Enhancement of epitope-specific cellular immune responses by immunization with HIV-1 peptides genetically conjugated to the B-subunit of recombinant cholera toxin.

As more HIV-1 infected patients receive anti-retroviral drug treatment, the occurrence of drug-resistant variants of the virus is increasing. We have previously shown that mutated HIV peptide sequences represent mutations induced by antiretroviral drugs are equally good and often better immunogens than wild type peptides. The non-toxic B subunit of cholera toxin (CTB) is an active substance in the oral cholera vaccine, and has been shown to bind ganglioside receptors and activate mucosal cells. By fusing mutant epitopes deriving from HIV-1 enzymes with the B subunit of cholera toxin, we aim is to induce cellular responses against virus harboring drug-induced mutations. We successfully created conjugates of HIV peptide sequences fused to rCTB. The immune response against the different peptides was strongly enhanced by the fusion to the toxin. Moreover, immunization with sequence containing drug-induced mutation triggered a cross-reactive immune response against the wild type epitope. Long-term follow-up of immunized animals revealed a persistence of cellular immune response for over 4 months, which could readily be boosted with an additional late immunization. By linking HIV-peptides to the B subunit of cholera toxin it is thus possible to stimulate a strong and long-lasting immune response, significantly better than that evoked by the peptide alone.

Temperature effects on the catalytic efficiency, rate enhancement, and transition state affinity of cytidine deaminase, and the thermodynamic consequences for catalysis of removing a substrate "anchor".

To obtain a clearer understanding of the forces involved in transition state stabilization by Escherichia coli cytidine deaminase, we investigated the thermodynamic changes that accompany substrate binding in the ground state and transition state for substrate hydrolysis. Viscosity studies indicate that the action of cytidine deaminase is not diffusion-limited. Thus, K(m) appears to be a true dissociation constant, and k(cat) describes the chemical reaction of the ES complex, not product release. Enzyme-substrate association is accompanied by a loss of entropy and a somewhat greater release of enthalpy. As the ES complex proceeds to the transition state (ES), there is little further change in entropy, but heat is taken up that almost matches the heat that was released with ES formation. As a result, k(cat)/K(m) (describing the overall conversion of the free substrate to ES is almost invariant with changing temperature. The free energy barrier for the enzyme-catalyzed reaction (k(cat)/K(m)) is much lower than that for the spontaneous reaction (k(non)) (DeltaDeltaG = -21.8 kcal/mol at 25 degrees C). This difference, which also describes the virtual binding affinity of the enzyme for the activated substrate in the transition state (S), is almost entirely enthalpic in origin (DeltaDeltaH = -20.2 kcal/mol), compatible with the formation of hydrogen bonds that stabilize the ES complex. Thus, the transition state affinity of cytidine deaminase increases rapidly with decreasing temperature. When a hydrogen bond between Glu-91 and the 3'-hydroxyl moiety of cytidine is disrupted by truncation of either group, k(cat)/K(m) and transition state affinity are each reduced by a factor of 10(4). This effect of mutation is entirely enthalpic in origin (DeltaDeltaH approximately 7.9 kcal/mol), somewhat offset by a favorable change in the entropy of transition state binding. This increase in entropy is attributed to a loss of constraints on the relative motions of the activated substrate within the ES complex. In an Appendix, some objections to the conventional scheme for transition state binding are discussed.

Self-rated imagery and encoding strategies in visual memory.

The value of self-rated vividness of imagery in predicting performance was investigated, taking into account the mnemonic strategies utilized among subjects performing a visual-memory task. Subjects classified as 'good' or 'poor' imagers, according to their scores in the Vividness of Visual Imagery Questionnaire (VVIQ; Marks, 1972), were to detect as rapidly as possible differences between pairs of similar pictures presented consecutively. No coding instructions were given and the mnemonic strategies used were analysed by studying subjective reports and objective performance measurements. The results indicated that the subjects utilized two main strategies--a detail or an image strategy. The detail strategy was the more efficient. In accordance with a previous study (Berger & Gaunitz, 1977), it was found that the VVIQ did not discriminate between performance by 'good' and 'poor' imagers. However, among subjects who used the image strategy, 'good' imagers performed more rapidly than 'poor' imagers. Self-rated imagery may then have some value in predicting performance among individuals shown to have utilized an image strategy.

Self-rated imagery and vividness of task pictures in relation to visual memory.

Marks (1973) and Gur & Hilgard (1975) have reported success in predicting performance in visual-memory tasks from scores in a questionnaire of self-rated vividness of imagery, i.e. the Vividness of Visual Imagery Questionnaire (VVIQ; Marks, 1973). The results obtained were attributed to the use of the VVIQ and pictures with a high degree of vividness in the memory task. These findings were disconfirmed in two experiments in which the VVIQ was used and vivid pictures were presented in the memory tasks. Subjects were to judge whether pairs of similar, successively presented pictures were identical or not. The results indicated that subjects rated as 'good' imagers did not perform differently from those rated as 'poor' imagers. The influences of demand characteristics in the previous experiments and differences in experimental procedures were referred to as possible causes of the observed inconsistencies in results. It is also suggested that questionnaires of self-rated visual imagery are ineffective as predictors of performance, since they only cover a limited aspect of imagery.