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INTRODUCTION: Magnetic resonance diffusion tensor imaging through the fraction of anisotropy allows evaluation of the integrity of the motor pathways after cerebral infarction. AIMS: To correlate the fraction of anisotropy with the clinical scales and the prognosis of cerebral infarction. SUBJECTS AND METHODS: Prospective study of patients with cerebral infarction to compare the fraction of anisotropy in different regions of interest with functional evaluations and with controls free of infarction. A subgroup of subjects with rehabilitation underwent an initial MRI scan and another at three months, with clinical follow-up for six months. RESULTS: Thirty-eight consecutive patients with middle cerebral artery infarction were included. The fraction of anisotropy values were lower in the ipsilateral corticospinal pathway than the fraction of anisotropy of the corticospinal pathway of the controls. The values of the fraction of anisotropy in the ipsilateral corticospinal pathway were associated with the value of the functional scale on admission. Changes in the fraction of anisotropy values between the initial MRI and the scan performed at three months correlated with the score on the functional scale and the modified Rankin scale at three and six months. CONCLUSIONS: The value of the fraction of anisotropy in the ipsilateral internal capsule is associated with the presence of a lesion and with its presenting symptoms. Changes in the fraction of anisotropy at three months suggest long-term clinical recovery.
TITLE: Imagen del tensor de difusión de la vía corticoespinal y su asociación con el pronóstico del infarto cerebral agudo: experiencia de una cohorte en México.Introducción. La imagen del tensor de difusión por resonancia magnética a través de la fracción de anisotropía permite evaluar la integridad de las vías motoras después de un infarto cerebral. Objetivo. Correlacionar la fracción de anisotropía con las escalas clínicas y el pronóstico del infarto cerebral. Sujetos y métodos. Estudio prospectivo de pacientes con infarto cerebral para comparar la fracción de anisotropía en diferentes regiones de interés con evaluaciones funcionales y con controles sin infarto. En un subgrupo con rehabilitación, se realizó una resonancia magnética inicial y a los tres meses, con un seguimiento clínico durante seis meses. Resultados. Se incluyó a 38 pacientes consecutivos con infarto cerebral de la arteria cerebral media. Los valores de la fracción de anisotropía fueron menores en la vía corticoespinal ipsilateral que en la vía corticoespinal de los controles. Los valores de la fracción de anisotropía en la vía corticoespinal ipsilateral se asociaron con el valor de la escala funcional en el momento de su admisión. Los cambios en los valores de la fracción de anisotropía entre la resonancia magnética inicial y a los tres meses se correlacionaron con la puntuación en la escala funcional y en la escala de Rankin modificada a los tres y a los seis meses. Conclusiones. El valor de la fracción de anisotropía en la cápsula interna ipsilateral se asocia a la presencia de lesión y a su presentación clínica. Los cambios en la fracción de anisotropía a los tres meses sugieren la recuperación clínica a largo plazo.
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Infarto Cerebral/diagnóstico por imagen , Imagen de Difusión Tensora , Tractos Piramidales/diagnóstico por imagen , Enfermedad Aguda , Anciano , Anisotropía , Femenino , Humanos , Infarto de la Arteria Cerebral Media , Masculino , México , Persona de Mediana Edad , Pronóstico , Estudios ProspectivosRESUMEN
The symbiotic relationships between bacteria of the genus Wolbachia (order Rickettsiales) and their arthropod hosts are diverse and can range from mutualism to parasitism. Whereas effects of Wolbachia on host biology are well investigated, little is known about diversity and abundance of Wolbachia in their natural hosts. The phloem-feeding Asian citrus psyllid, Diaphorina citri (Kuwayama) (Hemiptera: Liviidae), is naturally infected with Wolbachia (wDi). In the current study, we calculated the within-host density of Wolbachia in Florida D. citri populations using quantitative polymerase chain reaction for detection of the Wolbachia outer surface protein gene, wsp. Gene quantities were normalized to the D. citri wingless gene (Wg) to estimate Wolbachia abundance in individual D. citri. Using this method, significant geographic differences in Wolbachia densities were detected among Florida D. citri populations, with higher infection levels occurring in male versus female hosts.
