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The process by which transmissible spongiform encephalopathy (TSE) agents, or prions, infect cells is unknown. There are also no effective treatments available for TSE diseases. Studies of cultured cells persistently infected with TSE agents have greatly contributed to understanding these and many other aspects of TSE disease. New cell lines have been developed to increase the repertoire of TSE strains that can be investigated in ex vivo models. Candidates for TSE therapeutics have been identified. Initial events involving the internalization and trafficking of TSE agents and the effect of membranes on the infection process have been examined. Recent progress in these areas is discussed below, which together illustrate the value of cell culture models in the study of prion diseases.

Novel nuclear spin relaxation mechanism in proteins on a timesclae of micro to milliseconds has been proposed using the Berry-phase interference between periodic orbits within the semi-classical quantization formalism. In order to examine the proposed formalism, susceptibility has been represented by an analytical formulation as a function of the frequency of the periodic orbits, using a Riemann ζ function. Numerically obtained profile of the susceptibility has been quantitatively compared with that of experimentally obtained slow exchange rates in a stable globular protein, p8^MTCP1 and in a hamster prion protein. Behavior of amide nitrogen atoms in p8^MTCP1 was almost uniform between residues, and consistent with that of theoretically obtained ones. On the other hand, that of hamster prion was not uniform and quite different than that of theoretical one, suggesting that the slow dynamics of a hamster prion might not be well described by the periodical orbits around the unique saddle point. Local trajectories in the phase space around the native conformation of the hamster prion may be connected to the complicated geometry, corresponding to multiple conformational states, for instance, PrP^C, PrP* or PrP^Sc.

Chronic wasting disease (CWD) in cervids including elk, mule deer, and white-tailed deer, is a member of the transmissible spongiform encephalopathies (TSEs). CWD is a serious problem in North America. The detection of abnormal isoforms of prion protein (PrP^Sc) is a key factor for the diagnosis of CWD, similar to other TSEs. The surveillance program for TSEs in animals is conducted by the Ministry of Agriculture, Forestry, and Fishery (MAFF) and is targeted to sheep, goats, and deer. In Japan, several different anti-prion protein (PrP) monoclonal antibodies (mAbs) are utilized for bovine spongiform encephalopathy (BSE) confirmation. Since CWD does not occur naturally in Japan, the immunoreactivity of the antibodies against PrP^Sc found in deer was not known. In this study, we examined the immunoreactivities of these antibodies against PrP^Sc found in CWD. The protocols that are used in Japan for confirmation of BSE cases are Western blot (WB) and immunohistochemistry (IHC). We used these same protocols to examine CWD positive brain samples which were provided by Dr. A. Davis of the National Veterinary Service Laboratory, USA. Mabs. T1, T2, 44B1, and 72-5, were used successfully to detect PrP^Sc in CWD affected mule deer brains by WB. In IHC, PrP^Sc was detected with mAbs T2, 44B1, and polyclonal antibody B103. These results determined that the antibodies used for BSE confirmation are also applicable to CWD, as for scrapie. These same antibodies could detect PrP^C from Japanese deer by WB without proteinase digestion. The amino acid sequence of PrP of Japanese deer was found to be the same as sequence as the one reported for mule deer. These antibodies were then used for CWD surveillance in Japan. When 127 of hunter-killed deer from Hokkaido were examined, PrP^Sc was not detected in any of the animals.

Fluorescence correlation spectroscopy (FCS) is a detection technique for molecular parameters, such as concentration, diffusion constant and interaction in solution with single molecule level. FCS consists of a confocal optics with a well focused laser beam and detects fluorescent fluctuation that is caused by the diffusion of fluorescently labeled molecules through a tiny open volume element (< one femtoliter). Measured fluctuation can be used to calculate the lateral diffusion coefficient and thus the size of the molecule. We propose FCS as a newly method for the detection of prion diseases.

Bovine spongiform encephalopathy (BSE) is a disease of cattle that causes progressive neurodegeneration of the central nervous system. Infectivity of BSE agent is accompanied with an abnormal isoform of prion protein (PrPSc).

