progesterone receptor DNA binding domain and selected for mutants that gained the ability to bind to the ERE. We used a mutagenesis protocol based on degenerate oligonucleotides to create a large and diverse pool of mutants in which 10 nonconsensus amino acids in the DNA recognition helix of the progesterone receptor DNA binding domain were randomly mutated. After a single cycle of modified P22 challenge phage selection, 37 mutant proteins were identified, all of which lost the ability to bind to the progesterone response element. In gel mobility shift assays, approximately 70% of the genetically selected mutants bound to the consensus ERE with a >4-fold higher affinity than the naturally occurring estrogen receptor DBD. In the P-box region of the DNA recognition helix, the selected mutants contained the amino acids found in the wild-type estrogen receptor DBD, as well as other amino acid combinations seen in naturally occurring steroid/nuclear receptors that bind the aGGTCA half-site. We also obtained high affinity DBDs with Trp(585) as the first amino acid of the P-box, although this is not found in the known steroid/nuclear receptors. In the linker region between the two zinc fingers, G597R was by far the most common mutation. In transient transfections in mammalian cells using promoter interference assays, the mutants displayed enhanced affinity for the ERE. When linked to an activation domain, the transfected mutants activated transcription from ERE-containing reporter genes. We conclude that the P-box amino acids can display considerable variation and that the little studied linker amino acids play an important role in determining affinity for the ERE. This work also demonstrates that the P22 challenge phage genetic selection system, modified for use with a mammalian protein, provides a novel, single cycle selection for steroid/nuclear receptor DBDs with altered specificity and greatly enhanced affinity for their response elements.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Estrogênios/metabolismo , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Bactérias/metabolismo , Bacteriófago P22/genética , Sequência de Bases , Primers do DNA , Proteínas de Ligação a DNA/genética , Dados de Sequência Molecular , Mutagênese , Ligação Proteica , Receptores de Superfície Celular/genéticaRESUMO
Site-specific recombination by phages lambda and P22 is carried out by multiprotein-DNA complexes. Integration host factor (IHF) facilitates lambda site-specific recombination by inducing DNA bends necessary to form an active recombinogenic complex. Mutants lacking IHF are over 1,000-fold less proficient in supporting lambda site-specific recombination than wild-type cells. Although the attP region of P22 contains strong IHF binding sites, in vivo measurements of integration and excision frequencies showed that infecting P22 phages can perform site-specific recombination to its maximum efficiency in the absence of IHF. In addition, a plasmid integration assay showed that integrative recombination occurs equally well in wild-type and ihfA mutant cells. P22 integrative recombination is also efficient in Escherichia coli in the absence of functional IHF. These results suggest that nucleoprotein structures proficient for recombination can form in the absence of IHF or that another factor(s) can substitute for IHF in the formation of complexes.
Assuntos
Sítios de Ligação Microbiológicos , Proteínas de Bactérias/metabolismo , Bacteriófago P22/genética , Recombinação Genética , Salmonella typhimurium/virologia , Integração Viral/fisiologia , Bacteriófago P22/fisiologia , Escherichia coli/virologia , Fatores Hospedeiros de Integração , Lisogenia , Plasmídeos/genética , Ativação ViralRESUMO
The bacteriophage lambda excisionase (Xis) protein is required for excisive site-specific recombination. Xis is composed of 72 amino acids and binds cooperatively to two DNA sites (X1 and X2) that are arranged as direct repeats. Alternatively, Xis binds cooperatively with the host-encoded factor for inversion stimulation (FIS) protein at the X1 and F sites, respectively. Here we analyzed the effects of missense substitutions from codon 57 to the carboxyl end of the protein and nonsense mutations that truncate the protein at various positions from residues 60 to 69. We find that all of the mutant proteins promote excision to some extent and interact cooperatively with FIS. Some mutants have no detectible phenotype while others are altered in their abilities to promote excision or to interact cooperatively with integrase (Int). Computer modeling predicts that amino acids from residues 59 to 65 are in an alpha-helix conformation. Mutants with substitutions on one side of the helix at residues 57, 60, 63 and 64 as well as truncated mutants containing 60, 61 or 63 amino acids, fail to interact cooperatively with Int suggesting that this region of the protein forms the interface with Int. Mutants with substitutions at other positions in the putative helix have no detectible phenotype. Residues 66 to 68 may form a reverse turn and the last four amino acids (69 to 72) may not be crucial for the structure or function of the protein.
Assuntos
Bacteriófago lambda/enzimologia , DNA Nucleotidiltransferases/química , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Simulação por Computador , DNA Nucleotidiltransferases/genética , Proteínas de Ligação a DNA/química , Lisogenia/fisiologia , Dados de Sequência Molecular , Mutagênese/genética , Óperon/genética , Fragmentos de Peptídeos/genética , Fenótipo , Estrutura Secundária de Proteína , Recombinação Genética/genética , Proteínas Virais/químicaRESUMO
This study involved a review of the medical records of 367 patients treated surgically after femoral neck fracture. Linkage of these records with claims files from the Health Care Financing Administration allowed as many as 8 years of followup to analyze the rates of hospital readmission rates for revision, other postoperative complications and mortality. The results revealed: (1) a significantly higher revision rate was associated with internal fixation for the treatment of displaced femoral neck fractures in patients older than 80 years of age; no differences in revision rates were seen between internal fixation or hemiarthroplasty for the treatment of nondisplaced femoral neck fractures in this patient age group; (2) no differences in revision rates were found between internal fixation or hemiarthroplasty for the treatment of displaced femoral neck fractures in patients between the ages of 65 to 80 years; (3) a significantly higher mortality rate was associated with internal fixation than hemiarthroplasty for patients who were between the ages of 65 and 80 years; and (4) no differences in medical or surgical complications, revision rates, or other outcomes were found between unipolar and bipolar prostheses, or between anterior and posterior surgical approaches for hemiarthroplasty in patients who were age 65 years or older.