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Proteínas de la Membrana Bacteriana Externa/genética , Hemípteros/microbiología , Wolbachia/genética , Animales , Proteínas de la Membrana Bacteriana Externa/metabolismo , Secuencia de Bases , Citrus/crecimiento & desarrollo , Femenino , Florida , Masculino , Datos de Secuencia Molecular , Reacción en Cadena en Tiempo Real de la Polimerasa , Simbiosis , Wolbachia/aislamiento & purificación , Wolbachia/metabolismoRESUMEN
Chronic restraint stress impairs hippocampal-mediated spatial learning and memory, which improves following a post-stress recovery period. Here, we investigated whether brain-derived neurotrophic factor (BDNF), a protein important for hippocampal function, would alter the recovery from chronic stress-induced spatial memory deficits. Adult male Sprague-Dawley rats were infused into the dorsal hippocampal cornu ammonis (CA)3 region with an adeno-associated viral vector containing the sequence for a short hairpin RNA (shRNA) directed against BDNF or a scrambled sequence (Scr). Rats were then chronically restrained (wire mesh, 6 h/day for 21 days) and assessed for spatial learning and memory using a radial arm water maze (RAWM) either immediately after stressor cessation (Str-Imm) or following a 21-day post-stress recovery period (Str-Rec). All groups learned the RAWM task similarly, but differed on the memory retention trials. Rats in the Str-Imm group, regardless of adeno-associated viral contents, committed more errors in the spatial reference memory domain on the single retention trial during day 3 than did the non-stressed controls. Importantly, the typical improvement in spatial memory following the recovery from chronic stress was blocked with the shRNA against BDNF, as Str-Rec-shRNA performed worse on the RAWM compared with the non-stressed controls or Str-Rec-Scr. The stress effects were specific for the reference memory domain, but knockdown of hippocampal BDNF in unstressed controls briefly disrupted spatial working memory as measured by repeated entry errors on day 2 of training. These results demonstrated that hippocampal BDNF was necessary for the recovery from stress-induced hippocampal-dependent spatial memory deficits in the reference memory domain.
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Factor Neurotrófico Derivado del Encéfalo/metabolismo , Región CA3 Hipocampal/metabolismo , Memoria Espacial/fisiología , Estrés Psicológico/metabolismo , Animales , Regulación hacia Abajo , Masculino , Ratas , Ratas Sprague-Dawley , Restricción FísicaRESUMEN
Candidatus Liberibacter asiaticus (CLas) is a phloem-limited bacterium transmitted by the Asian citrus psyllid, Diaphorina citri, and the presumptive causal agent of citrus greening disease. The current method of detection for CLas within plant and insect samples is by a presence/absence qPCR assay using the CLas 16S rDNA gene target. Although qPCR is highly sensitive, low bacterial titers or suboptimal qPCR conditions can result in false-negatives. Using a nested qPCR assay, we determined the false-negative rate of the 16S presence/absence qPCR assay was greater than 50%. Studies to determine the performance parameters of the qPCR assays for CLas 16S and Wingless (Wg), the D. citri endogenous gene, using plasmid and psyllid DNA, revealed suboptimal and variable performance of the 16S assay in psyllid samples. Average efficiencies and sensitivity limits of the plasmid assays were 99.0% and 2.7 copies of template for Wg, respectively, and 98.5% and 2.2-22.1 copies for 16S, respectively. Variability in efficiency was significantly greater in psyllid samples for both gene targets compared to the corresponding plasmid assays, and efficiencies as low as 76% were obtained for 16S. A secondary structure analysis revealed the formation of two stem-loop structures that block the forward and probe binding sites in the 16S template, which could hinder amplification. In summary, our results suggest that suboptimal qPCR efficiency is not uncommon for the 16S presence/absence qPCR assay, which combined with lowCLas titers in some samples, could contribute significantly to the under-reporting of CLas infection in psyllid and plant samples.
