RESUMEN
Meiosis is a specialized form of cell division generating haploid gametes and is dependent upon protein ubiquitylation by the anaphase-promoting complex/cyclosome (APC/C). Accurate control of the APC/C during meiosis is important in all eukaryotic cells and is in part regulated by the association of coactivators and inhibitors. We previously showed that the fission yeast meiosis-specific protein Mes1 binds to a coactivator and inhibits APC/C; however, regulation of the Mes1-mediated APC/C inhibition remains elusive. Here we show how Mes1 distinctively regulates different forms of the APC/C. We study all the coactivators present in the yeast genome and find that only Slp1/Cdc20 is essential for meiosis I progression. However, Fzr1/Mfr1 is a critical target for Mes1 inhibition because fzr1Δ completely rescues the defect on the meiosis II entry in mes1Δ cells. Furthermore, cell-free studies suggest that Mes1 behaves as a pseudosubstrate for Fzr1/Mfr1 but works as a competitive substrate for Slp1. Intriguingly, mutations in the D-box or KEN-box of Mes1 increase its recognition as a substrate by Fzr1, but not by Slp1. Thus Mes1 interacts with two coactivators in a different way to control the activity of the APC/C required for the meiosis I/meiosis II transition.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/metabolismo , Anafase , Ciclosoma-Complejo Promotor de la Anafase , Proteínas Cdc20 , Proteínas Cdh1 , Meiosis , Mutación/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , UbiquitinaciónRESUMEN
Bacille Calmette Guérin substrain Moreau Rio de Janeiro is an attenuated strain of Mycobacterium bovis that has been used extensively as an oral tuberculosis vaccine. We assessed its potential as a challenge model to study clinical and immunological events following repeated mycobacterial gut infection. Seven individuals received three oral challenges with approximately 10(7) viable bacilli. Clinical symptoms, T-cell responses and gene expression patterns in peripheral blood were monitored. Clinical symptoms were relatively mild and declined following each oral challenge. Delayed T-cell responses were observed, and limited differential gene expression detected by microarrays. Oral challenge with BCG Moreau Rio de Janeiro vaccine was immunogenic in healthy volunteers, limiting its potential to explore clinical innate immune responses, but with low reactogenicity.
Asunto(s)
Inmunidad Innata , Enfermedades Intestinales , Infecciones por Mycobacterium , Mycobacterium bovis/inmunología , Linfocitos T/inmunología , Vacunas contra la Tuberculosis/administración & dosificación , Vacunas contra la Tuberculosis/inmunología , Adolescente , Adulto , Femenino , Humanos , Enfermedades Intestinales/sangre , Enfermedades Intestinales/inmunología , Enfermedades Intestinales/microbiología , Enfermedades Intestinales/terapia , Masculino , Persona de Mediana Edad , Infecciones por Mycobacterium/sangre , Infecciones por Mycobacterium/inmunología , Infecciones por Mycobacterium/microbiología , Infecciones por Mycobacterium/terapia , Linfocitos T/metabolismoRESUMEN
SC599 vaccine is a live Shigella dysenteriae 1 strain attenuated by deletion of invasion [icsA], iron chelation [ent, fep] and shiga toxin A subunit [stxA] genes. In a preliminary Phase 1 single dose prospective study, we showed that SC599 vaccine was well tolerated, and the maximum tolerable dose was greater than 10(8) CFU [Sadorge C, Ndiaye A, Beveridge N, Frazer S, Giemza R, Jolly N, et al. Phase 1 clinical trial of live attenuated Shigella dysenteriae type-1 DeltaicsA Deltaent Deltafep DeltastxA:HgR oral vaccine SC599 in healthy human adult volunteers. Vaccine 2008; 26(7):978-8]. In this Phase 2 trial, three groups of volunteers ingested a single dose of SC599 [10(5) CFU, n=38; 10(7) CFU, n=36] or placebo [n=37]. Both 10(5) and 10(7) CFU doses were immunogenic, inducing significant IgA and IgG LPS-specific ASCs and antibody responses, comparable in magnitude to those of other strains that prevented illness following experimental challenge. In the intention to treat analysis, 34.2% and 44.4% IgA ASC responders were detected in the 10(5) and 10(7) CFU groups respectively (p<0001 vs placebo for both groups), as well as 31.6% and 33.3% serum IgA responders (p<001 and p<0.001 vs placebo for 10(5) and 10(7) CFU groups, respectively). No difference between the two vaccine groups was observed. No stxB-specific antibody response was detected in the vaccines. SC599 excretion occurred in 23.7 and 30.6% of subjects in the 10(5) and 10(7) CFU groups, respectively. SC599 vaccine was well tolerated, and the reported adverse events were mainly digestive. These results indicate that a single oral immunization of SC599 vaccine elicits a significant circulating IgA ASC and serum antibody response that may confer protection against the most severe symptoms of Shigellosis in responders to the vaccine.
Asunto(s)
Vacunas contra la Shigella/efectos adversos , Vacunas contra la Shigella/inmunología , Shigella dysenteriae/genética , Shigella dysenteriae/inmunología , Administración Oral , Adulto , Anticuerpos Antibacterianos/sangre , Método Doble Ciego , Femenino , Humanos , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Masculino , Placebos/administración & dosificación , Vacunas Atenuadas/efectos adversos , Vacunas Atenuadas/inmunología , Adulto JovenRESUMEN
Human papillomavirus type 16 (HPV16) is the most common cause of cervical carcinoma. Cervical cancer develops from low-grade lesions that support the productive stages of the virus life cycle. The 16E1 wedge E4 protein is abundantly expressed in such lesions and can be detected in cells supporting vegetative viral genome amplification. Using an inducible mammalian expression system, we have shown that 16E1 wedge E4 arrests HeLa cervical epithelial cells in G(2). 16E1 wedge E4 also caused a G(2) arrest in SiHa, Saos-2 and Saccharomyces pombe cells and, as with HeLa cells, was found in the cytoplasm. However, whereas 16E1 wedge E4 is found on the keratin networks in HeLa and SiHa cells, in Saos-2 and S. pombe cells that lack keratins, 16E1 wedge E4 had a punctate distribution. Mutagenesis studies revealed a proline-rich region between amino acids 17 and 45 of 16E1 wedge E4 to be important for arrest. This region, which we have termed the "arrest domain," contains a putative nuclear localization signal, a cyclin-binding motif, and a single cyclin-dependent kinase (Cdk) phosphorylation site. A single point mutation in the putative Cdk phosphorylation site (T23A) abolished 16E1 wedge E4-mediated G(2) arrest. Arrest did not involve proteins regulating the phosphorylation state of Cdc2 and does not appear to involve the activation of the DNA damage or incomplete replication checkpoint. G(2) arrest was also mediated by the E1 wedge E4 protein of HPV11, a low-risk mucosal HPV type that also causes cervical lesions. The E1 wedge E4 protein of HPV1, which is more distantly related to that of HPV16, did not cause G(2) arrest. We conclude that, like other papillomavirus proteins, 16E1 wedge E4 affects cell cycle progression and that it targets a conserved component of the cell cycle machinery.