RESUMEN
We present further evidence in support of the notion that Borrelia burgdorferi is possibly involved in the pathogenesis of morphea and lichen sclerosus et atrophicus (LSA). Running a nested polymerase chain reaction (PCR) with a primer set specific for the flagellin gene of B. burgdorferi enabled us to demonstrate the presence of Borrelia DNA in skin biopsies of patients with morphea (nine of nine) of LSA (six of six). Biopsy specimens obtained from patients with erythema chronicum migrans (two patients, four of four samples) and acrodermatitis chronica atrophicans (one patient, one of one sample) also showed positive PCR results. By contrast, there was no amplification of Borrelia DNA in control biopsies either from patients with chronic eczema (three of three) or psoriasis (two of two) or from normal skin (three of three). Antibodies directed against B. burgdorferi were only detected in the serum of patients with erythema chronicum migrans (two of two) and acrodermatitis chronica atrophicans (one of one) but were not present in cases of morphea (five of five), LSA (three of three), or in control subjects (three of three). These data suggest that B. burgdorferi may play a role in the pathogenesis of both morphea and LSA. Furthermore, we conclude that PCR analysis provides an important diagnostic tool, even in seronegative Borrelia infections.
Asunto(s)
ADN Bacteriano/análisis , Erupciones Liquenoides/microbiología , Enfermedad de Lyme/complicaciones , Reacción en Cadena de la Polimerasa , Esclerodermia Localizada/microbiología , Adolescente , Adulto , Anciano , Secuencia de Bases , Grupo Borrelia Burgdorferi/genética , Femenino , Humanos , Erupciones Liquenoides/genética , Enfermedad de Lyme/genética , Masculino , Persona de Mediana Edad , Sondas Moleculares/genética , Datos de Secuencia Molecular , Esclerodermia Localizada/genéticaRESUMEN
No decision has been made yet as to whether or not the origin of the circumscribed scleroderma (morphea) is spirochetal. We describe a morphea-like skin lesion that developed after a tick bite 10 years ago. The histological investigation showed sclerodermal characteristics and necrobiosis lipoidica of the granulomatous type as well. No antibodies directed against Borrelia burgdorferi could be detected absorbance by a flagellin ELISA or by Western blot analysis. The VDRL, TPHA and FTA absorbance test, Warthin-Starry staining, and cultivation of Borrelia from skin biopsies were negative. The application of a nested polymerase chain reaction (PCR), relying on a combination of flagellin-gene-specific primers, demonstrated for the first time the presence of Borrelia DNA in a morphealike skin lesion. Immunohistological examination of the skin by a monoclonal antibody directed against flagellin was positive. Furthermore, in vitro GM-CSF secretion and lymphocyte proliferation upon stimulation with Borrelia-antigen was elevated and decreased significantly after 3 weeks of treatment with tetracyclines. In this case PCR analysis, immunohistochemistry and cellular immune response confirmed an infection with Borrelia, although no serum antibodies against spirochetal antigens could be detected.
Asunto(s)
Grupo Borrelia Burgdorferi/aislamiento & purificación , Eritema Crónico Migrans/patología , Enfermedad de Lyme/patología , Esclerodermia Localizada/patología , Adolescente , Anticuerpos Antibacterianos/análisis , Grupo Borrelia Burgdorferi/genética , Grupo Borrelia Burgdorferi/inmunología , Diagnóstico Diferencial , Femenino , Flagelina/inmunología , Humanos , Necrobiosis Lipoidea/patología , Reacción en Cadena de la Polimerasa , Piel/patologíaRESUMEN
CSF and serum specimens were consecutively obtained from three patients with neuroborreliosis (stage I, II and III), CSF protein content, cell counts and differential, IgG index, oligoclonal bands and anti-B. burgdorferi antibodies were measured. Cerebrospinal fluid (CSF) was tested for Borrelia-DNA being present prior to and after antibiotic treatment. While DNA could be identified before ceftriaxone was administered, there were no more amplification products afterwards. The goal of this study was to compare the usefulness of serodiagnostic methods and the detection of Borrelia burgdorferi-DNA in patients with clinically confirmed neuroborreliosis to test the efficiency of antibiotic therapy.
