We established that follicular dendritic cells (FDCs) are the site of abnormal prion protein (PrPCJD) accumulations in lymphoid tissues from mice infected with Creutzfeldt-Jakob disease. Evidence of positive FDC staining was observed in Creutzfeldt-Jakob disease-infected mice irrespective of the inoculation route, while no such staining was seen in the control mice. We also found that the severe combined immunodeficiency mouse trait is transmittable via the intracranial route but not via the intraperitoneal route. Mice with severe combined immunodeficiency did not have PrPCJD accumulation in FDCs.

The therapeutic efficacy of direct drug infusion into the brain, the target organ of transmissible spongiform encephalopathies, was assessed in transgenic mice intracerebrally infected with 263K scrapie agent. Pentosan polysulfate (PPS) gave the most dramatic prolongation of the incubation period, and amphotericin B had intermediate effects, but antimalarial drugs such as quinacrine gave no significant prolongation. Treatment with the highest dose of PPS at an early or late stage of the infection prolonged the incubation time by 2.4 or 1.7 times that of the control mice, respectively. PPS infusion decreased not only abnormal prion protein deposition but also neurodegenerative changes and infectivity. These alterations were observed within the brain hemisphere fitted with an intraventricular infusion cannula but not within the contralateral hemisphere, even at the terminal disease stage long after the infusion had ended. Therapeutic effects of PPS were also demonstrated in mice infected with either RML agent or Fukuoka-1 agent. However, at doses higher than that providing the maximal effects, intraventricular PPS infusion caused adverse effects such as hematoma formation in the experimental animals. These findings indicate that intraventricular PPS infusion might be useful for the treatment of transmissible spongiform encephalopathies in humans, providing that the therapeutic dosage is carefully evaluated.

Ca2+ handling in excitation-contraction coupling requires considerable O2 consumption (Vo 2) in cardiac contraction. We have developed an integrative method to quantify total Ca2+ handling in normal hearts. However, its direct application to failing hearts, where futile Ca2+ cycling via the Ca2+-leaky sarcoplasmic reticulum (SR) required an increased Ca2+handling Vo 2, was not legitimate. To quantify total Ca2+ handling even in such failing hearts, we combined futile Ca2+ cycling with Ca2+ handling Vo 2 and the internal Ca2+ recirculation fraction via the SR. We applied this method to the canine heart mechanoenergetics before and after intracoronary ryanodine at nanomolar concentrations. We found that total Ca2+ handling per beat was halved after the ryanodine treatment from ∼60 μmol/kg left ventricle before ryanodine. We also found that futile Ca2+ cycling via the SR increased to >1 cycle/beat after ryanodine from presumably zero before ryanodine. These results support the applicability of the present method to the failing hearts with futile Ca2+ cycling via the SR.

Transfer RNAs (tRNAs) contain a wide variety of posttranscriptional modifications that are important for accurate decoding. Mammalian mitochondrial tRNAs (mt-tRNAs) are modified by nuclear-encoded tRNA-modifying enzymes; however, the physiological roles of these modifications remain largely unknown. In this study, we report that Cdk5 regulatory subunit-associated protein 1 (Cdk5rap1) is responsible for 2-methylthio (ms(2)) modifications of mammalian mt-tRNAs for Ser(UCN), Phe, Tyr, and Trp codons. Deficiency in ms(2) modification markedly impaired mitochondrial protein synthesis, which resulted in respiratory defects in Cdk5rap1 knockout (KO) mice. The KO mice were highly susceptive to stress-induced mitochondrial remodeling and exhibited accelerated myopathy and cardiac dysfunction under stressed conditions. Furthermore, we demonstrate that the ms(2) modifications of mt-tRNAs were sensitive to oxidative stress and were reduced in patients with mitochondrial disease. These findings highlight the fundamental role of ms(2) modifications of mt-tRNAs in mitochondrial protein synthesis and their pathological consequences in mitochondrial disease.

