Enhanced detection of human immunodeficiency virus type 1-specific T-cell responses to highly variable regions by using peptides based on autologous virus sequences

The antigenic diversity of human immunodeficiency virus type 1 (HIV-1) represents a significant challenge for vaccine design as well as the comprehensive assessment of HIV-1-specific immune responses in infected persons. In this study we assessed the impact of antigen variability on the characterization of HIV-1-specific T-cell responses by using an HIV-1 database to determine the sequence variability at each position in all expressed HIV-1 proteins and a comprehensive data set of CD8 T-cell responses to a reference strain of HIV-1 in infected persons. Gamma interferon Elispot analysis of HIV-1 clade B-specific T-cell responses to 504 overlapping peptides spanning the entire expressed HIV-1 genome derived from 57 infected subjects demonstrated that the average amino acid variability within a peptide (entropy) was inversely correlated to the measured frequency at which the peptide was recognized (P = 6 x 10(-7)). Subsequent studies in six persons to assess T-cell responses against p24 Gag, Tat, and Vpr peptides based on autologous virus sequences demonstrated that 29% (12 of 42) of targeted peptides were only detected with peptides representing the autologous virus strain compared to the HIV-1 clade B consensus sequence. The use of autologous peptides also allowed the detection of significantly stronger HIV-1-specific T-cell responses in the more variable regulatory and accessory HIV-1 proteins Tat and Vpr (P = 0.007). Taken together, these data indicate that accurate assessment of T-cell responses directed against the more variable regulatory and accessory HIV-1 proteins requires reagents based on autologous virus sequences. They also demonstrate that CD8 T-cell responses to the variable HIV-1 proteins are more common than previously reported.     

Subunit interaction during catalysis. ATP modulation of catalytic steps in the succinyl-CoA synthetase reaction

A new approach for assessing of catalytic cooperativity may occur between subunits has been applied to succinyl-CoA synthetase. This is based on the extent of oxygen exchange between medium [18O]Pi and succinate per molecule of ATP cleaved during steady state succinyl-CoA synthesis. Suitable traps are used to remove succinyl-CoA and ADP as soon as they are released to the medium. With the Escherichia coli enzyme, which has an alpha 2 beta 2 structure, a pronounced increase in oxygen exchange per ATP cleaved occurs as ATP concentration is lowered. In contrast, when the CoA concentration is varied, the oxygen exchange per molecule of product formed remains constant. Also, with the pig heart enzyme, which is shown to retain its alpha beta structure during catalysis and thus has only one catalytic site, no modulation of oxygen exchange by ATP concentration is observed. These experimental findings show that the binding of an ATP either promotes the dissociation of bound succinyl-CoA or decreases its participation in exchange. Measurement of the distribution of [18O]Pi species found as exchange occurs shows that only one catalytic sequence is involved in exchange at various ATP concentrations. These observations along with other controls and results eliminate most other explanations of the ATP modulation of the exchange and suggest that binding of ATP at one catalytic site promotes catalytic site promotes catalytic events at an alternate catalytic site.     

Effect of Tetanus Toxin on Transmitter Release from the Substantia Nigra and Striatum In Vitro

Abstract: The effect of tetanus toxin on the uptake and release of radiolabelled transmitters from slices prepared from substantia nigra (SN) and striatum of rats has been investigated. Tetanus toxin-500–750 mouse lethal doses (MLD)-injected into the SN 6 h before preparing the slices significantly reduced the calcium-dependent, potassium-evoked release of [³H]GABA. Endogenous GABA levels in the SN and [³H]GABA uptake by nigral slices were unaffected by pretreatment with the toxin. Injections of tetanus toxin (1000–2000 MLD) into the striatum significantly reduced the calcium-dependent, potassium-evoked release of [¹⁴C]GABA and also [³H]dopamine, but had no effect on the K⁺-evoked release of [³H]5-hydroxytryptamine or [¹⁴C]acetylcholine. It is concluded that tetanus toxin inhibits GABA release directly and not by interference with synthesis or inactivation processes.     

Complement-dependent induction of DNA synthesis and proliferation of human diploid fibroblasts

The effects of fresh human serum (FHS) and heat-inactivated human serum (HHS) on the DNA synthesis and proliferation of human diploid fibroblasts were assessed. FHS activated significantly more quiescent fibroblasts to undergo DNA synthesis and proliferation than did HHS. The stimulatory effect occurred consistently over a serum concentration range of 0.1–10%. Using bromodeoxyuridine selective killing techniques, it was shown that this FHS stimulatory effect was on a specific subpopulation of fibroblasts unresponsive to HHS. The involvement of the complement system, and specifically of C1, was shown by the inability of Clq-depleted FHS to support enhanced DNA synthesis whereas Clq-depleted serum reconstituted with purified Clq was effective. Purified Clq did not restore activity when added to heated serum, nor was it mitogenic when tested in basal medium without serum. The addition of purified Clq to fresh serum inhibited the enhancement of DNA synthesis, and at Clq concentrations of 4γ/ml and greater, the fresh serum effects were abrogated. Thus, it appears that binding of the assembled C1 complex to the fibroblast surface was required for FHS-mediated enhancement of fibroblast proliferation, with Clq subcomponent serving as the recognition site. The results from several experiments indicated that antibody was not required for the complement-dependent fibroblast activation. FHS was not cytotoxic, and autologous serum was as effective as allogeneic sera. A 20-fold molar excess of Fab' from pooled human IgG did not alter the FHS effects. FHS from which IgG was more than 99% depleted was still effective. These results suggested an antibody-independent role for complement in the activation of a subpopulation of human diploid fibroblasts.     

Monte Carlo study of the effect of β2-microglobulin on the binding cleft of the HLA-A2 complex

Peptide recognition by class I products of the major histocompatibility complex requires association of the class I heavy chain with β₂-microglobulin. We present results of Monte Carlo simulations of the β-pleated sheet floor of the human class I MHC molecule, HLA-A2, with and without β₂-microglobulin. We find a significant effect of β₂-microglobulin on the side chains of residues near a region that would accommodate the C-terminus of a bound peptide. By modeling simultaneously each loop and its neighboring strand at either end of the class I cleft, we find that β₂-microglobulin restricts the conformational space of residues that are central to binding peptides. The effect is most pronounced for R97 and H114 and somewhat less important for Y99 and Y116, the latter forming strong hydrogen bonds with neighboring residues in the heavy chain itself.