Asunto(s)
Anticuerpos Antibacterianos/líquido cefalorraquídeo , Grupo Borrelia Burgdorferi/inmunología , Enfermedad de Lyme/inmunología , Enfermedades del Sistema Nervioso/inmunología , Anciano , Antibacterianos/uso terapéutico , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/aislamiento & purificación , Secuencia de Bases , Grupo Borrelia Burgdorferi/genética , ADN Bacteriano/líquido cefalorraquídeo , ADN Bacteriano/genética , Femenino , Humanos , Inflamación/inmunología , Enfermedad de Lyme/diagnóstico , Enfermedad de Lyme/tratamiento farmacológico , Persona de Mediana Edad , Datos de Secuencia Molecular , Enfermedades del Sistema Nervioso/diagnóstico , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Reacción en Cadena de la PolimerasaRESUMEN
Escherichia coli O157:H7 strains are newly recognized pathogens associated with haemorrhagic colitis and haemolytic-uremic syndrome. In addition to Shiga-like toxin types I and II known to be produced by E. coli O157 isolates, we have identified in E. coli O157:H7 strain 7279 a third toxin, designated SLT-IIvhc, that is neutralized neither by anti-SLT-I nor by anti-SLT-II antibodies. The genes for this toxin were isolated by using a PCR-mediated cell-free cloning technique. DNA sequence analysis revealed a high degree of homologies to SLT-II and several SLT-II variants. The predicted amino acid sequence of the A subunit of SLT-IIvhc differed from that of the O91:H7 toxin VT2ha and SLT-IIc from E. coli O157:H- strain E2511 by 2 and 3 amino acids, respectively. The amino acid sequence of its B subunit was identical to VT2ha and SLT-IIc but different from SLT-II and SLT-IIv. Immunological differences of SLT-IIvhc and SLT-II as well as their different toxicity to HeLa cells presumably resulted from the small deviations within the primary structure of the B subunit.
Asunto(s)
Toxinas Bacterianas/genética , ADN Bacteriano/química , Enterotoxinas/genética , Escherichia coli/genética , Secuencia de Aminoácidos , Toxinas Bacterianas/química , Secuencia de Bases , Clonación Molecular , Electroforesis en Gel de Agar , Enterotoxinas/química , Humanos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Sondas de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Toxina Shiga IIRESUMEN
To improve the serodiagnosis of erythema migrans, we evaluated how sensitivity and specificity of immunoblotting are influenced by antigen concentration and blocking conditions. We found that an antigen concentration of 0.5 micrograms per lane in concert with ovalbumin blocking of the nitrocellulose provided the best results. In this case, 81% of the erythema migrans had positive immunoglobulin M tests, whereas only 33% were positive in a flagellum enzyme-linked immunosorbent assay (ELISA) and 28% were positive in a sonicate ELISA.
Asunto(s)
Especificidad de Anticuerpos , Ensayo de Inmunoadsorción Enzimática , Eritema Crónico Migrans/diagnóstico , Inmunoglobulina M , Ovalbúmina , Sitios de Unión de Anticuerpos , Unión Competitiva , Grupo Borrelia Burgdorferi/inmunología , Eritema Crónico Migrans/sangre , Flagelos/inmunología , Humanos , Sensibilidad y Especificidad , Pruebas SerológicasRESUMEN
The prevalence of antibodies against Borrelia burgdorferi, the etiologic agent of Lyme borreliosis, was determined in a group of blood donors from the Würzburg area (Southern Germany). 26 of 472 donors (5.5%) tested positive in a hemagglutination test. When performing immunoblots only 13 donors (2.7%) gave rise to B. burgdorferi-specific antibodies. 9 of them were examined in more detail by anamnesis, physical examination, determination of inflammation parameters of the blood and polymerase chain reaction (PCR) analysis of urine. All persons were asymptomatic for Lyme borreliosis. One of 4, who remembered a tick bite, actually had suffered from an erythema migrans 5 years ago. Another one had been affected by fever, headaches and pains in the limbs, arthralgia and motoric disorder in both hands 6 months before examination. Analysis of the blood did not provide any evidence of an acute infection. In the urine of 2 donors we detected B. burgdorferi-specific DNA by PCR. No seroconversion due to blood transfusion could be observed, when 9 recipients of blood products provided by the 13 seropositive donors were serologically reexamined. PCR analysis of urine samples of 5 recipients was also negative.
Asunto(s)
Anticuerpos Antibacterianos/análisis , Donantes de Sangre/estadística & datos numéricos , Grupo Borrelia Burgdorferi/inmunología , Enfermedad de Lyme/epidemiología , Tamizaje Masivo , Transfusión Sanguínea , Estudios Transversales , Ensayo de Inmunoadsorción Enzimática , Alemania/epidemiología , Pruebas de Hemaglutinación , Humanos , Incidencia , Enfermedad de Lyme/diagnóstico , Enfermedad de Lyme/transmisión , Factores de RiesgoRESUMEN
The two genes encoding the class IIS restriction-modification system MboII from Moraxella bovis were cloned separately in two compatible plasmids and expressed in E. coli RR1 delta M15. The nucleotide sequences of the MboII endonuclease (R.MboII) and methylase (M.MboII) genes were determined and the putative start codon of R.MboII was confirmed by amino acid sequence analysis. The mboIIR gene specifies a protein of 416 amino acids (MW: 48,617) while the mboIIM gene codes for a putative 260-residue polypeptide (MW: 30,077). Both genes are aligned in the same orientation. The coding region of the methylase gene ends 11 bp upstream of the start codon of the restrictase gene. Comparing the amino acid sequence of M.MboII with sequences of other N6-adenine methyltransferases reveals a significant homology to M.RsrI, M.HinfI and M.DpnA. Furthermore, M.MboII shows homology to the N4-cytosine methyltransferase BamHI.
