RESUMEN
Porphyromonas gingivalis is a key periodontal pathogen that has been implicated in the aetiology of chronic adult periodontitis. The aim of this study was to characterize two potential vaccine candidates (PG32 and PG33) identified from a previous genomic sequence analysis. Gene knockout studies suggested that these proteins play an important role in bacterial growth and are transcriptionally linked. Analysis of 14 laboratory and clinical isolates of P. gingivalis found that in all strains, both genes were present with a high level of conservation and that the two proteins were also expressed in vitro. Truncated recombinant PG32 and PG33 proteins were produced in Escherichia coli in an attempt to increase the solubility of the proteins while retaining their native conformation. While most of the truncated proteins remained insoluble, two truncated proteins showed good solubility and high levels of protection in the P. gingivalis murine lesion model and may be considered as potential vaccine candidates for further testing in models of human periodontal disease.
Asunto(s)
Antígenos Bacterianos/aislamiento & purificación , Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Porphyromonas gingivalis/inmunología , Sustancias Protectoras/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas/inmunología , Secuencia Conservada , Modelos Animales de Enfermedad , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Silenciador del Gen , Vectores Genéticos , Humanos , Inmunización , Ratones , Ratones Endogámicos BALB C , Porinas/inmunología , Porinas/aislamiento & purificación , Porphyromonas gingivalis/genética , Proteínas Recombinantes , Solubilidad , Transformación Genética/genética , Vacunas Acelulares/inmunologíaRESUMEN
We report the characterization of a Porphyromonas gingivalis gene, designated omp28, encoding a protein that we have previously purified and characterized as a 28-kDa outer membrane protein. The deduced amino acid sequence of the omp28 open reading frame displayed an outer membrane leader sequence and lipoprotein attachment site but did not exhibit any significant overall sequence identity with protein sequences in the databases. A small stretch of amino acids (19 residues) exhibits 50% sequence identity with a segment of a fimbrial protein from Dichelobacter nodosus involved in adhesion, suggesting that Omp28 may be a surface adhesin/receptor of P. gingivalis. Using the pET-24 vector we expressed recombinant Omp28 (rOmp28) in Escherichia coli. Western blot analyses of purified rOmp28 with rabbit antisera to a P. gingivalis outer membrane preparation, protective rat anti-whole P. gingivalis antisera and pooled human sera from chronic periodontitis patients showed that the recombinant was recognized by all antisera. Further, anti-rOmp28 antisera exhibited strong reactivity with a panel of four laboratory strains and 10 clinical isolates of P. gingivalis from the United States, Sudan, Romania and Norway. These results suggest that Omp28 is expressed by a wide distribution of P. gingivalis strains.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Porphyromonas gingivalis/genética , Adhesinas Bacterianas/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Sitios de Unión/genética , Southern Blotting , Western Blotting , Cromatografía de Afinidad , Clonación Molecular , ADN Recombinante/genética , Dichelobacter nodosus/genética , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Expresión Génica , Vectores Genéticos/genética , Humanos , Lipoproteínas/genética , Sistemas de Lectura Abierta/genética , Periodontitis/sangre , Porphyromonas gingivalis/inmunología , Conejos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ProteínaRESUMEN
Porphyromonas gingivalis is a key periodontal pathogen which has been implicated in the etiology of chronic adult periodontitis. Our aim was to develop a protein based vaccine for the prevention and or treatment of this disease. We used a whole genome sequencing approach to identify potential vaccine candidates. From a genomic sequence, we selected 120 genes using a series of bioinformatics methods. The selected genes were cloned for expression in Escherichia coli and screened with P. gingivalis antisera before purification and testing in an animal model. Two of these recombinant proteins (PG32 and PG33) demonstrated significant protection in the animal model, while a number were reactive with various antisera. This process allows the rapid identification of vaccine candidates from genomic data.
Asunto(s)
Antígenos Bacterianos/inmunología , Vacunas Bacterianas/inmunología , Porphyromonas gingivalis/inmunología , Vacunas Sintéticas/inmunología , Secuencia de Aminoácidos , Animales , Proteínas de la Membrana Bacteriana Externa/inmunología , Western Blotting , Humanos , Ratones , Datos de Secuencia Molecular , Porphyromonas gingivalis/genética , Conejos , Ratas , Ratas Sprague-DawleyRESUMEN
Insomnia is a problem with complex and multifactorial etiologies that requires both standardized and individualized treatment interventions. Specific targets of treatment may include hyperarousal, poor sleep habits, underlying mood disorders, sedative overuse, pain and general medical problems, circadian dysrhythmias, sleep apnea, and restless legs syndrome. Optimal treatment also will incorporate stress management, coping strategies, enhancement of relationships, and promoting lifestyle changes that facilitate sleep.
Asunto(s)
Terapias Complementarias , Menopausia , Trastornos del Inicio y del Mantenimiento del Sueño/diagnóstico , Trastornos del Inicio y del Mantenimiento del Sueño/terapia , Femenino , Humanos , Sueño/fisiologíaRESUMEN
Biological circadian clocks oscillate with an approximately 24-hour period, are ubiquitous, and presumably confer a selective advantage by anticipating the transitions between day and night. The circadian rhythms of sleep, melatonin secretion and body core temperature are thought to be generated by the suprachiasmatic nucleus of the hypothalamus, the anatomic locus of the mammalian circadian clock. Autosomal semi-dominant mutations in rodents with fast or slow biological clocks (that is, short or long endogenous period lengths; tau) are associated with phase-advanced or delayed sleep-wake rhythms, respectively. These models predict the existence of familial human circadian rhythm variants but none of the human circadian rhythm disorders are known to have a familial tendency. Although a slight 'morning lark' tendency is common, individuals with a large and disabling sleep phase-advance are rare. This disorder, advanced sleep-phase syndrome, is characterized by very early sleep onset and offset; only two cases are reported in young adults. Here we describe three kindreds with a profound phase advance of the sleep-wake, melatonin and temperature rhythms associated with a very short tau. The trait segregates as an autosomal dominant with high penetrance. These kindreds represent a well-characterized familial circadian rhythm variant in humans and provide a unique opportunity for genetic analysis of human circadian physiology.