RESUMEN
Micelle-templated polyguaiacol nanowires were successfully prepared via polymerization oxidation of guaiacol (o-methoxy phenol) by peroxidase enzyme in the presence of hydrogen peroxide at mild reaction conditions. The dimensions of the prepared nanowires were controlled by tuning the size and shape of the micelle structure via changing and controlling the type, chain length and molar concentrations of the ionic surfactant. The progress of the reaction and estimation of the size of soft micellar templates were followed by UV-Vis spectroscopy and dynamic light scattering (DLS). The resulting micelle encapsulated or purified polyguaiacol nanowires were characterized using transmission electron microscopy (TEM).
Asunto(s)
Biocatálisis , Guayacol/análogos & derivados , Nanocables/química , Polímeros/síntesis química , Guayacol/síntesis química , Guayacol/química , Micelas , Nanocables/ultraestructura , TensoactivosRESUMEN
BACKGROUND: In the last 10 years type-I allergy against proteins from Hevea brasiliensis latex has become an acknowledged medical issue. Fruit-allergic patients represent one risk group for developing latex allergy. Class I chitinases have been identified from chestnut, avocado and banana as relevant allergens. The chitin binding (hevein) domain from these class I chitinases has been postulated to bear the important IgE binding epitopes. OBJECTIVE: To clone the cDNA of an allergenic latex class I chitinase, to express the recombinant protein and to determine its IgE cross-reactivity with hevein (Hev b 6.02). METHODS: A full-length cDNA coding for a class I chitinase has been isolated from Hevea latex RNA by reverse transcription followed by PCR. The chitinase encoding sequence has been subcloned into the pMAL expression vector and expressed in E. coli as a fusion protein to maltose binding protein. The highly enriched recombinant protein fraction has been tested for its IgE binding capacity in immunoblots and ELISA. Furthermore, the pathogenesis-related function of the recombinant protein was tested in a fungal growth inhibition assay. RESULTS: The Hevea brasiliensis latex chitinase, designated Hev b 11, displays 70% identity to the endochitinase from avocado and its hevein-domain 58% to hevein (Hev b 6.02). The recombinant Hev b 11-maltose binding protein is recognized by latex- and fruit-allergic patients with IgE binding in both, ELISA and immunoblots. Pre-incubation of sera with rHev b 11-maltose binding protein showed an overall 16% inhibition of subsequent binding to rHev b 6.02-maltose binding protein on solid phase. The growth of F. oxysporum was inhibited in a dose dependent manner by addition of rHev b 11-maltose binding protein to the culture. CONCLUSIONS: Hev b 11, a class I chitinase, is another allergen from Hevea latex with a chitin binding domain and displays a different IgE binding capacity compared with hevein.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Alérgenos/química , Alérgenos/inmunología , Alérgenos/aislamiento & purificación , Quitinasas/química , Quitinasas/inmunología , Quitinasas/aislamiento & purificación , Clonación Molecular , Proteínas de Escherichia coli , Hevea/inmunología , Proteínas de Transporte de Monosacáridos , Proteínas de Plantas/química , Secuencia de Aminoácidos , Antígenos de Plantas , Secuencia de Bases , Sitios de Unión/inmunología , Proteínas Portadoras/inmunología , Proteínas Portadoras/aislamiento & purificación , ADN Complementario/inmunología , Ensayo de Inmunoadsorción Enzimática , Hipersensibilidad a los Alimentos/inmunología , Frutas/inmunología , Humanos , Immunoblotting , Inmunoglobulina E/inmunología , Hipersensibilidad al Látex/inmunología , Proteínas de Unión a Maltosa , Datos de Secuencia Molecular , Proteínas de Plantas/inmunología , Proteínas de Plantas/aislamiento & purificación , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
The authors report two patients with more than 20 diopters of myopia, severely restricted abduction, and more than 90Delta of acquired esotropia. Marked axial elongation of the globes was present. Each underwent large bilateral medial rectus recessions and bilateral lateral rectus resections. The deviations were significantly reduced and abduction improved with combined horizontal recession-resection surgery on both eyes.
