Proteomics analyses for the global proteins in the brain tissues of different human prion diseases.

Proteomics changes of brain tissues have been described in different neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. However, the brain proteomics of human prion disease remains less understood. In the study, the proteomics patterns of cortex and cerebellum of brain tissues of sporadic Creutzfeldt-Jakob disease, fatal familial insomnia, and G114V genetic CJD were analyzed with isobaric tags for relative and absolute quantitation combined with multidimensional liquid chromatography and MS analysis, with the brains from three normal individuals as controls. Global protein profiling, significant pathway, and functional categories were analyzed. In total, 2287 proteins were identified with quantitative information both in cortex and cerebellum regions. Cerebellum tissues appeared to contain more up- and down-regulated proteins (727 proteins) than cortex regions (312 proteins) of Creutzfeldt-Jakob disease, fatal familial insomnia, and G114V genetic CJD. Viral myocarditis, Parkinson's disease, Alzheimer's disease, lysosome, oxidative phosphorylation, protein export, and drug metabolism-cytochrome P450 were the most commonly affected pathways of the three kinds of diseases. Almost coincident biological functions were identified in the brain tissues of the three diseases. In all, data here demonstrate that the brain tissues of Creutzfeldt-Jakob disease, fatal familial insomnia, and G114V genetic CJD have obvious proteomics changes at their terminal stages, which show the similarities not only among human prion diseases but also with other neurodegeneration diseases. This is the first study to provide a reference proteome map for human prion diseases and will be helpful for future studies focused on potential biomarkers for the diagnosis and therapy of human prion diseases.

Remarkable impairment of Wnt/ß-catenin signaling in the brains of the mice infected with scrapie agents.

Prion diseases are a group of neurodegenerative diseases characterized by neuronal loss and spongiform degeneration, astrogliosis and aggregation of scrapie prion protein (PrPSc ) in the central nervous system (CNS). The Wnt signaling pathway is a highly evolutionarily conserved pathway in eukaryotes that regulates cell proliferation, differentiation and survival. Impairment of Wnt/ß-catenin signaling has been reported in the CNS of various neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. To investigate the functional state of Wnt/ß-catenin signaling in the CNS tissues during the progression of prion disease, the components of Wnt/ß-catenin signaling in the brains of the scrapie agents 139A- and ME7-infected mice were evaluated. Compared with the normal controls, the brain levels of phosphor-ß-catenin (Ser33,37 and Thr41 ) in 139A- and ME7-infected mice were significantly increased, while those of cyclin D1, which is one of the target genes of Wnt signaling, were decreased. The levels of phosphor-glycogen synthase kinase-3ß (GSK-3ß) Ser9 were markedly reduced, representing an enhanced GSK-3ß activity in scrapie-infected mice. Both western blot and immunohistochemical assays revealed a remarkable increase of Dickkopf-1, the antagonist of Wnt/ß-catenin signaling, in the brains of scrapie-infected anim-als, which co-localized well with the remaining neurons in the immunofluorescent tests. We also observed slightly decreased Wnt-3 and unchanged disheveled-3 (Dvl-3) in the brains of the infected mice. Our data, here, strongly indicate an impairment of Wnt/ß-catenin pathway in the brains of prion disease, which shows a time-dependent progression along with the incubation period. Schematic for the impairment of canonical Wnt signaling during prion infection. The left and right parts represent the normal and prion-infected situations, respectively. Prion infection or PrPSc accumulation triggers the over-expression of Dickkopf WNT signaling pathway inhibitor 1 (DKK-1) and the enhancement of glycogen synthase kinase 3ß (GSK-3ß) activity, which subsequently promotes the phosphorylation and degradation of ß-catenin. As a result, the impairment of ß-catenin signaling leads to the down-regulation of Wnt target genes.

[A mechanistic review of how hypoxic mircroenvironment regulates mammalian ovulation].