) (21 and 19 kDa, respectively), reflecting differences in the proteinase K cleavage site (at residues 82 and 97, respectively) (2, 3). The genotype at codon 129 also influences the susceptibility to variant CJD (vCJD), iatrogenic CJD, and kuru (6 -11). A transmission study using transgenic mice expressing human PrP revealed that the congruency of the genotype at codon 129 between the inoculum and the inoculated transgenic mice determines the susceptibility to sCJD prions (12). Transmission of sCJD prions to mice with an incongruent genotype (referred to as cross-sequence transmission) results in a relatively long incubation period.The potential for cross-sequence transmission should be considered in the iatrogenic transmission of CJD via cadaveric pituitary hormones, dura mater and corneal grafts, or contaminated neurosurgical instruments. More than half of the reported cases of dura mater graft-associated CJD (dCJD) occurred in Japan, where 123 cases have been recognized as of February 2006 (13-16). The dural grafts used in Japan were manufactured by German companies (13-15). In Europe, 28.4% of sCJD patients are valine homozygotes (129V/V) or methionine/valine heterozygotes at codon 129 (129M/V) (5). Meanwhile, the population data show a high prevalence (91.6%) of methionine homozygosity (129M/M) in Japanese people (17). These data raise the possibility that part of the Japanese dCJD cases might have been caused by cross-sequence transmission of sCJD prions. In fact, there are two distinct phenotypes in dCJD, with the majority represented by a non-plaque type of dCJD (np-dCJD) and the minority by a plaque-type dCJD (p-dCJD) (18 -21). The clinicopathological features of np-dCJD are similar to those of sCJD with 129M/M and type 1 PrP Sc (sCJD-MM1) (14). In contrast, p-dCJD cases show unique features characterized by (i) the absence or late occurrence of myoclonus and periodic synchronous discharges on electroencephalogram; (ii) a long incubation period after grafting and a clinical course of long duration, and (iii) plaque-type PrP deposits in the brain (18 -25). Although we have classified p-dCJD cases into MM1, the clinicopathological features of p-dCJD are quite different from those of sCJD-MM1 or npdCJD (18 -21). The reason for the existence of two distinct phenotypes in dCJD has remained elusive.To identify the origin of p-dCJD, we inoculated sCJD prions into mice expressing human PrP with either 129M/M or 129V/V. Here we report that cross-sequence transmission of sCJD prions from a patient with 129V/V and type 2 PrP Sc

The clinicopathological phenotypes of sporadic Creutzfeldt-Jakob disease (sCJD) correlate with the allelotypes (M or V) of the polymorphic codon 129 of the human prion protein (PrP) gene and the electrophoretic mobility patterns of abnormal prion protein (PrP Sc ). Transmission of sCJD prions to mice expressing human PrP with a heterologous genotype (referred to as cross-sequence transmission) results in prolonged incubation periods. We previously reported that cross-sequence transmission can generate a new prion strain with unique transmissibility, designated a traceback phenomenon. To verify experimentally the traceback of sCJD-VV2 prions, we inoculated sCJD-VV2 prions into mice expressing human PrP with the 129M/M genotype. These 129M/M mice showed altered neuropathology and a novel PrP Sc type after a long incubation period. We then passaged the brain homogenate from the 129M/M mouse inoculated with sCJD-VV2 prions into other 129M/M or 129V/V mice. Despite cross-sequence transmission, 129V/V mice were highly susceptible to these prions compared to the 129M/M mice. The neuropathology and PrP Sc type of the 129V/V mice inoculated with the 129M/M mouse-passaged sCJD-VV2 prions were identical to those of the 129V/V mice inoculated with sCJD-VV2 prions. Moreover, we generated for the first time a type 2 PrP Sc -specific antibody in addition to type 1 PrP Sc -specific antibody and discovered that drastic changes in the PrP Sc subpopulation underlie the traceback phenomenon. Here, we report the first direct evidence of the traceback in prion infection.