Asunto(s)
Esotropía/complicaciones , Esotropía/cirugía , Miopía/complicaciones , Músculos Oculomotores/cirugía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Órbita/diagnóstico por imagen , Tomografía Computarizada por Rayos X , Resultado del Tratamiento , Agudeza VisualRESUMEN
Genetic modification of the vectorial capacity of mosquito vectors of human disease requires promoters capable of driving gene expression with appropriate tissue and stage specificity. We report on the characterization in transgenic Aedes aegypti of two mosquito gut-specific promoters. A 1.4-kb DNA fragment adjacent to the 5' end of the coding region of the Ae. aegypti carboxypeptidase (AeCP) gene and a corresponding 3.4-kb DNA fragment at the 5' end of the Anopheles gambiae carboxypeptidase (AgCP) gene were linked to a firefly luciferase reporter gene and introduced into the Ae. aegypti germ line by using Hermes and mariner (Mos1) transposons. Six independent transgenic lines were obtained with the AeCP construct and one with the AgCP construct. Luciferase mRNA and protein were abundantly expressed in the guts of transgenic mosquitoes in four of the six AeCP lines and in the AgCP line. Expression of the reporter gene was gut-specific and reached peak levels at about 24 h post-blood ingestion. The AeCP and AgCP promoters can be used to drive the expression of genes that hinder parasite development in the mosquito gut.
Asunto(s)
Aedes/genética , Animales Modificados Genéticamente , Carboxipeptidasas/genética , Regulación de la Expresión Génica , Animales , Genes Reporteros , Humanos , Regiones Promotoras GenéticasRESUMEN
In this report, we present a simple nonradioactive labeling procedure for DNA fragments of high specific labeling density that can be used for a variety of applications. The protocol is based on the universal mono-functional platinum reagent for chemical digoxigenin (DIG) labeling of nucleic acids. The labeling protocol was optimized for large DNA templates as complete bacterial artificial chromosomes (BAC). Variations of incubation time and temperature improved the labeling density such that about 30% of the nucleotides were DIG-modified within 30 min. Furthermore, the refined procedure generates in a single-tube reaction and without prior digestion-labeled DNA fragments of 0.5-4.0 kb from a 130-kb template. Hybridization experiments were performed on Southern and northern blots and allowed the detection of single copy genes in 2.5 micrograms genomic DNA from Arabidopsis thaliana, which has a haploid genome size of 0.13 pg (ca. 120 Mb) and medium expressed transcripts from 0.8 microgram poly(A)+ RNA, respectively. The extremely high specific labeling density, the stability and the universal application of the probe generated with the platinum reagent makes this method a useful alternative to classical radioactive nuclei acids labeling techniques.
Asunto(s)
Northern Blotting/métodos , Paseo de Cromosoma/métodos , ADN/análisis , Digoxigenina , Polimorfismo de Longitud del Fragmento de Restricción , Alelos , Arabidopsis/genética , Mapeo Contig , Hibridación de Ácido Nucleico/métodos , Polimorfismo Genético , ARN de Planta/aislamiento & purificaciónRESUMEN
Rapid and efficient procedures for the detection of sequence polymorphisms are essential for chromosomal walking and mutation detection analyses. While DNA chip technology and denaturing high-performance liquid chromatography (DHPLC) are the methods of choice for large scale facilities, small laboratories are dependent on simple ready-to-use techniques. We show that heteroduplex analysis on high resolution gel matrices efficiently detects sequence polymorphism differing as little as a single base pair (e.g. single-nucleotide polymorphism, SNP) with standard laboratory equipment. Furthermore, the matrices also discerned differences between homoduplexes, a prerequisite for co-dominant markers. The markers thus generated are referred to as duplex analysis markers. We designed PCR primers for 36 Arabidopsis thaliana loci ranging in length from 230 bp to 1000 bp. Among three ecotypes, more than half (n = 19) of the loci examined were polymorphic; five of which contained three different alleles. This simple, high resolution technique can be used to rapidly convert sequence tagged sites into co-dominant PCR-based molecular markers for fine-scale mapping studies and chromosomal walking strategies as well as for the detection of mutations in particular genes.