Natural and experimental oral infection of nonhuman primates by bovine spongiform encephalopathy agents

Experimental lemurs either were infected orally with the agent of bovine spongiform encephalopathy (BSE) or were maintained as uninfected control animals. Immunohistochemical examination for proteinase-resistant protein (prion protein or PrP) was performed on tissues from two infected but still asymptomatic lemurs, killed 5 months after infection, and from three uninfected control lemurs. Control tissues showed no staining, whereas PrP was detected in the infected animals in tonsil, gastrointestinal tract and associated lymphatic tissues, and spleen. In addition, PrP was detected in ventral and dorsal roots of the cervical spinal cord, and within the spinal cord PrP could be traced in nerve tracts as far as the cerebral cortex. Similar patterns of PrP immunoreactivity were seen in two symptomatic and 18 apparently healthy lemurs in three different French primate centers, all of which had been fed diets supplemented with a beef protein product manufactured by a British company that has since ceased to include beef in its veterinary nutritional products. This study of BSE-infected lemurs early in their incubation period extends previous pathogenesis studies of the distribution of infectivity and PrP in natural and experimental scrapie. The similarity of neuropathology and PrP immunostaining patterns in experimentally infected animals to those observed in both symptomatic and asymptomatic animals in primate centers suggests that BSE contamination of zoo animals may have been more widespread than is generally appreciated.

Cloning and expression of a cDNA encoding a bovine brain brefeldin A-sensitive guanine nucleotide-exchange protein for ADP-ribosylation factor

A 200-kDa guanine nucleotide-exchange protein (p200 or GEP) for ADP-ribosylation factors 1 and 3 (ARF1 and ARF3) that was inhibited by brefeldin A (BFA) was purified earlier from cytosol of bovine brain cortex. Amino acid sequences of four tryptic peptides were 47% identical to that of Sec7 from Saccharomyces cerevisiae, which is involved in vesicular trafficking in the Golgi. By using a PCR-based procedure with two degenerate primers representing sequences of these peptides, a product similar in size to Sec7 that contained the peptide sequences was generated. Two oligonucleotides based on this product were used to screen a bovine brain library, which yielded one clone that was a partial cDNA for p200. The remainder of the cDNA was obtained by 5′ and 3′ rapid amplification of cDNA ends (RACE). The ORF of the cDNA encodes a protein of 1,849 amino acids (≈208 kDa) that is 33% identical to yeast Sec7 and 50% identical in the Sec7 domain region. On Northern blot analysis of bovine tissues, a ≈7.4-kb mRNA was identified that hybridized with a p200 probe; it was abundant in kidney, somewhat less abundant in lung, spleen, and brain, and still less abundant in heart. A six-His-tagged fusion protein synthesized in baculovirus-infected Sf9 cells demonstrated BFA-inhibited GEP activity, confirming that BFA sensitivity is an intrinsic property of this ARF GEP and not conferred by another protein component of the complex from which p200 was originally purified.

Purification and characterization of a guanine nucleotide-exchange protein for ADP-ribosylation factor from spleen cytosol.

ADP-ribosylation factors (ARFs) are 20-kDa guanine nucleotide-binding proteins and are active in the GTP-bound state and inactive with GDP bound. ARF-GTP has a critical role in vesicular transport in several cellular compartments. Conversion of ARF-GDP to ARF-GTP is promoted by a guanine nucleotide-exchange protein (GEP). We earlier reported the isolation from bovine brain cytosol of a 700-kDa protein complex containing GEP activity that was inhibited by brefeldin A (BFA). Partial purification yielded an approximately 60-kDa BFA-insensitive GEP that enhanced binding of ARF1 and ARF3 to Golgi membranes. GEP has now been purified extensively from rat spleen cytosol in a BFA-insensitive, approximately 55-kDa form. It activated class I ARFs (ARFs 1 and 3) that were N-terminally myristoylated, but not nonmyristoylated ARFs from class-I, II, or III. GEP activity required MgCl2. In the presence of 0.6-0.8 mM MgCl2 and 1 mM EDTA, binding of guanosine 5'-[gamma[35S]thio]triphosphate ([35S]GTP gamma S) by ARF1 and ARF3 was equally high without and with GEP. At higher Mg2+ concentrations, binding without GEP was much lower; with 2-5 mM MgCl2, GEP-stimulated binding was maximal. The rate of GDP binding was much less than that of GTP gamma S with and without GEP. Phospholipids were necessary for GEP activity; phosphatidylinositol was more effective than phosphatidylserine, and phosphatidic acid was less so. Other phospholipids tested were ineffective. Maximal effects required approximately 200 microM phospholipid, with half-maximal activation at 15-20 microM. Release of bound [35S]GTP gamma S from ARF3 required the presence of both GEP and unlabeled GTP or GTP gamma S; GDP was much less effective. This characterization of the striking effects of Mg2+ concentration and specific phospholipids on the purified BFA-insensitive ARF GEP should facilitate experiments to define its function in vesicular transport.