Michael A. Tranulis

Distribution of prion protein in the ileal Peyer's patch of scrapie-free lambs and lambs naturally and experimentally exposed to the scrapie agent

A sensitive immunohistochemical procedure was used to investigate the presence of prion protein (PrP) in the ileal Peyers patch of PrP-genotyped lambs, including scrapie-free lambs and lambs naturally and experimentally exposed to the scrapie agent. The tyramide signal amplification system was used to enhance the sensitivity of conventional immunohistochemical procedures to show that PrP was widely distributed in the enteric nervous plexus supplying the gut wall. In scrapie-free lambs, PrP was also detected in scattered cells in the lamina propria and in the dome and interfollicular areas of the Peyers patch. In the follicles, staining for PrP was mainly confined to the capsule and cells associated with vascular structures in the light central zone. In lambs naturally exposed to the scrapie agent, staining was prominent in the dome and neck region of the follicles and was also found to be associated with the follicle-associated epithelium. Similar observations were made in lambs that had received a single oral dose of scrapie-infected brain material from sheep with a homologous and heterologous PrP genotype 1 and 5 weeks previously. These studies show that the ileal Peyers patch in young sheep may be an important site of uptake of the scrapie agent and that the biology of this major gut-associated lymphoid tissue may influence the susceptibility to oral infection in sheep. Furthermore, these studies suggest that homology or heterology between PrP genotypes or the presence of PrP genotypes seldom associated with disease does not impede uptake of PrP.

Salmonid T cells assemble in the thymus, spleen and in novel interbranchial lymphoid tissue

In modern bony fishes, or teleost fish, the general lack of leucocyte markers has greatly hampered investigations of the anatomy of the immune system and its reactions involved in inflammatory responses. We have previously reported the cloning and sequencing of the salmon CD3 complex, molecules that are specifically expressed in T cells. Here, we generate and validate sera recognizing a peptide sequence of the CD3ε chain. Flow cytometry analysis revealed high numbers of CD3ε+ or T cells in the thymus, gill and intestine, whereas lower numbers were detected in the head kidney, spleen and peripheral blood leucocytes. Subsequent morphological analysis showed accumulations of T cells in the thymus and spleen and in the newly discovered gill‐located interbranchial lymphoid tissue. In the latter, the T cells are embedded in a meshwork of epithelial cells and in the spleen, they cluster in the white pulp surrounding ellipsoids. The anatomical organization of the salmonid thymic cortex and medulla seems to be composed of three layers consisting of a sub‐epithelial medulla‐like zone, an intermediate cortex‐like zone and finally another cortex‐like basal zone. Our study in the salmonid thymus reports a previously non‐described tissue organization. In the intestinal tract, abundant T cells were found embedded in the epithelium. In non‐lymphoid organs, the presence of T cells was limited. The results show that the interbranchial lymphoid tissue is quantitatively a very important site of T cell aggregation, strategically located to facilitate antigen encounter. The interbranchial lymphoid tissue has no resemblance to previously described lymphoid tissues.

A glucokinase-like-enzyme in the liver of Atlantic salmon (Salmo salar).

An enzyme with properties similar to rat liver glucokinase (Hexokinase IV or D) is present in salmon liver in addition to low-Km hexokinase(s). The specific activity of this enzyme increases about 1.6 fold, comparing activities after feeding diets with 25% and 0% digestive energy from starch. The enzyme has a low affinity for glucose, S0.5 = 25.2-26.8 mM (95% confidence interval) and a low activity with fructose, approximately 8% of the activity with glucose. Its molecular mass was estimated to 50.7 +/- 0.6 kDa (SEM. n = 3) by gel filtration, and it displays positive cooperativity with respect to glucose. The Hill constant = 1.73-1.81 (95% confidence interval). The enzyme is competitively inhibited by N-acetyl glucosamine, K(i) approximately 0.28 mM.

Influence of the prion protein gene, Prnp, on scrapie susceptibility in sheep

Natural scrapie in sheep occurs through a complex interplay between host genetic elements and various strains of the infectious scrapie agent. Scrapie‐related polymorphisms in the coding region of the prion protein (PrP) gene, Prnp, have been studied in a number of breeds. The disease‐promoting V136 allele, and the susceptibility‐reducing R171 allele, have proved to be most important. However, variation in the coding region of Prnp cannot alone explain the diverse patterns of scrapie susceptibility in various breeds. For instance, in many breeds plagued with scrapie, the V136 allele appears to be a rarity. The R171 allele greatly reduces scrapie susceptibility. This lays the molecular foundation for marker‐assisted breeding for reduced scrapie susceptibility now underway in many countries. Although potentially important, and still under investigation, variable expression level and pattern of the ovine Prnp appears to be of little importance for the occurrence of natural scrapie. Studies of scrapie in mice also indicate that genetic elements other than Prnp may have a strong influence on scrapie incubation time, and hence susceptibility. Narrowing down the search to focus on these elements and identification of candidate genes are important tasks for future research in sheep scrapie.

Genomic organization, comparative analysis, and genetic polymorphisms of the bovine and ovine prion Doppel genes (PRND).