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Hemípteros/microbiología , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Rhizobiaceae/aislamiento & purificación , Animales , Citrus/microbiología , ADN Bacteriano/genética , ADN Ribosómico/genética , Humanos , Insectos/microbiología , Plásmidos , ARN Ribosómico 16S/genética , Rhizobiaceae/genética , Sensibilidad y EspecificidadRESUMEN
INTRODUCTION: In nursing homes animal-assisted therapy has been applied in demented elderly patients for several years. There are no studies on this treatment in hospitals, especially in geriatric departments. METHODS: From September 2010 to November 2011 105 in-patients, among them 77 female, participated in a 30 min dog-assisted group therapy (mean age 84,4 ± 6,56 years). The patients had cognitive and functional impairments (mean MMSE 18 points, mean Barthel Index 34,6 points). RESULTS: Adverse events were not observed. Thirteen patients discontinued the treatment early, due to different reasons. The psychologist, who attended the treatment, observed an improvement of mood in 58 % and an improvement in activity in 54 %. DISCUSSION AND CONCLUSION: Animal assisted therapy can be safely established in a hospital among patients with cognitive impairment. The data support the hypothesis that animal assisted therapy improves mood, communication and activity in patients with cognitive impairment.
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Terapia Asistida por Animales/estadística & datos numéricos , Demencia/epidemiología , Demencia/enfermería , Servicios de Salud para Ancianos/estadística & datos numéricos , Hospitalización/estadística & datos numéricos , Anciano , Anciano de 80 o más Años , Demencia/psicología , Femenino , Alemania/epidemiología , Humanos , Masculino , Prevalencia , Resultado del TratamientoRESUMEN
OBJECTIVE: This study aimed at investigating the diagnostic value of ¹8F-FDG PET/CT in cases of suspected spondylodiscitis after inconclusive results in initial diagnostic imaging. PATIENTS, METHODS: We analysed 38 consecutive cases of suspected spondylodiscitis (mean age: 67 ± 14 years) with chronic back pain referred to our Department during a four-year-period after inconclusive results in MRI or other conventional modalities. Clinical histories were retrospectively worked up and results of ¹8F-FDG PET/CT and MRI were analysed and related to the results of biopsy, blood culture and a one-year clinical follow-up. ¹8F-FDG PET/CT was analysed qualitatively by visual analysis and quantitatively. We measured the maximum standardized uptake value (SUV(max)) in the region of back pain and in a corresponding reference region (RR) in each patient and calculated a SUV(max-ratio). RESULTS: 22/38 patients had confirmed spondylodiscitis, while 16 were negative. ¹8F-FDG PET/CT established a correct diagnosis in 34 out of 38 patients by visual analysis. ¹8F-FDG PET/CT reached a sensitivity, specificity and accuracy of 81.8%, 100%, 89.5% and a PPV and NPV of 100% and 80%. MRI, performed in 27 patients reached a sensitivity, specificity and accuracy of 75%, 71.4%, 74.1% and a PPV and NPV of 88.2% and 50%. Patients with confirmed spondylodiscitis showed a significantly (p < 0.05) higher SUV(max) of 5.1 ± 1.9 and SUV(max)-ratio of 1.9 ± 0.8 than patients without it (SUV(max) (3.8 ± 1.5), SUV(max-ratio) (1.2 ± 0.3). CONCLUSION: ¹8F-FDG PET/CT provided diagnostic information in most patients with chronic back pain and suspected spondylodiscitis. It was helpful in establishing a correct diagnosis in challenging cases of spondylodiscitis with mostly unclear findings in previous MRI.
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Dolor de Espalda/diagnóstico , Dolor de Espalda/etiología , Dolor Crónico/diagnóstico , Dolor Crónico/etiología , Discitis/complicaciones , Discitis/diagnóstico , Imagen Multimodal/métodos , Tomografía de Emisión de Positrones , Tomografía Computarizada por Rayos X , Femenino , Fluorodesoxiglucosa F18 , Humanos , Masculino , Persona de Mediana Edad , Radiofármacos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: The diagnosis of the isolated leptomeningeal involvement of a primary central nervous system B-cell lymphoma without parenchyma lesions may be difficult. Patients with leptomeningeal meningeosis lymphomatosa can present with various neurologic deficits. AIMS OF THE STUDY: To demonstrate the impact of cerebrospinal fluid (CSF) flow cytometry in the diagnosis of an isolated leptomeningeal manifestation of B-cell lymphoma by presenting an interesting case report. METHODS: Flow cytometric analysis of B-cell monoclonality of the CSF was performed as complementary diagnostic procedure in addition to CSF cytology. Final diagnosis was confirmed by necropsy. RESULTS: We suspected isolated leptomeningeal manifestation of B-cell lymphoma with palsy of the VI and VII cranial nerves in a 79-year-old male, because of mononuclear pleocytosis in CSF. Interestingly, the decisive diagnostic hint was given by implementation of flow cytometry of the CSF. Diagnosis was confirmed by postmortem autopsy. CONCLUSION: Our case shows that flow cytometry of the CSF in addition to conventional CSF cytology has the potential to accelerate diagnosis of lymphomeningeal infiltration of B-cell lymphoma.
