RESUMEN
AIMS: Patients undergoing femoral lengthening by external fixation tolerate treatment less well when compared to tibial lengthening. Lengthening of the femur with an intramedullary device may have advantages. PATIENTS AND METHODS: We reviewed all cases of simple femoral lengthening performed at our unit from 2009 to 2014. Cases of nonunions, concurrent deformities, congenital limb deficiencies and lengthening with an unstable hip were excluded, leaving 33 cases (in 22 patients; 11 patients had bilateral procedures) for review. Healing index, implant tolerance and complications were compared. RESULTS: In 20 cases (15 patients) the Precice lengthening nail was used and in 13 cases (seven patients) the LRS external fixator system. The desired length was achieved in all cases in the Precice group and in 12 of 13 cases in the LRS group. The mean healing index was 31.3 days/cm in the Precice and 47.1 days/cm in the LRS group (p < 0.001). This was associated with an earlier ability to bear full weight without aids in the Precice group. There were more complications with LRS lengthening, including pin site infections and regenerate deformity. Implant tolerance and the patients' perception of the cosmetic result were better with the Precice treatment. CONCLUSION: Femoral lengthening with the Precice femoral nail achieved excellent functional results with fewer complications and greater patient satisfaction when compared with the LRS system in our patients. Cite this article: Bone Joint J 2016;98-B:1382-8.
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Clavos Ortopédicos , Fijadores Externos , Fémur/cirugía , Diferencia de Longitud de las Piernas/cirugía , Osteogénesis por Distracción/métodos , Tibia/cirugía , Adolescente , Adulto , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Factores de Tiempo , Resultado del Tratamiento , Adulto JovenRESUMEN
Guillain-Barré syndrome (GBS) is a post-infectious disease in which the human peripheral nervous system is affected after infection by specific pathogenic bacteria, including Campylobacter jejuni. GBS is suggested to be provoked by molecular mimicry between sialylated lipooligosaccharide (LOS) structures on the cell envelope of these bacteria and ganglioside epitopes on the human peripheral nerves, resulting in autoimmune-driven nerve destruction. Earlier, the C. jejuni sialyltransferase (Cst-II) was found to be linked to GBS and demonstrated to be involved in the biosynthesis of the ganglioside-like LOS structures. Apart from a role in pathogenicity, we report here that Cst-II-generated ganglioside-like LOS structures confer efficient bacteriophage resistance in C. jejuni. By bioinformatic analysis, it is revealed that the presence of sialyltransferases in C. jejuni and other potential GBS-related pathogens correlated significantly with the apparent degeneration of an alternative anti-virus system: type II Clusters of Regularly Interspaced Short Palindromic Repeat and associated genes (CRISPR-Cas). Molecular analysis of the C. jejuni CRISPR-Cas system confirmed the bioinformatic investigation. CRISPR degeneration and mutations in the cas genes cas2, cas1 and csn1 were found to correlate with Cst-II sialyltransferase presence (p < 0.0001). Remarkably, type II CRISPR-Cas systems are mainly found in mammalian pathogens. To study the potential involvement of this system in pathogenicity, we inactivated the type II CRISPR-Cas marker gene csn1, which effectively reduced virulence in primarily cst-II-positive C. jejuni isolates. Our findings indicate a novel link between viral defence, virulence and GBS in a pathogenic bacterium.