Mammalian ovulation is a complicated process that includes development of follicles, ovulation, formation of corpus luteum and luteolysis. The three different stages of the ovulation activity are affected by hypoxic microenvironment and hypoxia-induced factors (HIF), which play a crucial role in physiologyical processes, such as angiogenesis and inflammation. Although the process of ovulation has been well elucidated, the molecular mechanism regulated by hypoxia needs an in depth study. In this review, we summarize how hypoxic and HIF regulate gene expression during mammalian ovulation in order to provide a better understanding of ovulation mechanism, which may lay a theoretical basis for prevention and therapy of various ovarian diseases.

[The mechanism of miRNA-mediated PGR signaling pathway in regulating female reproduction].

MicroRNAs (miRNAs) are involved in several physiological processes as important post-transcriptional regulators. Progesterone (P4), an important steroid hormone, produces physiological effect through binding specific receptor progesterone receptors (PGR) which regulates functions of both reproductive and non-reproductive tissues as a member of the nuclear receptor superfamily. P4/PGR and miRNAs could regulate female reproduction independently, however, it is still unclear how miRNAs and P4/PGR interaction regulates female reproductive activities such as ovulation in female reproduction. In this review, we summarize the possible ways in which miRNAs regulate P4 production and PGR gene expression as well as P4/PGR regulate miRNAs expression, which provide a theoretical basis for further studying the role of miRNAs and P4/PGR in female reproduction.

Application of epigenetic markers in molecular breeding of the swine.

Livestock phenotypes are determined by the interaction of a variety of factors, including the genome, the epigenome and the environment. Epigenetics refers to gene expression changes without DNA sequence alterations. Epigenetic markers mainly include DNA methylation, histone modifications, non-coding RNAs, and imprinting genes. More and more researches show that epigenetic markers play an important role in the traits of pigs by modulating phenotype changes via gene expression. However, the role of epigenetic markers has caught little attention in swine breeding. The mechanism that influences important traits of swine has not been analyzed in detail, and it still lacks adequate scientific basis for practical applications. From the aspects of nutrition, diseases, important economic traits and trans-generational inheritance, we summarize the research, application prospects and challenges in the field of utilizing epigenetic markers in molecular breeding of pigs, thus providing a more comprehensive theoretical basis to promote more rapid research development in this field.

Remarkable reductions of PAKs in the brain tissues of scrapie-infected rodent possibly linked closely with neuron loss.

Prion diseases are irreversible progressive neurodegenerative diseases characterized in the brain by PrP(Sc) deposits, neuronal degeneration, gliosis and by cognitive, behavioral and physical impairments, leading to severe incapacity and inevitable death. Proteins of the p21-activated kinase (PAK) family are noted for roles in gene transcription, cytoskeletal dynamics, cell cycle progression and survival signaling. In the present study, we aimed to identify the potential roles of PAKs during prion infection, utilizing the brains of scrapie agent-infected hamsters. Western blots and immunohistochemical assays showed that brain levels of PAK3 and PAK1, as well as their upstream activator Rac/cdc42 and downstream substrate Raf1, were remarkably reduced at terminal stage. Double-stained immunofluorescent assay demonstrated that PAK3 was expressed mainly in neurons. Dynamic analyses of the brain samples collected at the different time points during the incubation period illustrated successive decreases of PAK3, PAK1 and Raf1, especially phosphor Raf1, which correlated well with neuron loss. Rac/cdc42 in the brain tissues increased at early stage and reached to the top at mid-late stage, but diminished at final stage. Unlike the alteration of PAKs in vivo, PAK3 and PAK1, as well as Rac/cdc42 and Raf1 in the prion-infected cell line SMB-S15 remained unchanged compared with those of its normal cell line SMB-PS. Our data here indicate that the functions of PAKs and their associated signaling pathways are seriously affected in the brains of prion disease, which appear to associate closely with the extensive neuron loss.

Mouse-adapted scrapie strains 139A and ME7 overcome species barrier to induce experimental scrapie in hamsters and changed their pathogenic features.

