Prion protein (PrP)^Sc, the only known component of the prion, is present mostly in the brains of animals and humans affected with prion diseases. We now show that a protease-resistant PrP isoform can also be detected in the urine of hamsters, cattle, and humans suffering from transmissible spongiform encephalopathies. Most important, this PrP isoform (UPrP^Sc) was also found in the urine of hamsters inoculated with prions long before the appearance of clinical signs. Interestingly, intracerebrally inoculation of hamsters with UPrP^Sc did not cause clinical signs of prion disease even after 270 days, suggesting it differs in its pathogenic properties from brain PrP^Sc. We propose that the detection of UPrP^Sc can be used to diagnose humans and animals incubating prion diseases, as well as to increase our understanding on the metabolism of PrP^Sc in vivo.

Virtually all human granular lymphocytes expressed the HNK-1 differentiation antigen when examined in lymphoid compartments from adults, neonates, and fetuses. The HNK-1+ cells were distinguishable into three subsets having distinct antigenic phenotypes: HNK+T3-M1-, HNK+T3+M1-, and HNK+T3-M1+. Thus, greater than 70% of the HNK-1+ cells from 13-17 wk fetuses (less than 0.2% of nucleated cells) lacked T cell antigens (e.g., T3, T8, T4, and T6) and the M1 myeloid antigen. Morphologically, the HNK+T3-M1- cells consisted of three different types: small granular lymphocytes (less than 10% of HNK-1+ cells), agranular small lymphocytes with a narrow rim of cytoplasm (70-80%), and agranular giant cells (greater than 15 micrometers) with considerable neutrophilic cytoplasm (15%). The purified fetal HNK-1+ cells exhibited a low level of cytotoxicity against K562 target cells. On the other hand, almost all of HNK-1+ cells in neonatal tissues as well as adult bone marrow, lymph node, and thymus, exhibited the HNK+T3+M1- phenotype, contained sparse cytoplasmic granules, and had an intermediate level of NK functional activity. Only adult blood and spleen contained a majority of mature HNK-1+ cells. These cells had an HNK+T3-M1- phenotype, abundant cytoplasmic granules, and maximum NK function. We propose that human NK cells may generate from a separate cell lineage and that they alter their phenotype, morphology, and functional capability during differentiation.

Pharmacological inhibition of Hsp90 is an exciting option for cancer therapy. The clinical efficacy of Hsp90 inhibitors is, however, less than expected. Binding of the co-chaperone p23 to Hsp90 and induced overexpression of anti-apoptotic proteins Hsp70 and Hsp27 are thought to contribute to this outcome. Herein, we report that the natural product gedunin may provide a new alternative to inactivate the Hsp90 machine. We show that gedunin directly binds to p23 and inactivates it, without overexpression of Hsp27 and relatively modest induction of Hsp70. Using molecular docking and mutational analysis, we mapped the gedunin-binding site on p23. Functional analysis shows that gedunin inhibits the p23 chaperoning activity, blocks its cellular interaction with Hsp90, and interferes with p23-mediated gene regulation. Cell treatment with gedunin leads to cancer cell death by apoptosis through inactivation of p23 and activation of caspase 7, which cleaves p23 at the C terminus. These results provide important insight into the molecular mechanism of action of this promising lead compound.

The human aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator protein (ARNT) were coexpressed in the yeast Saccharomyces cerevisiae to create a system for the study of this heterodimeric transcription factor. Specific transcriptional activation mediated by AHR/ARNT heterodimer, which is a functional indicator of receptor expression, was assessed by β-galactosidase activity produced from a reporter plasmid. Yeast expressing AHR and ARNT displayed constitutive transcriptional activity that was not augmented by addition of AHR agonists in strains that required exogenous tryptophan for viability. In contrast, strains with an intact pathway for tryptophan biosynthesis responded to AHR agonists and had lower levels of background β-galactosidase activity. Hexachlorobenzene, benzo(a)pyrene, and β-naphthoflavone were effective AHR agonists in the yeast system, and had EC₅₀ values of 200, 40, and 20 nm, respectively, for β-galactosidase activity induction. Tryptophan, indole, indole acetic acid, and tryptamine activated transcription in yeast coexpressing AHR and ARNT (EC₅₀ values ∼300 μm). Indole-3-carbinol was an exceptionally potent AHR agonist (EC₅₀ ∼10 μm) in yeast. This yeast system is useful for the study of AHR/ARNT protein complexes, and may be generally applicable to the investigation of other multiprotein complexes.

The pBEVY (bi-directional expression vectors for yeast) plasmids were designed with constitutive and galactose-induced bi-directional promoters to direct the expression of multiple proteins in Saccharomyces cerevisiae . Using human estrogen receptor as a test gene, relatively balanced expression levels from each side of a bi-directional promoter were observed. Expression of a functional heterodimeric transcription factor composed of human aryl hydrocarbon receptor (Ahr) and aryl hydrocarbon receptor nuclear translocator (Arnt) proteins was accomplished using a single pBEVY plasmid. Previous studies suggest that inhibitory cross-talk between the estrogen receptor and the Ahr/Arnt complex may occur and that Hsp90-Ahr complex formation is important for Ahr-mediated signal transduction. Evidence for functional interaction among these proteins was investigated using pBEVY plasmids in a yeast system. No inhibitory cross-talk was observed in signaling assays performed with yeast that co-expressed Ahr, Arnt and estrogen receptor. In contrast, Ahr/Arnt-mediated signal transduction was reduced by 80% in a temperature-sensitive Hsp90 strain grown under non-permissive conditions. We conclude that pBEVY plasmids facilitate the examination of multiple protein interactions in yeast model systems.