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Bacteriófagos/crecimiento & desarrollo , Infecciones por Campylobacter/complicaciones , Campylobacter jejuni/patogenicidad , Gangliósidos/metabolismo , Síndrome de Guillain-Barré/microbiología , Factores de Virulencia/metabolismo , Infecciones por Campylobacter/inmunología , Infecciones por Campylobacter/microbiología , Campylobacter jejuni/genética , Campylobacter jejuni/inmunología , Campylobacter jejuni/virología , Biología Computacional , ADN Bacteriano/genética , Gangliósidos/inmunología , Humanos , Factores de Virulencia/inmunologíaRESUMEN
Members of the genus Campylobacter are frequently responsible for human enteric disease with occasionally very serious outcomes. Much of this disease burden is thought to arise from consumption of contaminated poultry products. More than 80% of poultry in the UK harbour Campylobacter as a part of their intestinal flora. To address this unacceptably high prevalence, various interventions have been suggested and evaluated. Among these is the novel approach of using Campylobacter-specific bacteriophages, which are natural predators of the pathogen. To optimize their use as therapeutic agents, it is important to have a comprehensive understanding of the bacteriophages that infect Campylobacter, and how they can affect their host bacteria. This review will focus on many aspects of Campylobacter-specific bacteriophages including: their first isolation in the 1960s, their use in bacteriophage typing schemes, their isolation from the different biological sources and genomic characterization. As well as their use as therapeutic agents to reduce Campylobacter in poultry their future potential, including their use in bio-sanitization of food, will be explored. The evolutionary consequences of naturally occurring bacteriophage infection that have come to light through investigations of bacteriophages in the poultry ecosystem will also be discussed.
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Bacteriófagos/aislamiento & purificación , Terapia Biológica/métodos , Infecciones por Campylobacter/prevención & control , Campylobacter/virología , Pollos/virología , Animales , Bacteriófagos/clasificación , Bacteriófagos/genética , Campylobacter/aislamiento & purificación , Infecciones por Campylobacter/terapia , Infecciones por Campylobacter/veterinaria , Pollos/microbiología , Contaminación de Alimentos/prevención & control , Humanos , Carne/microbiologíaRESUMEN
BACKGROUND: Antineutrophil cytoplasm antibodies (ANCA) are used as diagnostic markers for small-vessel vasculitis of the Wegener Granulomatosis-microscopic polyangiitis (WG-MPA) spectrum, but if testing is applied indiscriminately, its value is diminished. The authors measured the effect of a targeted ANCA testing policy introduced in our institution in an attempt to improve the diagnostic value of testing in patients with suspected vasculitis. METHODS: The authors measured the rate of ANCA requests at a single regional centre in the year prior to and following the introduction of clinical guidelines to ensure appropriate test usage. The authors also audited clinical outcomes in patients in whom ANCA testing was declined. RESULT: Following implementation of the antineutrophil cytoplasm antibodies (ANCA) gating policy, the number of monthly ANCA tests carried out fell from 287+/-30 to 143+/-18 (p<0.0001) and was associated with an increased rate of positivity, from 18.5% (95% CI 17.0 to 20.1%) to 30.3% (27.5 to 33.1%; p<0.0001). The authors undertook a careful review of the case records from 263 patients in whom testing was declined according to the gating policy over an 8-month period. After 6 months' follow-up, no diagnoses of small-vessel vasculitis of the WG-MPA spectrum were reached. CONCLUSIONS: The rational use of ANCA testing to aid in the diagnosis of vasculitis should include a clinical gating policy to improve diagnostic performance. Adherence to a gating policy for ANCA testing coupled with close liaison between clinician and laboratory does not result in either a missed or delayed diagnosis of small-vessel vasculitis belonging to the WG-MPA spectrum.