Abstract. The doppel protein (Dpl) is a prion-like protein encoded by the gene PRND, which has been found downstream of the prion gene, PRNP, in human and mouse. This paper describes the isolation and structural organization of the bovine and ovine PRND genes, which are composed of two exons compared with the three of human and mouse. Intergenic distances between PRNP and PRND were covered by means of long-range PCR and found to be 16.8 and 20 kb, in cattle and sheep respectively. The 5′ and 3′ untranslated regions (UTR) were analyzed to identify transcription regulatory sequences and compared with those from the PRND and PRNP sequences published for other species. Three polymorphisms (R50H, N110H, and R132Q) were revealed in the cattle coding region; two synonymous substitutions (I12I, A26A) were found in sheep. None of the polymorphisms was significantly associated with either Bovine Spongiform Encephalopathy (BSE) in cattle or scrapie in sheep.

The PrP-like protein Doppel gene in sheep and cattle: cDNA sequence and expression

Abstract. cDNAs encoding the ovine and bovine prion protein-like protein Doppel (Dpl) have been cloned. Sequencing revealed cDNAs of 2.85 and 3.31 kb from ovine and bovine testicular tissue, in accordance with observations of single transcripts of 3.2 and 3.6 kb on Northern blots. Sequence alignments showed a very high degree of identity between the sheep and cattle Dpl cDNAs, except for a 0.4-kb stretch in the bovine 3′ untranslated region and the terminal 3′ end of the sequences. The expression pattern of the Dpl gene (Prnd) in adult tissues from both species was compared by Northern blot and RT-PCR analyses. The Prnd gene was expressed strongly in testicular tissue, while low levels of expression were seen in other tissues. The open reading frame of the ovine and bovine sequences encodes a 178-amino acid protein with 95% sequence identity between the two species. Predicted structural features are in close agreement with previous reports for mouse, human, and rat Dpl.

Adaptable hexokinase with low affinity for glucose in the liver of atlantic salmon (Salmo salar)

Abstract 1. 1. The results show the presence of an adaptable hexokinase (glucokinase) with low affinity for glucose in salmon liver. The activity of this enzyme increased gradually when dietary starch was increased from 0 to 30% at the expense of dietary protein. 2. 2. The change in dietary carbohydrate/protein had minor, but statistically significant effects on hepatic glucose dehydrogenase (EC 1.1.1.47), glucose-6-phosphate dehydrogenase (EC 1.1.1.49), malic enzyme (NADP malate dehydrogenase, EC 1.1.1.40), citrate lyase (EC 4.1.3.6), and fructose-1,6-bisphosphatase (EC 3.1.3.11). 3. 3. The activity of glucose-6-phosphate dehydrogenase was maximal after feeding 10% dietary starch which also was the optimal amount as judged from energetic factors.

Glucose dehydrogenase, glucose-6-phosphate dehydrogenase and hexokinase in liver of rainbow trout (Salmo gairdneri). Effects of starvation and temperature variations.

1. Activities of trout liver glucose dehydrogenase (GDH, EC 1.1.1.47) and glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49) were increased after a sudden drop in water temperature, but not in long-time cold acclimated as compared with warm acclimated trout. 2. Possibly, the activities of GDH and G6PD were temporarily increased in connection with metabolic adaptation to the lower temperature. 3. The activities of GDH and G6PD were not changed by the stress of handling. 4. Partially purified trout liver GDH has a lower activation energy with glucose than with glucose-6-phosphate as substrate, and the Km (glucose) decreases with decreasing assay temperature. 5. At low temperatures, the activity of trout liver GDH with glucose as substrate may be comparable to that of glucose-6-phosphate. 6. Partially purified beef liver GDH has a high activation energy with glucose as substrate, and the Km (glucose) does not change with the assay temperature. 7. Hexokinase (HK, EC 2.7.1.1) and GDH activities were unchanged when trout were deprived of food for 4 weeks. Apparently, the trout liver glucose utilization did not adapt to the starvation.

Healthy goats naturally devoid of prion protein

Prion diseases such as scrapie in small ruminants, bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt-Jakob disease (CJD) in man, are fatal neurodegenerative disorders. These diseases result from the accumulation of misfolded conformers of the host-encoded prion protein (PrP) in the central nervous system. To date naturally-occurring PrP free animals have not been reported. Here we describe healthy non-transgenic animals, Norwegian Dairy Goats, lacking prion protein due to a nonsense mutation early in the gene. These animals are predicted to be resistant to prion disease and will be valuable for research and for production of prion-free products.

Characterization of the prion protein 3F4 epitope and its use as a molecular tag

The monoclonal antibody (MAb) 3F4 has for nearly two decades been one of the most commonly used tools in prion research. This MAb has contributed significantly to our understanding of the normal cell biology of the prion protein (PrP(C)), as well as the disease related abnormalities occurring in prion diseases. The 3F4 antibody binds strongly to human and hamster PrP, with a specific requirement of two Met residues at positions 109 and 112 in the human PrP. Other species in which PrP lack one of the Met residues, like cattle and sheep, or both, like rat and mouse, do not react with the 3F4 antibody. These and other observations have led to the commonly accepted notion that the 3F4 epitope consists of the tetra-peptide Met-Lys-His-Met. In this study, we have identified the minimal epitope for 3F4 by studying its binding to synthetic peptides and by analysis of mutated ovine PrP::GFP constructs expressed in cell culture. We have found that the 3F4 epitope consists of a hepta-peptide (Lys-Thr-Asn-Met-Lys-His-Met), which in sheep encompass residues 109-115. We found that Lys 109 is critically important for 3F4 binding, as omission, or substitution of this residue to Ala resulted in no binding. We also demonstrate that the hepta-peptide constituting the minimal 3F4 epitope, can be used as a discrete, moveable high-affinity molecular tag. Thus, the 3F4 antibody can find its use beyond prion research.