BACKGROUND: Transmissible spongiform encephalopathy (TSE) diseases are known to be zoonotic diseases that can infect different kinds of animals. The transmissibility of TSE, like that of other infectious diseases, shows marked species barrier, either being unable to infect heterologous species or difficult to form transmission experimentally. The similarity of the amino acid sequences of PrP among species is believed to be one of the elements in controlling the transmission TSE interspecies. Other factors, such as prion strains and host's microenvironment, may also participate in the process. METHODS: Two mouse-adapted strains 139A and ME7 were cerebrally inoculated to Golden hamsters. Presences of scrapie associate fibril (SAF) and PrPSc in brains of the infected animals were tested by TEM assays and Western blots dynamically during the incubation periods. The pathogenic features of the novel prions in hamsters, including electrophoretic patterns, glycosylating profiles, immunoreactivities, proteinase K-resistances and conformational stabilities were comparatively evaluated. TSE-related neuropathological changes were assayed by histological examinations. RESULTS: After long incubation times, mouse-adapted agents 139A and ME7 induced experimental scrapie in hamsters, respectively, showing obvious spongiform degeneration and PrPSc deposits in brains, especially in cortex regions. SAF and PrPSc in brains were observed much earlier than the onset of clinical symptoms. The molecular characteristics of the newly-formed PrPSc in hamsters, 139A-ha and ME7-ha, were obviously distinct from the original mouse agents, however, greatly similar as that of a hamster-adapted scrapie strain 263 K. Although the incubation times and main disease signs of the hamsters of 139A-ha and ME7-ha were different, the pathogenic characteristics and neuropathological changes were highly similar. CONCLUSIONS: This finding concludes that mouse-adapted agents 139A and ME7 change their pathogenic characteristics during the transmission to hamsters. The novel prions in hamsters' brains obtain new molecular properties with hamster-specificity.

Heat shock protein 70 selectively mediates the degradation of cytosolic PrPs and restores the cytosolic PrP-induced cytotoxicity via a molecular interaction.

BACKGROUND: Although the aggregation of PrPSc is thought to be crucial for the neuropathology of prion diseases, there is evidence in cultured cells and transgenic mice that neuronal death can be triggered by the accumulation of cytosolic PrPs, leading to the hypothesis that the accumulation of PrPs in the cytosol of neurons may be a primary neurotoxic culprit. Hsp70, a molecular chaperone involved in protein folding/refolding and degradation in the cytoplasm, has a protective effect in some models of neurodegenerative diseases, e.g., Alzheimer's and Parkinson's diseases, but its role in prion diseases remains unclear. RESULTS: To study the role of Hsp70 in prion diseases, we used immunoprecipitation to first identify a molecular interaction between Hsp70 and PrPs. Using immunofluorescence, we found that Hsp70 colocalized with cytosolic PrPs in HEK293 cells transiently transfected with plasmids for Cyto-PrP and PG14-PrP but not with wild-type PG5-PrP or endoplasmic reticulum (ER)-retained PrPs (3AV-PrP and ER-PrP). Using western blot analysis and apoptosis assays of cultured cells, we found that the overexpression of Hsp70 by transfection or the activation of Hsp70 by geldanamycin selectively mediated the degradation of cytosolic PrPs and restored cytosolic PrP-induced cytotoxicity. Moreover, we found that Hsp70 levels were up-regulated in cells expressing Cyto-PrP and in hamster brains infected with the scrapie agent 263K. CONCLUSION: These data imply that Hsp70 has central role in the metabolism of cytosolic PrPs.

The diversities of PrP(Sc) distributions and pathologic changes in various brain regions from a Chinese patient with G114V genetic CJD.

Human genetic Creutzfeldt-Jakob disease (gCJD; one of the prion diseases) is caused by point mutations and insertions in the prion protein gene (PRNP). Previously we have reported a Chinese gCJD case with a substitution of valine (V) for glycine (G) at codon 114. To investigate the detailed pathogenic and pathologic characteristics of G114V gCJD, 10 different brain regions were thoroughly analyzed. PrP-specific Western blots and immunohistochemical (IHC) assays identified larger amounts of PrP(Sc) in the regions of brain cortex. Assays of the transcriptions of PrP-specific mRNA by RT-PCR and real-time PCR showed comparable levels in 10 brain regions. In line with the distribution of PrP(Sc) , typical vacuolations in brains, markedly in four cortex regions, were detected. Contrast to the distributing features of spongiform and of PrP(Sc) , massive gliosis was detected in all brain regions by GFAP-specific IHC tests. Moreover, two-dimensional gel immunoblots found three major sets of PrP(Sc) spots, indicating that PrP(Sc) in brain tissues was a mixture of molecules with different biochemical properties. The data here provide the pathogenic and neuropathological features of G114V gCJD.