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Anticuerpos Anticitoplasma de Neutrófilos/sangre , Enfermedades Autoinmunes/diagnóstico , Vasculitis/diagnóstico , Biomarcadores/sangre , Granulomatosis con Poliangitis/diagnóstico , Humanos , Inmunoensayo/estadística & datos numéricos , Auditoría Médica , Selección de PacienteRESUMEN
Technicians from one hundred and eighteen Human Tissue Authority (HTA) approved mortuaries licensed to perform post-mortems in England completed a telephone interview. All were questioned on whether they had contact with reusable external fixators, who was responsible for the removal, the number removed annually, and the destination of the fixator post-removal. Opinion was sought on how the return of the equipment could be better facilitated. Seventy-four of the technicians interviewed could remember seeing external fixation devices, but were unable to quantify how many were removed annually. Sixty-one of those questioned stated that they personally removed the fixator, three always requested an Orthopaedic surgeon to remove the device and five contacted a Nurse Specialist. Forty-eight stated that they returned the devices to their local Sterile Services Department or Orthopaedic department. Nine technicians always discarded the fixators, eight always left them with the body and two stored them in the mortuary. Many reusable external fixation devices are inappropriately disposed of each year due to a lack of knowledge and communication with Orthopaedic departments. Confusion also exists among some technicians over whether external fixation components should be treated as 'implants'. There is a need for clear guidelines to raise awareness and ensure the appropriate return of these high cost devices.
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Equipo Médico Durable/estadística & datos numéricos , Fijadores Externos , Prácticas Mortuorias/métodos , Equipo Médico Durable/economía , Inglaterra , Equipo Reutilizado/economía , Equipo Reutilizado/estadística & datos numéricos , Guías como Asunto , Humanos , Comunicación Interdisciplinaria , Prácticas Mortuorias/estadística & datos numéricos , Investigación CualitativaRESUMEN
OBJECTIVES: The aim of the current study was to determine the contribution of interleukin (IL)1 gene cluster polymorphisms previously implicated in susceptibility for ankylosing spondylitis (AS) to AS susceptibility in different populations worldwide. METHODS: Nine polymorphisms in the IL1 gene cluster members IL1A (rs2856836, rs17561 and rs1894399), IL1B (rs16944), IL1F10 (rs3811058) and IL1RN (rs419598, the IL1RA VNTR, rs315952 and rs315951) were genotyped in 2675 AS cases and 2592 healthy controls recruited in 12 different centres in 10 countries. Association of variants with AS was tested by Mantel-Haenszel random effects analysis. RESULTS: Strong association was observed with three single nucleotide polymorphisms (SNPs) in the IL1A gene (rs2856836, rs17561, rs1894399, p = 0.0036, 0.000019 and 0.0003, respectively). There was no evidence of significant heterogeneity of effects between centres, and no evidence of non-combinability of findings. The population attributable risk fraction of these variants in Caucasians is estimated at 4-6%. CONCLUSIONS: This study confirms that IL1A is associated with susceptibility to AS. Association of the other IL1 gene complex members could not be excluded in specific populations. Prospective meta-analysis is a useful tool in confirmation studies of genes associated with complex genetic disorders such as AS, providing sufficiently large sample sizes to produce robust findings often not achieved in smaller individual cohorts.
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Interleucina-1/genética , Polimorfismo de Nucleótido Simple , Espondilitis Anquilosante/genética , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Interleucina-1alfa/genética , Familia de Multigenes , Estudios Prospectivos , Espondilitis Anquilosante/inmunologíaRESUMEN
OBJECTIVE: Ankylosing spondylitis (AS) is a debilitating chronic inflammatory condition with a high degree of familiality (lambda(s) = 82) and heritability (>90%) that primarily affects spinal and sacroiliac joints. Whole genome scans for linkage to AS phenotypes have been conducted, although results have been inconsistent between studies and all have had modest sample sizes. One potential solution to these issues is to combine data from multiple studies in a retrospective meta-analysis. METHODS: The International Genetics of Ankylosing Spondylitis Consortium combined data from three whole genome linkage scans for AS (n = 3744 subjects) to determine chromosomal markers that show evidence of linkage with disease. Linkage markers typed in different centres were integrated into a consensus map to facilitate effective data pooling. We performed a weighted meta-analysis to combine the linkage results, and compared them with the three individual scans and a combined pooled scan. RESULTS: In addition to the expected region surrounding the HLA-B27 gene on chromosome 6, we determined that several marker regions showed significant evidence of linkage with disease status. Regions on chromosome 10q and 16q achieved 'suggestive' evidence of linkage, and regions on chromosomes 1q, 3q, 5q, 6q, 9q, 17q and 19q showed at least nominal linkage in two or more scans and in the weighted meta-analysis. Regions previously associated with AS on chromosome 2q (the IL-1 gene cluster) and 22q (CYP2D6) exhibited nominal linkage in the meta-analysis, providing further statistical support for their involvement in susceptibility to AS. CONCLUSION: These findings provide a useful guide for future studies aiming to identify the genes involved in this highly heritable condition.