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Neoplasias del Sistema Nervioso Central/patología , Citometría de Flujo , Linfoma no Hodgkin/patología , Neoplasias Meníngeas/diagnóstico , Neoplasias Meníngeas/secundario , Anciano , Neoplasias del Sistema Nervioso Central/líquido cefalorraquídeo , Técnicas Citológicas , Humanos , Linfoma no Hodgkin/líquido cefalorraquídeo , Masculino , Neoplasias Meníngeas/líquido cefalorraquídeo , Necrosis/diagnósticoRESUMEN
cDNAs encoding two gut laccase isoforms (RfLacA and RfLacB) were sequenced from the termite Reticulitermes flavipes. Phylogenetic analyses comparing translated R. flavipes laccases to 67 others from prokaryotes and eukaryotes indicate that the R. flavipes laccases are evolutionarily unique. Alignments with crystallography-verified laccases confirmed that peptide motifs involved in metal binding are 100% conserved in both isoforms. Laccase transcripts and phenoloxidase activity were most abundant in symbiont-free salivary gland and foregut tissue, verifying that the genes and activities are host-derived. Using a baculovirus-insect expression system, the two isoforms were functionally expressed with histidine tags and purified to near homogeneity. ICP-MS (inductively coupled plasma - mass spectrometry) analysis of RfLacA identified bound metals consisting mainly of copper (â¼4 copper molecules per laccase protein molecule and â¼3 per histidine tag) with lesser amounts of calcium, manganese and zinc. Both recombinant enzyme preparations showed strong activity towards the lignin monomer sinapinic acid and four other phenolic substrates. By contrast, both isoforms displayed much lower or no activity against four melanin precursors, suggesting that neither isoform is involved in integument formation. Modification of lignin alkali by the recombinant RfLacA preparation was also observed. These findings provide evidence that R. flavipes gut laccases are evolutionarily distinct, host-derived, produced in the salivary gland, secreted into the foregut, bind copper, and play a role in lignocellulose digestion. These findings contribute to a better understanding of termite digestion and gut physiology, and will assist future translational studies that examine the contributions of individual termite enzymes in lignocellulose digestion.
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Proteínas de Insectos/metabolismo , Isópteros/enzimología , Lacasa/metabolismo , Fenol/metabolismo , Animales , Proteínas de Insectos/genética , Intestinos/enzimología , Isópteros/clasificación , Isópteros/genética , Isópteros/metabolismo , Lacasa/genética , Datos de Secuencia Molecular , Oxidación-Reducción , FilogeniaRESUMEN
Tango is a transposon of the Tc1 family and was originally discovered in the African malaria mosquito, Anopheles gambiae. Here we report a systematic analysis of the genome sequence of the yellow fever mosquito, Aedes aegypti, which uncovered three distinct Tango transposons. We name the only An. gambiae Tango transposon AgTango1 and the three Ae. aegypti Tango elements AeTango1-3. Like AgTango1, AeTango1 and AeTango2 elements both have members that retain characteristics of autonomous elements such as intact open reading frames and terminal inverted repeats (TIRs). AeTango3 is a degenerate transposon with no full-length members. All full-length Tango transposons contain subterminal direct repeats within their TIRs. AgTango1 and AeTango1-3 form a single clade among other Tc1 transposons. Within this clade, AgTango1 and AeTango1 are closely related and share approximately 80% identity at the amino acid level, which exceeds the level of similarity of the majority of host genes in the two species. A survey of Tango in other mosquito species was carried out using degenerate PCR. Tango was isolated and sequenced in all members of the An. gambiae species complex, Aedes albopictus and Ochlerotatus atropalpus. Oc. atropalpus contains a rich diversity of Tango elements, while Tango elements in Ae. albopictus and the An. gambiae species complex all belong to Tango1. No Tango was detected in Culex pipiens quinquefasciatus, Anopheles stephensi, Anopheles dirus, Anopheles farauti or Anopheles albimanus using degenerate PCR. Bioinformatic searches of the Cx. p. quinquefasciatus (~10 x coverage) and An. stephensi (0.33 x coverage) databases also failed to uncover any Tango elements. Although other evolutionary scenarios cannot be ruled out, there are indications that Tango1 underwent horizontal transfer among divergent mosquito species.