Little is known about the mechanisms responsible for the adaptation and changes in the capillary network of hindlimb unweighting (HU)-induced atrophied skeletal muscle, especially the coupling between functional and structural alterations of intercapillary anastomoses and tortuosity of capillaries. We hypothesized that muscle atrophy by HU leads to the apoptotic regression of the capillaries and intercapillary anastomoses with their functional alteration in hemodynamics. To clarify the three-dimensional architecture of the capillary network, contrast medium-injected rat soleus muscles were visualized clearly using a confocal laser scanning microscope, and sections were stained by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) and with anti-von Willebrand factor. In vivo, the red blood cell velocity of soleus muscle capillaries were determined with a pencil-lens intravital microscope brought into direct contact with the soleus surface. After HU, the total muscle mass, myofibril protein mass, and slow-type myosin heavy chain content were significantly lower. The number of capillaries paralleling muscle fiber and red blood cells velocity were higher in atrophied soleus. However, the mean capillary volume and capillary luminal diameter were significantly smaller after HU than in the age-matched control group. In addition, we found that the number of anastomoses and the tortuosity were significantly lower and TUNEL-positive endothelial cells were observed in atrophied soleus muscles, especially the anastomoses and/or tortuous capillaries. These results indicate that muscle atrophy by HU generates structural alterations in the capillary network, and apoptosis appears to occur in the endothelial cell of the muscle capillaries. intercapllary anastomosis; tortuosity; capillary volume; capillary lumen; erythrocyte velocity; disuse atrophy; endothelial terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling SKELETAL MUSCLE CAPILLARIES run tortuously along muscle fibers in the relaxed resting state (2,4,18,20,33). These capillaries are connected with anastomoses, which run orthogonally to muscle fiber direction like parallel rungs of ladder (12, 33). Capillary-to-fiber (C/F) ratio is frequently used to evaluate O 2 supply capacity in skeletal muscle (27). In fact, the C/F ratio is higher in rat soleus muscle, which mainly contains slow oxidative fibers (type I), than in extensor digitorum longus, which is predominantly comprised of fast glycolytic fibers (type IIb) (1, 8, 13). C/F ratio was increased by the augmentation of muscle activity, e.g., exercise or electrical stimulation (9,14,20,25), and was decreased by disuse (14, 31, 33). Histological sections from earlier studies of capillary remodeling in skeletal muscle with disuse atrophy demonstrate a decrease in capillary luminal diameter (16,36) and in the C/F ratio (13, 31, 32) despite an increase in capillary density (13,31,32).Although the regression of anastomoses in atrophied skeletal muscle has not been well studied, it wa...

Atypical bovine spongiform encephalopathy (BSE) has recently been identified in Europe, North America, and Japan. It is classified as H-type and L-type BSE according to the molecular mass of the disease-associated prion protein (PrPSc). To investigate the topographical distribution and deposition patterns of immunolabeled PrPSc, H-type BSE isolate was inoculated intracerebrally into cattle. H-type BSE was successfully transmitted to 3 calves, with incubation periods between 500 and 600 days. Moderate to severe spongiform changes were detected in the cerebral and cerebellar cortices, basal ganglia, thalamus, and brainstem. H-type BSE was characterized by the presence of PrP-immunopositive amyloid plaques in the white matter of the cerebrum, basal ganglia, and thalamus. Moreover, intraglial-type immunolabeled PrPSc was prominent throughout the brain. Stellate-type immunolabeled PrPSc was conspicuous in the gray matter of the cerebral cortex, basal ganglia, and thalamus, but not in the brainstem. In addition, PrPSc accumulation was detected in the peripheral nervous tissues, such as trigeminal ganglia, dorsal root ganglia, optic nerve, retina, and neurohypophysis. Cattle are susceptible to H-type BSE with a shorter incubation period, showing distinct and distinguishable phenotypes of PrPSc accumulation.