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Ligamiento Genético , Espondilitis Anquilosante/genética , Cromosomas Humanos Par 2/genética , Cromosomas Humanos Par 22/genética , Femenino , Predisposición Genética a la Enfermedad , Genoma Humano , Humanos , Masculino , Estudios RetrospectivosRESUMEN
OBJECTIVE: To test the association of interleukin 1 (IL1) gene family members with ankylosing spondylitis (AS), previously reported in Europid subjects, in an ethnically remote population. METHODS: 200 Taiwanese Chinese AS patients and 200 ethnically matched healthy controls were genotyped for five single nucleotide polymorphisms (SNPs) and the IL1RN.VNTR, markers previously associated with AS. Allele, genotype, and haplotype frequencies were compared between cases and controls. RESULTS: Association of alleles and genotypes of the markers IL1F10.3, IL1RN.4, and IL1RN.VNTR was observed with AS (p<0.05). Haplotypes of pairs of these markers and of the markers IL1RN.6/1 and IL1RN.6/2 were also significantly associated with AS. The strongest associations observed were with the marker IL1RN.4, and with the two-marker haplotype IL1RN.4-IL1RN.VNTR (both p = 0.004). Strong linkage disequilibrium was observed between all marker pairs except those involving IL1B-511 (D' 0.4 to 0.9, p<0.01). CONCLUSIONS: The IL1 gene cluster is associated with AS in Taiwanese Chinese. This finding provides strong statistical support that the previously observed association of this gene cluster with AS is a true positive finding. These authors contributed equally to the study.
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Pueblo Asiatico/genética , Interleucina-1/genética , Polimorfismo Genético , Espondilitis Anquilosante/genética , Estudios de Casos y Controles , Cromosomas Humanos Par 2 , Femenino , Frecuencia de los Genes , Marcadores Genéticos , Genotipo , Haplotipos , Humanos , Desequilibrio de Ligamiento , Masculino , Espondilitis Anquilosante/etnología , Taiwán/etnologíaRESUMEN
OBJECTIVE: The ank/ank mouse develops a phenotype similar to ankylosing spondylitis (AS) in humans. ANKH, the human homolog of the mutated gene in the ank/ank mouse, has been implicated in familial autosomal-dominant chondrocalcinosis and autosomal-dominant craniometaphyseal dysplasia. This study was undertaken to investigate the role of ANKH in susceptibility to and clinical manifestations of AS. METHODS: Sequence variants were identified by genomic sequencing of the 12 ANKH exons and their flanking splice sites in 48 AS patients; variants were then screened in 233 patients and 478 controls. Linkage to the ANKH locus was assessed in 185 affected-sibling-pair families. RESULTS: Five single-nucleotide polymorphisms were identified within the coding region and flanking splice sites. No association between either susceptibility to AS or its clinical manifestations and these novel polymorphisms, or between disease susceptibility and 3 known promoter variants, was seen. No linkage between the ANKH locus and AS was observed. Multipoint exclusion mapping rejected the hypothesis of a locus of a magnitude lambda>/=1.4 (logarithm of odds score <-2) (equivalent to a genetic contribution of >10% to the AS sibling recurrence risk ratio) within this area contributing to AS. CONCLUSION: These findings indicate that ANKH is not significantly involved in susceptibility to or clinical manifestations of AS.