Assuntos
Fraturas do Colo Femoral/cirurgia , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Artroplastia de Quadril/efeitos adversos , Artroplastia de Quadril/métodos , Centers for Medicare and Medicaid Services, U.S. , Estudos de Coortes , Feminino , Seguimentos , Fixação Interna de Fraturas/efeitos adversos , Fixação Interna de Fraturas/métodos , Prótese de Quadril/efeitos adversos , Humanos , Luxações Articulares/cirurgia , Masculino , Registro Médico Coordenado , Avaliação de Resultados em Cuidados de Saúde , Readmissão do Paciente , Complicações Pós-Operatórias , Desenho de Prótese , Reoperação , Estudos Retrospectivos , Taxa de Sobrevida , Resultado do Tratamento , Estados UnidosAssuntos
Deficiências do Desenvolvimento/reabilitação , Instituições para Cuidados Intermediários/normas , Satisfação do Paciente , Deficiências do Desenvolvimento/psicologia , Estudos de Avaliação como Assunto , Pesquisas sobre Atenção à Saúde , Humanos , Entrevistas como Assunto , Avaliação de Resultados em Cuidados de Saúde , Inquéritos e Questionários , Estados UnidosRESUMO
The Council on Quality and Leadership in Supports for People With Disabilities (The Council), formerly known as The Accreditation Council, altered its definition of quality from "compliance with organizational process" to "responsiveness to people." Council representatives conducted focus group and individual meetings with people who have disabilities to identify priority outcomes they expect from services and supports. The 1993 Outcome Based Performance Measures was used in 447 interviews as part of 54 accreditation reviews. Staff analysis and factor analysis of the outcome interviews using a principle components extraction and varimax rotation resulted in 24 variables loading onto seven major factors (Identity, Autonomy, Affiliation, Attainment, Rights, Health, and Safeguards), which form the basis of The Council 1997 Personal Outcome Measures.
Assuntos
Deficiência Intelectual , Serviços de Saúde Mental/normas , Qualidade da Assistência à Saúde , HumanosRESUMO
Bacteriophage lambda site-specific recombination is catalyzed by the phage-encoded integrase (Int) protein. Using a collection of 21 recombination-defective Int mutants, we performed a second-site reversion analysis. One of the primary mutants contained a valine-to-glutamic acid change at position 175 (V175E), and a pseudorevertant with a lysine change at this site (V175K) was also isolated. Relative to the wild-type protein, the V175E protein was defective in its ability to form the attL complex and to catalyze excision in vivo and in vitro. A mutant containing an alanine substitution (V175A) was made by site-directed mutagenesis, and it was more efficient than the V175K protein in forming the attL complex and promoting excision. These results indicate that a nonpolar side chain at residue 175 is required for function. The second primary mutant contained a proline-to-leucine change at position 243 (P243L). A true second-site revertant was isolated that contained a glutamic acid-to-lysine change (E218K). The P243L-E218K protein promoted recombination and bound arm-type sites more efficiently than the original P243L protein but not as efficiently as the protein containing the E218K substitution alone. The E218K substitution also restored activity to a mutant with a threonine-to-isoleucine substitution at position 270 (T270I). This result showed that suppression by the E218K change is not allele specific and suggests that the substitution improves an inherent activity of Int rather than directly compensating for the defect caused by the primary substitutions. Results with challenge phages carrying attL sites with altered core sites indicate that the E218K change may improve binding to the core site.
Assuntos
Bacteriófago lambda/genética , Integrases/fisiologia , Integrases/química , Integrases/genética , Mutação , Recombinação Genética , Relação Estrutura-AtividadeRESUMO
RNA challenge phages are derivatives of bacteriophage P22 that enable direct genetic selection for a specific RNA-protein interaction. The bacteriophage P22 R17 encodes a wild-type R17 operator site and undergoes lysogenic development following infection of susceptible bacterial strains that express the R17/MS2 coat protein. A P22 R17 derivative with an OcRNA site (P22 R17 [A(-10)U]) develops lytically following infection of these strains. RNA challenge phages can be used to isolate second-site coat protein suppressors that recognize an OcRNA sequence by selecting for lysogens with a P22 R17 [Oc] phage derivative. The bacteriophage derivative P22 R17 [A(-10)U] was used in one such scheme to isolate two classes of genes that encode R17 coat proteins with altered capsid assembly properties and expanded RNA-binding characteristics. These mutations map outside the RNA-binding surface and include amino acid substitutions that interfere with interactions between coat protein dimers in the formation of the stable phage capsid. One class of mutants encodes substitutions at the highly conserved first and second positions of the mature coat protein. N-terminal sequence analysis of these mutants reveals that coat proteins with substitutions only at position 1 are defective in post-translational processing of the initiator methionine. All selected proteins possess expanded RNA-binding properties since they direct efficient lysogen formation for P22 R17 and P22 R17 [A(-10)U]; however, bacterial strains that express the protein mutants remain sensitive to lytic infection by other P22 R17 [Oc] bacteriophages. The described selection strategy provides a novel genetic approach to dissecting protein structure within RNA-binding proteins.