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Aedes/genética , Anopheles/genética , Elementos Transponibles de ADN/genética , Evolución Molecular , Genómica , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas de Insectos/química , Proteínas de Insectos/genética , Datos de Secuencia Molecular , Filogenia , Especificidad de la EspecieRESUMEN
Tc1 is a family of DNA transposons found in diverse organisms including vertebrates, invertebrates and fungi. Tc1 belongs to the IS630-Tc1-mariner superfamily, which is characterized by common 'TA' target site and conserved D(Asp)DE(Glu) or DDD catalytic triad. All functional Tc1-like transposons contain a transposase with a DD34E catalytic triad. We conducted a systematic analysis of DD34E transposons in the African malaria mosquito, Anopheles gambiae, using a reiterative and exhaustive search program. In addition to previously described Tc1-like elements, we uncovered 26 new DD34E transposons including a novel family that we named gambol. Designation of family status to gambol is based on phylogenetic analyses of transposase sequences that showed gambol and Tc1 transposons as distinct clades that were separated by mariner and other families of the IS630-Tc1-mariner superfamily. The distinction between Tc1 and gambol is also consistent with the unique TIRs in gambol elements and the presence of a 'W[I/L/V]DEDC' signature near their N-termini. This signature is predicted as part of the 'RED' domain, a component of the 'PAI' and 'RED' DNA binding domains in Tc1 and possibly mariner. Although gambol appears to be related to a few DD34E transposons from cyanobacteria and fungi, no gambol has been reported in any other insects or animals thus far. Several gambol and Tc1 elements have intact ORFs and different genomic copies with high sequence identity, which suggests that they may have been recently active.
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Anopheles/genética , Elementos Transponibles de ADN/genética , Variación Genética , Proteínas de Insectos/genética , Familia de Multigenes/genética , Secuencia de Aminoácidos , Animales , Mapeo Cromosómico , Proteínas de Unión al ADN/genética , Datos de Secuencia Molecular , Filogenia , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
Both aging and diabetes are characterized by the formation of advanced glycation end products (AGEs). Both exhibit other similarities including deficits in wound healing that are associated with higher rates of fibroblast apoptosis. In order to investigate a potential mechanism for enhanced fibroblast apoptosis in diabetes and aged individuals, experiments were carried out to determine whether the predominant advanced glycation end product in skin, N-epsilon-(carboxymethyl) lysine (CML)-collagen, could induce fibroblast apoptosis. In vivo experiments established that CML-collagen but not unmodified collagen induced fibroblast apoptosis and that apoptosis was dependent upon caspase-3, -8, and -9 activity. In vitro experiments demonstrated that CML-collagen but not control collagen induced a time- and dose-dependent increase in fibroblast apoptosis. By use of blocking antibodies, apoptosis was shown to be mediated through receptor for AGE signaling. AGE-induced apoptosis was largely dependent on the effector caspase, caspase-3, which was activated through both cytoplasmic (caspase-8-dependent) and mitochondrial (caspase-9) pathways. CML-collagen had a global effect of enhancing mRNA levels of pro-apoptotic genes that included several classes of molecules including ligands, receptors, adaptor molecules, mitochondrial proteins, and others. However, the pattern of expression was not identical to the pattern of apoptotic genes induced by tumor necrosis factor alpha.
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Apoptosis , Citoplasma/metabolismo , Fibroblastos/patología , Productos Finales de Glicación Avanzada/fisiología , Lisina/análogos & derivados , Mitocondrias/metabolismo , Animales , Caspasa 3 , Caspasa 8 , Caspasa 9 , Inhibidores de Caspasas , Caspasas/metabolismo , Células Cultivadas , Colágeno/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Fibroblastos/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Humanos , Etiquetado Corte-Fin in Situ , Lisina/metabolismo , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Antibodies, proteins produced in animals that bind with high specificity and affinity to a seemingly limitless variety of biomolecules, have an essential role in clinical medicine and basic biological research. Methods to produce antibody fragments on the surface of bacterial viruses were surveyed for their ability to replace animal-dependent antibodies. The use of filamentous phage to display antibody fragments derived from semisynthetic antibody genes was found to produce proteins that bind to antigens with a variety, specificity, and affinity similar to those produced in animal systems.