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Proteínas de la Membrana/genética , Espondilitis Anquilosante/genética , Adulto , Femenino , Frecuencia de los Genes , Ligamiento Genético , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Masculino , Fenotipo , Proteínas de Transporte de Fosfato , Polimorfismo GenéticoAsunto(s)
Condrocalcinosis/genética , Anciano , Anciano de 80 o más Años , Pirofosfato de Calcio/metabolismo , Enfermedades de los Cartílagos/genética , Enfermedades de los Cartílagos/metabolismo , Enfermedades de los Cartílagos/patología , Cartílago Articular/metabolismo , Cartílago Articular/patología , Condrocalcinosis/metabolismo , Condrocalcinosis/patología , Cristalización , Femenino , Predisposición Genética a la Enfermedad , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Hereditary haemochromatosis (HH) is the most common lethal monogenic human disease, affecting roughly 1 in 300 white northern Europeans. Homozygosity for the C282Y polymorphism within the HFE gene causes more than 80% of cases, with compound heterozygosity of the C282Y and H63D polymorphism also increasing susceptibility to disease. The aim of this study was to determine the frequency of the C282Y and H63D polymorphisms in the disease, and to assess the risk of HH in heterozygotes for the C282Y polymorphism. 128 patients were recruited because of either radiographic chondrocalcinosis (at least bicompartmental knee disease or joints other than the knee involved) or CPPD pseudogout. Genotyping of the HFE C282Y and H63D mutations was performed using PCR/SSP and genotypes for the C282Y polymorphism confirmed by PCR/RFLP. Historical white European control data were used for comparison. Two previously undiagnosed C282Y homozygotes (1.6%), and 16 C282Y heterozygotes (12.5%), including four (3.1%) C282Y/H63D compound heterozygotes were identified. This represents a significant overrepresentation of C282Y homozygotes (relative risk 3.4, p=0.037), but the number of heterozygotes was not significantly increased. At a cost per test of pound1 for each subject, screening all patients with chondrocalcinosis using the above ascertainment criteria costs only pound64 for each case of haemochromatosis identified, clearly a highly cost effective test given the early mortality associated with untreated haemochromatosis. Routine screening for haemochromatosis in patients with appreciable chondrocalcinosis is recommended.
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Condrocalcinosis/genética , Hemocromatosis/genética , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Frecuencia de los Genes , Pruebas Genéticas/métodos , Heterocigoto , Homocigoto , Humanos , Masculino , Persona de Mediana Edad , Mutación/genética , Polimorfismo Genético , Factores de RiesgoRESUMEN
Ankylosing spondylitis (AS) is a common inflammatory arthritis predominantly affecting the axial skeleton. Susceptibility to the disease is thought to be oligogenic. To identify the genes involved, we have performed a genomewide scan in 185 families containing 255 affected sibling pairs. Two-point and multipoint nonparametric linkage analysis was performed. Regions were identified showing "suggestive" or stronger linkage with the disease on chromosomes 1p, 2q, 6p, 9q, 10q, 16q, and 19q. The MHC locus was identified as encoding the greatest component of susceptibility, with an overall LOD score of 15.6. The strongest non-MHC linkage lies on chromosome 16q (overall LOD score 4.7). These results strongly support the presence of non-MHC genetic-susceptibility factors in AS and point to their likely locations.
