No abstract

The fidelity of protein synthesis is substantially greater than the specificity of codon-anticodon recognition that would be expected from'the known energetics of base-pairing in solution. To test the suggestion that the specificity of recQgnition may be increased by "kinetic proofreading" associated with GTP hydrolysis UJ. 1. Hopfield (1974) Proc. Nat]. Acad. Sci.USA 71, we have studied the interaction of ternary comjiexes of polypeptide elongation factor Tu, aminoacyltRNA, and GTP with poly(U).programed ribosomes. With most noncognate ternary complexes, including two that pair correctly with the 5' and 3' bases of UUU, rejection occurred without GTP hydrolysis, presumably by the reverse of the initial binding reaction. However, with complexes containing Leu-or Ile-tRNAs, which may pair correctly with the 3' and middle bases, GTP hydrolysis was stimulated though the aa-tRNA was not retained on the ribosome. These results demonstrate the existence of a GTP-dependent proofreading step in aminoacyl-tRNA recognition on ribosomes. They also sugest that the 5' base of the codon is more prone than the middle base to errors that can be corrected by proofreading. The fidelity of translation of mRNA on the ribosome requires that a trinucleotide codon specify the binding of an aminoacyl-tRNA (aa-tRNA) with a complementary anticodon but the rejection of aa-tRNAs with only two, or fewer, complementary bases in their anticodons. Some estimate of the potential specificity of base pairing, and therefore of the pairing of complementary trinucleotides, can be gained from investigations of the stability of double helices formed by synthetic oligonucleotides. These investigations have shown that, of all the possible standard pairs, that between G and U comes closest in binding energy to the standard pairs. The free energy of formation of a G-U pair differs from that of the correct A-U and G-C pairs by only 2-3 keal/mol (1 cal = 4.18 J) (1). The specificity of U for A and of G for C is therefore far from being'absolute: the minimum frequency of G-U errors in a simple trinucleotide double helix will be 1-2%, i.e., greater than or equal to exp-(AG/RT), where AG is the free energy difference between G-U and a standard base pair.With the exception of the 3' base of the codon, where "wobble" permits G-U to be equivalent to a standard base pair in the genetic code (2), the pairing of U with G will almost always result in the'binding of the wrong aa-tRNA. Hence if the specificity of codon-anticodon pairing on the ribosome were derived entirely from the specificity of formation of a threenucleotide segment of simple double helix, over half the codons would be translated with an accuracy of less than 99%. This is clearly below the actual fidelity of translation required for the reliable formation of a specific chain of several hundred amino acid residues. The ribosome, and its associated factors, must therefore increase the specificity of codon-anticodon pairing through improving the accuracy of base-pairing over that provided by ...

Cervical Cancer is the second leading cause of cancer–related deaths in women worldwide and is associated with Human Papillomavirus (HPV) infection, creating a unique opportunity to treat cervical cancer through anti-viral vaccination. Although a prophylactic vaccine may be available within a year, millions of women, already infected, will continue to suffer from HPV-related disease, emphasizing the need to develop therapeutic vaccination strategies. A majority of clinical trials examining therapeutic vaccination have shown limited efficacy due to examining patients with more advanced-stage cancer who tend to have decreased immune function. Current trends in clinical trials with therapeutic agents examine patients with pre-invasive lesions in order to prevent invasive cervical cancer. However, longer follow-up is necessary to correlate immune responses to lesion regression. Meanwhile, preclinical studies in this field include further exploration of peptide or protein vaccination, and the delivery of HPV antigens in DNA-based vaccines or in viral vectors. As long as pre-clinical studies continue to advance, the prospect of therapeutic vaccination to treat existing lesions seem good in the near future. Positive consequences of therapeutic vaccination would include less disfiguring treatment options and fewer instances of recurrent or progressive lesions leading to a reduction in cervical cancer incidence.

The recent finding that Th17 infiltration of ovarian tumors positively predicts patient outcomes suggests that Th17 responses play a protective role in ovarian tumor immunity. This observation has led to the question of whether Th17 cells could be induced or expanded to therapeutic advantage by tumor vaccination. In this study, we show that treatment of ovarian tumor antigen-loaded, cytokine-matured human dendritic cells (DC) with a combination of IL-15 and a p38 MAP kinase inhibitor offers potent synergy in antagonism of CD4+ Treg induction and redirection toward CD4+ Th17 responses that correlate with strong CD8+ cytotoxic T lymphocyte (CTL) activation. Ovarian tumor antigen-specific CD4+ T cells secrete high levels of IL-17 and show reduced expression of CTLA-4, PD-1, and Foxp3 following activation with IL-15/p38 inhibitor-treated DC. We further show that modulation of p38 MAPK signaling in DC is associated with reduced expression of B7-H1 (PD-L1), loss of indoleamine 2,3-dioxygenase activity, and increased phosphorylation of ERK 1/2 MAPK. These observations may allow the development of innovative DC vaccination strategies to boost Th17 immunity in ovarian cancer patients.