Contamination of live virus during tissue homogenizing by ultrasonic processor and tissue disperser.

OBJECTIVE: To quantitatively evaluate the contamination area and risk of a live pathogen during tissue homogenization by either ultrasonic processor or tissue disperser. METHODS: A recombinant Herpes Simplex Virus (rHSV) containing GFP gene was used as the index virus, and fresh liver tissue from healthy mice was used as simulated specimen. After 10% liver homogenate was mixed with rHSV (100 TCID50/0.1 mL) in a 5 mL tube, the stability of rHSV in liver homogenate and influences of an ultrasonic processor and a tissue disperser on viral infectivity were determined by GFP expressions in cell cultures. The contaminating areas of live viruses during homogenization were evaluated by a cell culture-based sedimentary. The contamination radii were counted by measurement of the distance between the operator and the farthest GFP positive well. RESULTS: The infectivity of rHSV in 10% liver homogenate maintained almost unchanged after it was incubated at room temperature for 30 min. Treatment with an ultrasonic processor clearly dropped down the virus infectivity, while a disperser not. Obvious spills and slashes of live viruses were observed in processes of homogenization with those two apparatuses. The contamination radii are positively related with sample volume, output energy of operator and handling time. CONCLUSION: Homogenizing infectious samples with an ultrasonic processor and a tissue disperser at commonly used conditions caused obvious spills and splashes of live viruses, which possesses high risk to induce Laboratory acquired infections (LAIs).

[The neuroendocrine regulatory mechanisms of mammalian seasonal reproduction].

The seasonal reproduction of mammal means the reproduction experiences an annual period from quiescence to renaissance. Studies have shown that kisspeptin and RFRP play an important role in the reproductive seasonality. The non-breeding season is characterized by an increase in the negative feedback effect of estrogen on GnRH, and this effect is transmitted by kisspeptin neurons, which may be an important factor affecting the reproduction activities. The expression of RFRP depends on melatonin secretion, and shows an apparent inhibition on reproduction in non-breeding season. In addition, thyroid hormones influence termination of the breeding season. Dopaminergic neuron A14/A15 also contributes to the seasonal changes in estrogen negative feedback. These neural systems may synergistically modulate the seasonal changes of reproductive function with the photoperiod. This review makes a systematic expatiation on the relationship between seasonal reproduction and these neural systems.

[Progesterone receptor-mediated molecular mechanisms on mammalian female reproduction].

The steroid hormone, progesterone, plays a critical role in regulation of mammalian female reproductive activities. Besides the non-genomic activity of progesterone on target cells, its main physiological effect is caused through genomic action by the ligand-dependent nuclear progesterone receptor. The genomic and non-genomic effects of progesterone collectively mediate various female reproductive functions, including ovulation, embryo implantation, maintenance of pregnancy, initiation of parturition, and development of mammary gland. Although a large number of candidate genes regulated by progesterone have been identified by gene chip technology, the traditional progesterone response elements located in the promoter region of downstream target genes havenot been detected. Accordingly, it was suggested thatthe mechanism of nuclear progesterone receptors regulating transcription may be different from other nuclear steroid receptors. In this review, we summarized the mechanisms of progesterone receptors mediating the physiological effects in various female re-productive activities.

[Effects of RFRP-3 on reproductive function and energy balance in mammals].