Asunto(s)
Desoxirribonucleasas de Localización Especificada Tipo II/genética , Moraxella/enzimología , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/genética , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Codón , ADN Bacteriano , Desoxirribonucleasas de Localización Especificada Tipo II/aislamiento & purificación , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Escherichia coli/genética , Exones , Expresión Génica , Genes Bacterianos , Datos de Secuencia Molecular , Moraxella/genética , Plásmidos , Homología de Secuencia de Ácido Nucleico , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/aislamiento & purificación , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/metabolismoRESUMEN
Mutants of lactose permease of Escherichia coli with amino acid changes (Gly-24----Glu; Gly-24----Arg; Pro-28---Ser; Gly-24, Pro-28----Glu-Ser and Gly-24, Pro-28----Arg-Ser) within a putative membrane-spanning alpha-helix (Phe-Gly-Leu-Phe-Phe-Phe-Phe-Tyr-Phe-Phe-Ile-Met-Gly- Ala-Tyr-Phe-Pro-Phe-Phe-Pro-Ile) are incorporated into the cytoplasmic membrane. The mutant proteins retain the ability to bind galactosides, and the affinity for several substrates is actually increased. However, the rate of active transport is decreased to 0.01% of the wild-type rate in the mutants carrying Arg-24 or Arg-24, Ser-28. Kinetic analysis demonstrates that the two mutants require 10 min to cause occupied binding sites for galactoside and H+ to change their exposure from the periplasm to the cytoplasm as compared to 50 ms in the wild type. The effect is less pronounced when these sites are unoccupied.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/enzimología , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Monosacáridos , Simportadores , Translocación Genética , Secuencia de Aminoácidos , Transporte Biológico , ADN Bacteriano/análisis , Galactósidos/metabolismo , Cinética , Proteínas de Transporte de Membrana/metabolismo , MutaciónRESUMEN
The DNA sequence of several functionally interesting lac permease mutants of Escherichia coli has been determined. The phenotypes of the mutant permeases were described by Mieschendahl et al. (1981). The following exchanges are noteworthy: tyr to asp in codon 26 in Y-K MUB 7; thr to ile in codon 266 in Y-K AJ 33; gly to asp in codon 262 in Y-D 3 and in Y-D 4.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Lactosa/metabolismo , Maltosa/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Monosacáridos , Simportadores , Secuencia de Aminoácidos , Proteínas Bacterianas/fisiología , Secuencia de Bases , Codón , Escherichia coli/metabolismo , Proteínas de Transporte de Membrana/fisiologíaRESUMEN
The nucleotide sequence of three lacZ- -Y+ fusions found among spontaneous lacY+ revertants of the lacY- mutant MAB16 in Escherichia coli is reported. MAB16 is a frameshift mutation in codon 6 of the lacY gene. DNA sequence analysis of the fusions shows the first eight N-terminal codons of the lacY gene can be replaced by 3, 39 or 804/805 N-terminal codons of the lacZ gene without impairing lac permease activity qualitatively.
Asunto(s)
ADN Bacteriano/aislamiento & purificación , Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Monosacáridos , Simportadores , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Codón/aislamiento & purificación , Escherichia coli/enzimología , MutaciónRESUMEN
The purified lactose carrier of Escherichia coli (product of the lac Y gene) is shown to be a monomer in detergent micelles of dodecyl-O-beta-D-maltoside. The negative-dominant phenotype of mutant carriers (lacY-d mutants) could not be verified by measurements of the rate of galactoside transport in lacY+/Y-d diploid strains. It is proposed that the membrane-embedded carrier functions as a monomer in galactoside-H+ symport.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Monosacáridos , Simportadores , Transporte Biológico , Escherichia coli/genética , Galactósidos/metabolismo , Sustancias Macromoleculares , Proteínas de Transporte de Membrana/genética , Micelas , Peso Molecular , MutaciónRESUMEN
Mutations in the lacY gene of Escherichia coli have been used to analyze the functional organization of lactose permease. Deletions suggest that the NH2 terminus of lactose permease is not essential and can be replaced by residues of the cytoplasmic enzyme beta-galactosidase. Negative dominant mutations in the lacY gene can be explained by the assumption that membrane-associated lactose permease is active as a dimer or oligomer. The map positions of these mutations and other point mutations that lower or alter the sugar specificity define regions of lactose permease involved in sugar or proton binding and transport.