Virion capture assays, in which immobilized antibodies (Abs) capture virus particles, have been used to suggest that nonneutralizing Abs bind effectively to human immunodeficiency virus type 1 (HIV-1) primary viruses. Here, we show that virion capture assays, under conditions commonly reported in the literature, give a poor indication of epitope expression on the surface of infectious primary HIV-1. First, estimation of primary HIV-1 capture by p24 measurements shows a very poor correlation with an estimation based on infectivity measurements. Second, virion capture appears to require relatively low Ab affinity for the virion, as shown by the ability of a monoclonal Ab to capture a wild-type and a neutralization escape variant virus equally well. Nevertheless, in a more interpretable competition format, it is shown that nonneutralizing anti-CD4 binding site (CD4bs) Abs compete with a neutralizing anti-CD4bs Ab (b12) for virus capture, suggesting that the nonneutralizing anti-CD4bs Abs are able to bind to the envelope species that is involved in virion capture in these experiments. However, the nonneutralizing anti-CD4bs Abs do not inhibit neutralization by b12 even at considerable excess. This suggests that the nonneutralizing Abs are unable to bind effectively to the envelope species required for virus infectivity. The results were obtained for three different primary virus envelopes. The explanation that we favor is that infectious HIV-1 primary virions can express two forms of gp120, an accessible nonfunctional form and a functional form with limited access. Binding to the nonfunctional form, which needs only to be present at relatively low density on the virion, permits capture but does not lead to neutralization.

We have reported previously that PX-478 (S-2-amino-3-[4 ¶-N,N,-bis(chloroethyl)amino]phenyl propionic acid N-oxide dihydrochloride) has potent antitumor activity against a variety of human tumor xenografts associated with the levels of the hypoxia-inducible factor-1A (HIF-1A) within the tumor. We now report that PX-478 inhibits HIF-1A protein levels and transactivation in a variety of cancer cell lines. Hypoxia-induced vascular endothelial growth factor formation was inhibited by PX-478, whereas baseline levels of vascular endothelial growth factor in normoxia were unaffected. Studies of the mechanism of PX-478 action showed that HIF-1A inhibition occurs in both normoxia and hypoxia and does not require pVHL or p53. In addition, PX-478 decreases levels of HIF-1A mRNA and inhibits translation as determined by 35 S labeling experiments and reporter assays using the 5 ¶ untranslated region of HIF-1A. Moreover, to a lesser extent, PX-478 also inhibits HIF-1A deubiquitination resulting in increased levels of polyubiquitinated HIF-1A. The inhibitory effect of PX-478 on HIF-1A levels is primarily due to its inhibition of translation because HIF-1A translation continues in hypoxia when translation of most proteins is decreased. We conclude that PX-478 inhibits HIF-1A at multiple levels that together or individually may contribute to its antitumor activity against HIF-1A-expressing tumors. [Mol Cancer Ther 2008;7(1):90 -100]

Dendritic cells (DCs) are a heterogeneous population of cells that are specialized for Ag processing and presentation. These cells are believed to derive from both myeloid- and lymphoid-committed precursors. Normal human PBMC-derived, human CD14+ cell (monocyte)-derived, and mouse hematopoietic progenitor-derived DCs were shown to express the hematopoietic cell-restricted, ets family transcription factor PU.1. These populations represent myeloid progenitor-derived DCs. Hematopoietic progenitor cells from PU.1 gene-disrupted (null) mice were unable to generate MHC class IIhigh, CD11c+ myeloid-derived DCs in vitro. Mouse thymic DCs are proposed to be derived from a committed lymphoid progenitor cell that can give rise to T cells as well as DCs. Previously, we showed that CD4 and CD8 T cells developed in PU.1 null mice in a delayed manner and in reduced number. We examined the thymus of 10- to 12-day-old PU.1 null mice and found no evidence of DEC-205+, MIDC-8+ DCs in this tissue. Our findings indicate that PU.1 regulates the development of both thymic and myeloid progenitor-derived populations of DCs, and expand its known role in hematopoietic development.

A major challenge in protein design is to create stable scaffolds into which tailored functions can be introduced. Here we present the design, synthesis and characterization of a 61-residue all-beta protein: the minibody. We used a portion of the heavy chain variable domain of an immunoglobulin as a template, obtaining a molecule with a novel beta-sheet scaffold and two regions corresponding to the hypervariable loops H1 and H2. To exploit the potential for creating functional centres in the minibody, we engineered a metal-binding site into it. This site is formed by one histidine in H1 and two in H2. The protein is folded, compact and able to bind metal, thus representing the first designed beta-protein with a novel fold and a tailored function. By randomizing the sequence of the hypervariable loops, we are using the minibody scaffold to construct a conformationally constrained peptide library displayed on phage.

A strategy that combines limited proteolysis experiments and mass spectrometric analysis of the fragments generated has been developed to probe protease-accessible sites on the protein surface. This integrated approach has been employed to investigate the tertiary structure of the Minibody, a de novo designed 64-residue protein consisting of a P-sheet scaffold based on the heavy-chain variable-domain structure of a mouse immunoglobulin and containing two segments corresponding to the hypervariable HI and H2 regions. The low solubility of the protein prevented a detailed characterization by NMR and/or X-ray. Different proteases were used under strictly controlled conditions and the cleavage sites were mapped onto the anticipated Minibody model, leading to the identification of the most exposed regions. A single-residue mutant was constructed and characterized, following the same procedure, showing a slightly higher correspondence with the predicted model. This strategy can be used to effectively supplement NMR and X-ray investigations of protein tertiary structure, where these procedures cannot provide definitive data, or to verify and refine protein models.