The hypothalamo-pituitary-gonadal (HPG) axis integrates internal and external cues via a balance of stimulatory and inhibitory neurochemical systems to regulate reproductive function in mammals. However, RFRP-3 is a unique inhibitor of HPG axis at the hypothalamuic level in mammals to date. A large number of previous studies have confirmed that RFamide-related peptide (RFRP-3) suppresses gonadotropin-releasing hormone (GnRH) system and luteinizing hormone (LH) secretion, thereby affecting the reproduction. However, whether the inhibition of LH secretion by RFRP-3 occurs at the pituitary level or the hypothalamus level is not clear. It is interesting that RFRP-3 is also related to signal pathway of melatonin modulating mammal seasonal reproduction, but little is known about the effects of melatonin on the RFRP-3 neuron up to now. In addition, RFRP-3 also plays an important role in the regulation of energy balance and behavior. The regulatory mechanism of RFRP-3 in HPG axis and role of RFRP-3 in modulating mammalian energy balance, as well as behavior, are systematically elaborated and the remaining unsolved problems are also discussed in this paper.

Establishment of hamster- and human-PRNP transgenic mice.

OBJECTIVE: To create transgenic mice expressing hamster- and human-PRNP as a model for understanding the physiological function and pathology of prion protein (PrP), as well as the mechanism of cross-species transmission of transmissible spongiform encephalopathies (TSEs). METHODS: Hamster and human-PRNP transgenic mice were established by conventional methods. The copy number of integrated PRNP in various mouse lines was mapped by real-time PCR. PRNP mRNA and protein levels were determined by semi-quantitative RT-PCR, real-time RT-PCR, and western blot analysis. Histological analyses of transgenic mice were performed by hematoxylin and eosin (H & E) staining and immunohistochemical (IHC) methods. RESULTS: Integrated PRNP copy number in various mouse lines was 53 (Tg-haPrP1), 18 (Tg-huPrP1), 3 (Tg-huPrP2), and 16 (Tg-huPrP5), respectively. Exogenous PrPs were expressed at both the transcriptional and translational level. Histological assays did not detect any abnormalities in brain or other organs. CONCLUSION: We have established one hamster-PRNP transgenic mouse line and three human-PRNP transgenic mouse lines. These four transgenic mouse lines provide ideal models for additional research.

Changes of tau profiles in brains of the hamsters infected with scrapie strains 263 K or 139 A possibly associated with the alteration of phosphate kinases.

BACKGROUND: Phospho-tau deposition has been described in a rare genetic human prion disease, Gerstmann-Sträussler-Scheinker syndrome, but is not common neuropathological picture for other human and animal transmissible spongiform encephalopathies (TSEs). This study investigated the possible changes of tau and phosphorylated tau (p-tau, at Ser396, Ser404, and Ser202/Thr205) in scrapie experimental animals. METHODS: The profiles of tau and p-tau (p-tau, at Ser396, Ser404, and Ser202/Thr205) in the brain tissues of agents 263K- or 139A-infected hamsters were evaluated by Western blots and real-time PCR. Meanwhile, the transcriptional and expressive levels of GSK3beta and CDK5 in the brains were tested. RESULTS: The contents of total tau and p-tau at Ser202/Thr205 increased, but p-tau at Ser396 and Ser404 decreased at the terminal stages, regardless of scrapie strains. Transcriptional levels of two tau isoforms were also increased. Additionally, it showed higher CDK5, but lower GSK3beta transcriptional and expressive levels in the brains of scrapie-infected animals. Analysis of brain samples collected from different times after inoculated with agent 263 K revealed that the changes of tau profiles and phosphate kinases were time-relative events. CONCLUSION: These data suggest that changes of profiles of p-tau at Ser396, Ser404 and Ser202/Thr205 are illness-correlative phenomena in TSEs, which may arise of the alteration of phosphate kinases. Alteration of tau, p-tau (Ser396, Ser404, and Ser202/Thr205), GSK3beta and CDK5 were either intermediate or consequent events in TSE pathogenesis and proposed the potential linkage of these bioactive proteins with the pathogenesis of prion diseases.

PrP(Sc) of scrapie 263K propagates efficiently in spleen and muscle tissues with protein misfolding cyclic amplification.