A Prion Disease Research Center at the National Institute of Animal Health (NIAH) was established to conduct comprehensive research on BSE in response to the discovery of bovine spongiform encephalopathy (BSE) in Japan. A new research facility for the center has been constructed. It was designed as a biosafety level (BSL) 3 facility with the capacity to inoculate and hold experimental animals. Experiments have begun to infect cattle with BSE orally. This route of inoculation simulates the feeding of contaminated meat and bone meal that caused pandemic occurrence of BSE in the UK. An abnormal isoform of the prion protein (PrP^Sc) accumulates in BSE affected cattle. The purpose of this study is to examine the spread of the abnormal prion from digestive tract to the central nervous system and to describe the pathological changes in cattle during the course of infection. Atypical BSE and young BSE cases have been found in the abattoir surveillance program. As a result the cattle used in this experiment were imported from Australia, a country free of BSE, to exclude the possibility of prior BSE infection before inoculation. Each calf (Holstein heifer, 10-months old) was inoculated orally with 5g of brain stem from BSE infected cattle (courtesy provided by Veterinary Laboratory Agency, Weybridge, UK) into the rumen with a catheter. The cattle will be euthanized at intervals during the clinical stages of disease. Infectivity from different tissues at different stages of clinical disease as well as the deposition of PrP^Sc will be analyzed. Other factors that may be related to the pathogenesis of the BSE prion will be investigated.

It is very difficult to obtain cattle infected with bovine spongiform encephalopathy (BSE) because there is no means of diagnosing BSE in live animals. This has been the obstacle of the BSE research. It is pressing need to make sure of enough cattle and bovine tissues infected with BSE for it. An intracerebral (i.c.) inoculation is the most efficient route of a prion disease transmission in each animal. However there are few reports about methods of i.c. inoculation to cattle. We developed the efficient and safe method of an i.c. inoculation to calves. In addition, we challenged BSE brain tissues to calves. It is the first BSE experimental transmission to cattle in Japan.

The causative agent of transmissible spongiform encephalopathies, prion, is thought to be composed mainly of abnormal isoform of prion protein (PrP^Sc). Although prion is devoid of agent-specific nucleic acid, there exist prion strains that are characterized biologically. Distribution of neuropathological lesions is one of the phenotyes of prion strains, however, there are only a few reports that addressed the linkage of the clinical manifestations to neuropathological lesions. In this study, we compared the biological, biochemical, and neuropathological differences among three mouse adapted-scrapie strains, G1, I3/I5, and Obihiro. G1 exhibited longer incubation periods (∼330 days) than others in mice carrying PrP^A/A allotype. Eighty percent of G1 strain-infected ICR mice showed severe obesity and polyuria. Diffuse deposition of PrP^Sc was widespread in cerebral cortex, hippocampus of I3/I5 and Obihiro strain-infected mice, while in G1 strain-infected mice, deposition of PrP^Sc was rather restricted in the thalamus and hypothalamus and large PrP amyloid plaques were observed in cerebral cortex. PrP^Sc of three strains could also be distinguished in combination of relative proteinase K resistance and glycoform patterns. Serum concentration insulin and leptin levels were remarkable high in G1-infected ICR mice with obesity, suggesting endocrinopathy and/or carbohydrate metabolism failure. PrP^Sc-deposition and neuronal vacuolation were observed the regions in hypothalamus including suprachiasmatic nucleus, supraoptic nucleus and paraventricular nucleus. Kinetic analysis indicated that vasopressin-positive cells in these nuclei decreased along with the progression of the neuronal degeneration. In contrast, vasopressin-positive cells in these nuclei were detected even at the terminal stage of G1-infected C57BL/J6 mice that did not exhibit polyuria. These results suggest that G1-affected ICR mice developed hypothalamic diabetes insipidus. The particular characteristics of G1 strain would be useful for further analyzing the target cell specificity of prion and pathogenesis of prion diseases.