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Predisposición Genética a la Enfermedad/genética , Pruebas Genéticas , Genoma Humano , Complejo Mayor de Histocompatibilidad/genética , Espondilitis Anquilosante/genética , Mapeo Cromosómico , Cromosomas Humanos/genética , Estudios de Cohortes , Femenino , Genotipo , Humanos , Escala de Lod , Masculino , Análisis por Apareamiento , Núcleo Familiar , Programas Informáticos , Estadísticas no ParamétricasRESUMEN
The appearance over many days of Lac(+) frameshift mutations in Escherichia coli strain FC40 incubated on lactose selection plates is a classic example of apparent "adaptive" mutation in an episomal gene. We show that endogenously overproduced carotenoids reduce adaptive mutation under selective conditions by a factor of around two. Carotenoids are known to scavenge singlet oxygen suggesting that the accumulation of oxidative base damage may be an integral part of the adaptive mutation phenomenon. If so, the lesion cannot be 7,8-dihydro-8-oxoguanine since adaptive mutation in FC40 is unaffected by mutM and mutY mutations. If active oxygen species such as singlet oxygen are involved in adaptive mutation then they should also induce frameshift mutations in FC40 under non-selective conditions. We show that such mutations can be induced under non-selective conditions by protoporphyrin photosensitisation and that this photodynamic induction is reduced by a factor of just over two when endogenous carotenoids are present. We argue that the involvement of oxidative damage would in no way be inconsistent with current understanding of the mechanism of adaptive mutation and the role of DNA polymerases.
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Carotenoides/farmacología , Escherichia coli/genética , Mutación del Sistema de Lectura/efectos de los fármacos , Mutación del Sistema de Lectura/efectos de la radiación , Reparación del ADN/genética , Evolución Molecular Dirigida , Escherichia coli/efectos de los fármacos , Escherichia coli/efectos de la radiación , Fluorescencia , Mutación del Sistema de Lectura/genética , Lactosa/genética , Oxígeno/metabolismo , Oxígeno/farmacología , Fotoquímica , Especies Reactivas de Oxígeno/metabolismo , Oxígeno Singlete , Factores de TiempoRESUMEN
Busulfan has been previously only available in an oral formulation due to its poor water solubility. We report the results of a phase I study of multiple escalating doses of intravenous busulfan (Spartaject Busulfan, Orphan Europe, Paris, France) for myeloablation prior to stem cell transplantation (SCT) in 12 patients with chronic myeloid leukemia, acute myeloid leukemia or acute lymphocytic leukemia. One patient received allogeneic SCT; the other 11 patients received autologous SCT. The first six patients received i.v. busulfan diluted in 50 ml of 0.9% normal saline and the last six patients received busulfan in a 500-ml 5% dextrose solution. All patients experienced profound myelosuppression and all but one demonstrated hematopoietic engraftment. Toxicity was mild or moderate and there were no toxic deaths attributable to busulfan. Of note, there were no cases of veno-occlusive disease of the liver. Busulfan plasma concentrations were determined by gas chromatography with electron capture detection and showed little intrapatient variability. In most cases there was no significant difference between the first and last dose PK parameters. These data suggest that dose adjustment based on first dose PK data could allow uniformity of busulfan dosing for patients receiving SCT.
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Busulfano/administración & dosificación , Trasplante de Células Madre Hematopoyéticas , Inmunosupresores/administración & dosificación , Acondicionamiento Pretrasplante/métodos , Enfermedad Aguda , Adulto , Busulfano/efectos adversos , Enfermedades Cardiovasculares/inducido químicamente , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Ciclofosfamida/administración & dosificación , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Femenino , Enfermedades Gastrointestinales/inducido químicamente , Rechazo de Injerto/mortalidad , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Humanos , Inmunosupresores/efectos adversos , Infusiones Intravenosas , Leucemia Mielógena Crónica BCR-ABL Positiva/terapia , Leucemia Mieloide/terapia , Masculino , Persona de Mediana Edad , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Seguridad , Trasplante Autólogo , Trasplante Homólogo , Resultado del TratamientoRESUMEN
In Escherichia coli trpA23 bacteria lacking the MutY glycosylase and incubated on plates in the absence of tryptophan, tryptophan-independent mutants continue to arise during incubation over many days. Their appearance is enhanced in umuD+,C+ strains in comparison with strains carrying a deletion through the umu operon and the umuD,C-dependent mutants were greater in number in uvrA bacteria (lacking nucleotide excision repair) than in uvr+ bacteria. Sequencing of mutations occurring in uvrA bacteria revealed the presence of G:C to C:G transversions but only in umuD+,C+ strains. There is thus a pathway in starved bacteria that generates G:C to C:G transversions and requires the inducible UmuD,C proteins. The data are consistent with the occurrence of a lesion, probably 8-oxoguanine, against which guanine may be incorporated during DNA synthesis by "dNTP stabilised" misalignment against the downstream template base. Upon realignment the configuration is substrate for MutY glycosylase which can remove the unmodified guanine. It is hypothesised that UmuD,C proteins are required for primer extension from the mismatch once formed.
