Experimental transmission of bovine spongiform encephalopathy (BSE) to cynomolgus monkeys (Macaca fascicularis) is an animal model for variant Creutzfeldt–Jakob disease (vCJD). The presence of 14-3-3 proteins in cerebrospinal fluid (CSF) samples indicates neuronal destruction and is therefore used as a clinical biomarker. However, time-course studies using 14-3-3 proteins have not been performed until now in simian vCJD. The main goals of this study were to determine isoform patterns, to examine kinetics and to correlate the clinical course with the occurrence of this biomarker in simian vCJD. In monkeys dosed intracerebrally with BSE, the earliest clinical sign of illness was a drop in body weight that was detected months before the onset of mild neurological signs. Macaques dosed orally or intracerebrally with BSE developed neurological signs 4.3 (3.7–4.6) and 4.8 (2.9–6.0) years post-infection, respectively. 14-3-3β- and -γ-positive CSF samples were found around the time of onset of mild neurological signs, but not earlier. In contrast, 14-3-3ϵ and -η isoforms were not detectable. 14-3-3 levels increased with time and were positively correlated with the degree of neurological symptoms. Post-mortem examination of brain samples revealed a positive correlation between PrPres and 14-3-3ϵ levels. Interestingly, florid plaques characteristic of human vCJD could not be detected in diseased monkeys. It was concluded that analysis of 14-3-3 proteins in CSF is a reliable tool to characterize the time course of brain degeneration in simian vCJD. However, there are differences in the clinical course between orally and intracerebrally infected animals that may influence the detection of other biomarkers.

The cellular prion protein (PrP(C)), a highly conserved glycoprotein predominantly expressed by neuronal cells, can convert into an abnormal isoform (PrP(Sc)) and provoke a transmissible spongiform encephalopathy. In spite of many studies, the physiological function of PrP(C) remains unknown. Recent findings suggest that PrP(C) is a multifunctional protein participating in several cellular processes. Using recombinant human PrP as a probe, we performed far-Western immunoblotting (protein overlay assay) to detect cellular PrP(C) interactors. Brain extracts of wild-type and PrP knockout mice were screened by far-Western immunoblotting for PrP-specific interactions. Subsequently, putative ligands were isolated by 2-DE and identified by MALDI-TOF MS, enabling identification of heterogeneous nuclear ribonucleoprotein A2/B1 and aldolase C as novel interaction partners of PrP(C). These data provide the first evidence of a molecule indicating a mechanism for the predicted involvement of PrP(C) in nucleic acid metabolisms. In summary, we have shown the successful combination of 2-DE with far-Western immunoblotting and MALDI-TOF MS for identification of new cellular binding partners of a known protein. Especially the application of this technique to investigate other neurodegenerative diseases is promising.

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Infectious molecular clones of the human immunodeficiency virus type 2 (HIV-2) will be valuable tools for the study of regulatory gene functions and the development of an animal model for the human acquired immunodeficiency syndrome (AIDS). To this end, we have cloned and sequenced a novel HIV-2 isolate, HIV-2BEN. One clone, designated MK6, is infectious for various human T-cell lines and for human and macaque peripheral blood lymphocytes (PBL), allowing molecular studies of HIV-2 infection and replication. Since MK6 is highly cytopathic in MT-2 and Molt-4 clone 8 cells, antiviral agents and neutralizing sera may be tested. Cluster analysis of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) env and gag genes revealed that HIV-2BEN yielded the earliest node of phylogenetic divergence for all reported HIV-2 sequences. Noise analysis showed that, with the current data, no specification of any branching order can be made among the four groups of primate lentiviruses, HIV-1, HIV-2/SIVSMM/MAC, SIVAGM, and SIVMND.

The conformational conversion of the normal cellular prion protein (PrPC) into the pathology-associated PrPSc isoform is a key event in TSEs (transmissible spongiform encephalopathies). The host PrPC molecule contains two N-linked glycosylation sites and binds copper under physiological conditions. In contrast with PrPC, PrPSc is insoluble in non-ionic detergents and does not bind to Cu2+ ions. Hence, we utilized copper binding to separate and characterize both PrP isoforms. Infected and uninfected murine brain and bovine stem brain specimens were treated with the mild non-ionic detergent n-octyl-beta-D-glucopyranoside (octylglucoside) to maintain the native PrP conformations during isolation. The solubilized homogenates were loaded on to Cu2+-saturated IMAC (immobilized metal affinity chromatography) columns and eluted using the chelating agent EDTA. Fractions were separated by SDS/PAGE and analysed by immunoblotting using anti-PrP monoclonal antibodies for glycosylation profiling. Whereas native PrPC and denatured PrPSc were retained by a Cu2+-loaded resin, native PrPSc and PrPres [PK (proteinase K)-resistant PrP] passed through the column. We demonstrate here that the IMAC technique is appropriate to isolate and partially purify PrPC from healthy brains in its native-like and biologically relevant glycosylated copper-binding forms. The IMAC technique is also well suited for the separation of native PrPC from aggregated PrPSc in infected brains. Our results indicate that in contrast with PrPSc in uninfected as well as infected brains, PrPC is predominantly present in the glycosylated forms.