RESUMEN
The interaction of the Ferric Uptake Regulation (Fur) protein with the backbone of operator DNA was analyzed by hydroxyl radical footprinting and the ethylation interference assay. Comparison of the contacts made by Fur and those made by proteins containing the helix-turn-helix or related motifs shows that the mode of DNA binding by this repressor is unique. Ethylation interference experiments demonstrate that there are relatively few phosphate contacts of unique disposition while hydroxyl radical footprinting demonstrates that Fur-operator contacts are segregated on one face of the helix and span nearly three successive major grooves.
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Proteínas Bacterianas/metabolismo , ADN Bacteriano/metabolismo , Hierro/metabolismo , Proteínas Represoras/metabolismo , Alquilación , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión/genética , ADN Bacteriano/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Secuencias Hélice-Asa-Hélice/genética , Radical Hidroxilo , Datos de Secuencia Molecular , Regiones Operadoras Genéticas , Fosfatos/química , Proteínas Represoras/química , Proteínas Represoras/genética , Yersinia pestis/genética , Yersinia pestis/metabolismoRESUMEN
The 12 histidine and four cysteine residues of the Fur repressor of Escherichia coli were changed, respectively, to leucine and serine by site-directed mutagenesis of the fur gene. The affects of these mutations were measured in vivo by ligation of the mutated genes to a wild-type fur promoter followed by measurement of the ability of these plasmids to regulate expression of a lacZ fusion in the aerobactin operon. In vitro affects were assayed by insertion of the mutated genes in the expression vector pMON2064 attended by isolation of the altered Fur proteins and appraisal of their capacity to bind to operator DNA. The results suggest that cysteine residues at positions 92 and 95 are important for the activity of the Fur protein.
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Proteínas Bacterianas/genética , Escherichia coli/genética , Proteínas Represoras/genética , Proteínas Bacterianas/biosíntesis , Mutagénesis Sitio-Dirigida , Proteínas Represoras/biosíntesisRESUMEN
Proteolytic enzymes were used to detect metal-induced conformational changes in the ferric uptake regulation (Fur) protein of Escherichia coli K12. Metal binding results in enhanced cleavage of the N-terminal region of Fur by trypsin and chymotrypsin. Activation of both trypsinolysis sensitivity and DNA binding have similar metal ion specificity and concentration dependencies, suggesting that the conformational change detected is required for operator DNA binding. Isolation and characterization of biochemically generated fragments of Fur as well as other data indicate that the N-terminal region is necessary for the interaction of the repressor with DNA and that a C-terminal domain is sufficient for binding to metal ions.
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Proteínas Bacterianas/metabolismo , Cloruros , Hierro/metabolismo , Compuestos de Manganeso , Proteínas Represoras/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Cadmio/farmacología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Escherichia coli/análisis , Hidrólisis , Manganeso/farmacología , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Relación Estructura-Actividad , Tripsina , Zinc/farmacologíaRESUMEN
The enzyme N epsilon-hydroxylysine acetylase has been isolated from Escherichia coli 294 carrying recombinant plasmid ABN11. Activity of the enzyme was followed by measurement of the rate of appearance of 2-nitro-5-thiobenzoate, the product of cleavage of 5,5'-dithiobis(2-nitrobenzoate) by free coenzyme A released from its acetyl derivative. The enzyme bound firmly to Reactive Blue 2-Sepharose CL-6B and was eluated with 1.5 M KCl. The protein gave a single band, corresponding to a Mr of 33,000, on polyacrylamide gel electrophoresis in sodium dodecyl sulfate. In contrast, gel filtration of the native enzyme gave a Mr of 150,000-200,000. A sequence analysis of the DNA at the junction of the first and second genes in the aerobactin operon, considered in conjunction with the N-terminal amino acid sequence of the isolated protein, enabled the conclusion that the acetylase is specified by the second gene in the complex. The enzyme transfers the acetyl moiety from acetyl coenzyme A to a variety of hydroxylamines, with N epsilon-hydroxylysine as the preferred substrate. In agreement with the results found by affinity chromatography, Coomassie Blue was observed to act as a potent inhibitor.