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Proteínas Bacterianas/metabolismo , Citosina/metabolismo , ADN Glicosilasas , Proteínas de Escherichia coli , Escherichia coli/genética , Guanina/metabolismo , N-Glicosil Hidrolasas/genética , ADN Polimerasa Dirigida por ADNRESUMEN
Escherichia coli contains at least two iron storage proteins, a ferritin (FtnA) and a bacterioferritin (Bfr). To investigate their specific functions, the corresponding genes (ftnA and bfr) were inactivated by replacing the chromosomal ftnA and bfr genes with disrupted derivatives containing antibiotic resistance cassettes in place of internal segments of the corresponding coding regions. Single mutants (ftnA::spc and bfr::kan) and a double mutant (ftnA::spc bfr::kan) were generated and confirmed by Western and Southern blot analyses. The iron contents of the parental strain (W3110) and the bfr mutant increased by 1.5- to 2-fold during the transition from logarithmic to stationary phase in iron-rich media, whereas the iron contents of the ftnA and ftnA bfr mutants remained unchanged. The ftnA and ftnA bfr mutants were growth impaired in iron-deficient media, but this was apparent only after the mutant and parental strains had been precultured in iron-rich media. Surprisingly, ferric iron uptake regulation (fur) mutants also had very low iron contents (2.5-fold less iron than Fur+ strains) despite constitutive expression of the iron acquisition systems. The iron deficiencies of the ftnA and fur mutants were confirmed by Mössbauer spectroscopy, which further showed that the low iron contents of ftnA mutants are due to a lack of magnetically ordered ferric iron clusters likely to correspond to FtnA iron cores. In combination with the fur mutation, ftnA and bfr mutations produced an enhanced sensitivity to hydroperoxides, presumably due to an increase in production of "reactive ferrous iron." It is concluded that FtnA acts as an iron store accommodating up to 50% of the cellular iron during postexponential growth in iron-rich media and providing a source of iron that partially compensates for iron deficiency during iron-restricted growth. In addition to repressing the iron acquisition systems, Fur appears to regulate the demand for iron, probably by controlling the expression of iron-containing proteins. The role of Bfr remains unclear.
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Proteínas Bacterianas , Grupo Citocromo b/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/genética , Ferritinas/genética , Hierro/metabolismo , Aerobiosis , Cromosomas Bacterianos/genética , Grupo Citocromo b/metabolismo , Escherichia coli/efectos de los fármacos , Ferritinas/metabolismo , Genotipo , Cinética , Modelos Biológicos , Mutagénesis , Mutagénesis Sitio-Dirigida , Ácido Pentético/farmacología , Plásmidos , Mapeo Restrictivo , Eliminación de Secuencia , Espectroscopía de Mossbauer , Factores de TiempoRESUMEN
Under starvation conditions, a variety of stationary phase genes are up-regulated under the control of the stationary phase sigma factor RpoS including at least two peroxidases and a protective DNA binding protein Dps. Previous work suggested that the reversion to prototrophy of certain amino acid auxotrophs of Escherichia coli that occurs when the bacteria are starved of a required amino acid results from the accumulation of oxidative damage to guanine residues in DNA. We report here that three strains lacking RpoS are indistinguishable from wild type in their ability to undergo this starvation-associated mutation, suggesting that basal levels of catalase activity are more than adequate in these strains, and that the induction of catalases and other proteins controlled by rpoS does not contribute to the protection of the DNA, at least in cells starved in early stationary phase. In comparison, the introduction of a plasmid specifying the production of singlet oxygen scavengers (carotenoids) in stationary phase cells led to a roughly twofold reduction in mutant yield. The results suggest that singlet oxygen may be an important endogenously produced mutagen in resting cells.
