RESUMO
Enhancing the immunogenicity of an antitumour vaccine still poses a major challenge. It depends upon the selected antigen and the mode of its presentation. We here describe a fully synthetic antitumour vaccine, which addresses both aspects. For the antigen, a tumour-associated MUC1 glycopeptide as B-cell epitope was synthesised and linked to the immunostimulating T-cell epitope P2 derived from tetanus toxoid. The MUC1-P2 conjugate is presented multivalently on a hyperbranched polyglycerol to the immune system. In comparison to a related vaccine of lower multivalency, this vaccine exposing more antigen structures on the hyperbranched polymer induced significantly stronger immune responses in mice and elicited IgG antibodies of distinctly higher affinity to epithelial tumour cells.
Assuntos
Vacinas Anticâncer/imunologia , Glicerol/imunologia , Glicopeptídeos/imunologia , Mucina-1/imunologia , Animais , Vacinas Anticâncer/química , Vacinas Anticâncer/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Glicerol/química , Glicopeptídeos/química , Glicopeptídeos/genética , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Mucina-1/química , Mucina-1/genética , Polímeros/químicaRESUMO
We isolated and sequenced the two major tRNA(Gln) isoacceptors with CUG and UmUG anticodons from the cytoplasm of Nicotiana rustica. These are the first tRNAs(Gln) of nuclear origin characterized in plants. The tRNA(Gln) sequences were used to design probes for the isolation of the corresponding genes from a nuclear DNA library of N. rustica. The two cloned Nicotiana tRNA(Gln) genes, coding for either of the two isoacceptors, are efficiently transcribed in HeLa cell nuclear extract. In vitro translation in the presence of purified Nicotiana tRNAs(Gln) was carried out in a wheat germ extract partially depleted of endogenous tRNAs. Cytoplasmic (cyt) tRNA(Gln)CUG and to a lesser extent cyt tRNA(Gln)UmUG stimulated readthrough over the UAG stop codon present in the tobacco mosaic virus-specific context. The two tRNA(Gln) isoacceptors are the second class of natural UAG suppressors identified in plants, in addition to cyt tRNA(Tyr)GpsiA which has previously been characterized as the first natural UAG suppressor.
Assuntos
Anticódon , Genes de Plantas/genética , Nicotiana/genética , Plantas Tóxicas , RNA de Transferência de Glutamina/genética , Sequência de Bases , Núcleo Celular/química , Núcleo Celular/metabolismo , Códon , Citoplasma/química , Citoplasma/metabolismo , Células HeLa , Humanos , Dados de Sequência Molecular , RNA de Transferência de Glutamina/isolamento & purificação , Nicotiana/química , Transcrição Gênica , Zeína/genética , Zeína/metabolismoRESUMO
Three glutamine tRNA isoacceptors are known in Tetrahymena thermophila. One of these has the anticodon UmUG which reads the two normal glutamine codons CAA and CAG, whereas the two others with CUA and UmUA anticodons recognize UAG and UAA, respectively, which serve as termination codons in other organisms. We have employed these tRNA(Gln)-isoacceptors as tools for studying unconventional base interactions in a mRNA- and tRNA-dependent wheat germ extract. We demonstrate here (i) that tRNA(Gln)UmUG suppresses the UAA as well as the UAG stop codon, involving a single G:U wobble pair at the third anticodon position and two simultaneous wobble base pairings at the first and third position, respectively, and (ii) that tRNA(Gln)CUA, in addition to its cognate codon UAG, reads the UAA stop codon which necessitates a C:A mispairing in the first anticodon position. These unorthodox base interactions take place in a codon context which favours readthrough in tobacco mosaic virus (TMV) or tobacco rattle virus (TRV) RNA, but are not observed in a context that terminates zein and globin protein synthesis. Furthermore, our data reveal that wobble or mispairing in the middle position of anticodon-codon interactions is precluded in either context. The suppressor activities of tRNAs(Gln) are compared with those of other known naturally occurring suppressor tRNAs, i.e., tRNA(Tyr)G psi A and tRNA(Trp)CmCA. Our results indicate that a 'leaky' context is neither restricted to a single stop codon nor to a distinct tRNA species.
Assuntos
Anticódon , Códon , Globinas/biossíntese , RNA de Transferência de Glutamina/metabolismo , Tetrahymena/genética , Zeína/biossíntese , Aminoacil-tRNA Sintetases/metabolismo , Animais , Composição de Bases , Sequência de Bases , DNA de Protozoário , Glutamina/metabolismo , Dados de Sequência Molecular , RNA de Protozoário/isolamento & purificação , RNA de Protozoário/metabolismo , RNA de Transferência de Glutamina/isolamento & purificação , Regiões Terminadoras GenéticasRESUMO
Fifty TMR adults participated in a three-stage job-training and placement program over a 5-year period. Vocational inventoried interest and expressed interest were correlated, as were relationships between inventoried interest and such variables as CA and IQ. The results showed that (a) TMR adults can discriminate between occupational likes and dislikes when using pictorial inventoried interest assessment techniques, (b) a positive relationship exists between expressed and inventoried interest, (c) inventoried interest is independent of CA and IQ, (d) there was a general consistency in inventoried occupational choice over a 6-month interval, and (e) the Reading-Free Vocational Interest Inventory has predictive and status validity, as evidenced by job incumbents' success and their inventory cluster profiles.