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Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Carotenoides/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Mutación , Oxígeno/metabolismo , Factor sigma/genética , Factor sigma/metabolismo , Aminoácidos/metabolismo , Carotenoides/genética , Catalasa/metabolismo , Daño del ADN , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Escherichia coli/crecimiento & desarrollo , Depuradores de Radicales Libres/metabolismo , Genes Bacterianos , Mutágenos/metabolismo , Plásmidos/genética , Especies Reactivas de Oxígeno/metabolismo , Oxígeno SingleteRESUMEN
Genetic analysis of a mouse model of major histocompatability complex (MHC)-associated autoimmune type 1 (insulin-dependent) diabetes mellitus (IDDM) has shown that the disease is caused by a combination of a major effect at the MHC and at least ten other susceptibility loci elsewhere in the genome. A genome-wide scan of 93 affected sibpair families (ASP) from the UK (UK93) indicated a similar genetic basis for human type 1 diabetes, with the major genetic component at the MHC locus (IDDM1) explaining 34% of the familial clustering of the disease (lambda(s)=2.5; refs 3,4). In the present report, we have analysed a further 263 multiplex families from the same population (UK263) to provide a total UK data set of 356 ASP families (UK356). Only four regions of the genome outside IDDM1/MHC, which was still the only major locus detected, were not excluded at lambda(s)=3 and lod=-2, of which two showed evidence of linkage: chromosome 10p13-p11 (maximum lod score (MLS)=4.7, P=3x10(-6), lambda(s)=1.56) and chromosome 16q22-16q24 (MLS=3.4, P=6.5x10(-5), lambda(s)=1.6). These and other novel regions, including chromosome 14q12-q21 and chromosome 19p13-19q13, could potentially harbour disease loci but confirmation and fine mapping cannot be pursued effectively using conventional linkage analysis. Instead, more powerful linkage disequilibrium-based and haplotype mapping approaches must be used; such data is already emerging for several type 1 diabetes loci detected initially by linkage.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Adolescente , Adulto , Mapeo Cromosómico , Predisposición Genética a la Enfermedad , Humanos , Reino UnidoRESUMEN
When 3 x 10(8) bacteria of the Escherichia coli tyrA14(oc) leu308(am) strain WU3610 are plated on glucose salts agar supplemented with leucine only, colonies of slow-growing Tyr+ suppressor mutants begin to appear after about a week and increase in numbers roughly linearly with time thereafter (stationary phase or starvation-associated mutation). From a library constructed from two of these mutants, a clone was obtained that suppressed the tyrosine requirement of WU3610 when present on a multicopy plasmid. The activity was identified to an open reading frame we call tas, the sequence for which has homology with a variety of known genes with aldo-keto reductase activity. The activity of tas complements the prephenate dehydrogenase dysfunction of tyrA14 (the chorismate mutase activity of tyrA possibly being still functional). A strain deleted for tas showed no spontaneous mutation under starvation conditions. Whereas neither tas+ nor tas bacteria showed any increase in viable or total count when plated under conditions of tyrosine starvation at 3 x 10(8) cells per plate, at lower density (approximately 10(7) per plate) tas+ but not tas bacteria showed considerable residual growth. We suggest that the single copy of tas present in WU3610 allows cryptic cell or DNA turnover under conditions of tyrosine starvation and that this is an essential prerequisite for starvation-associated mutation in this system. The target gene for mutation is not tas, although an increase in the expression of this gene, for example, resulting from a suppressor mutation affecting supercoiling, could be responsible for the slow-growing Tyr+ phenotype.