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are transmissible neurodegenerative disorders of protein conformation. This group of diseases is caused by infectious agents, termed prions, which can convert normal conformation (PrP(C)) into misfolded protein (PrP(Sc)). The infectivity of non-neuronal tissues has been wildly addressed, but the propagating features and the biochemical properties of prion generated from these tissues are only partially settled. In this study, utilizing protein misfolding cyclic amplification (PMCA), the in vitro conversion of PrP(C) into PrP(Sc) in spleen and muscle tissues can be induced by PrP(Sc) produced in vivo. The further propagation of newly formed PrP(Sc) in normal brain and some of the biochemical properties of new PrP(Sc) are similar as the brain-derived prions, implying the naturally infectious pathway of prion from peripheral generation to neuro-invasion. However, compared with the brain-derived PrP(Sc), the weaker resistance of new PrP(Sc) to some inactivated agents, i.e. sodium hydroxide and thermal inactivation, are observed. Our data provide the reliable evidence that the brain-derived PrP(Sc) can utilize the PrP(C) from non-neuronal tissues for its propagation. Similarity of the replicative ability in PMCA in vitro and the infectivity in vivo highlights the possibility to use PMCA instead of bioassay to investigate the propagation of prion.

Establishment of a stable PrP(Sc) panel from brain tissues of experimental hamsters with scrapie strain 263K.

OBJECTIVE: To establish a stable PrP(Sc) panel from brain tissues of experimental hamsters infected with scrapie agent 263K for evaluating diagnostic techniques of human and animals' prion diseases. METHODS: Thirty brain tissue samples from hamsters intracerebrally infected with scrapie strain 263K and another 30 samples from normal hamsters were selected to prepare 10%, 1%, and 0.5% brain homogenates, which were aliquoted into stocks. PrP(Sc) in each brain homogenate was determined by proteinase K digestions followed by Western blot assay and partially by immunohistochemistry. Stability and glycoforms of PrP(Sc) were repeatedly detected by PrP(Sc)-specific Western blots in half a year and 3 years later. RESULTS: PrP(Sc) signals were observed in all 10% brain homogenates of infected hamsters. Twenty out of 30 stocks and 19 out of 30 stocks were PrP(Sc) positive in 1% and 0.5% brain homogenatesof infected hamsters, respectively. Twenty-seven out of 30 stocks presented three positive bands in 10% brain homogenates, whereas none of 1% and 0.5% homogenates contained 3 bands. The detection of PrP(Sc)-specific signals stored in half a year and 3 years later demonstrated that the ratio of PrP(Sc) positive samples and glycoforms was almost unchanged. All normal hamsters' brain homogenates were PrP(Sc) negative. CONCLUSION: A PrP(Sc) panel of prion disease can be established, which displays reliably stable PrP(Sc)-specific signals and glycoforms.

[Advances on related genes with sexual precocity in mammals].

In mammals and humans, reproductive capacity is attained at puberty as the end-point of a complex series of developmental and neuroendocrine events that lead to true sexual maturity. As for humans, sexual precocity looks like a pathologic status. While for some animals, sexual precocity may be a valuable quantitative character. For some species, the character of sexual precocity was developed in the evolutionary process and stably transmitted to future generations. Sexual precocity is a complex character determined by polygenes. This review introduced the association between KiSS-1, GPR54, LHR, FSHR, CYP, ER, TGFa, IGF-, GNAS1, HSD3B2, SHBG, VDR genes and sexual precocity in mammals.

The N-terminus of PrP is responsible for interacting with tubulin and fCJD related PrP mutants possess stronger inhibitive effect on microtubule assembly in vitro.

Microtubule dynamics is essential for many vital cellular processes such as in intracellular transport, metabolism, and cell division. Some evidences demonstrate that PrP may associate with microtubular cytoskeleton and its major component, tubulin. In the present study, the molecular interaction between PrP and tubulin was confirmed using pull-down assays, immunoprecipitation and ELISA. The interacting regions within PrP with tubulin were mapped in the N-terminus of PrP spanning residues 23-50 and 51-91. PrP octapeptide repeats are critical for the binding activity with tubulin, that the binding activity of PrP with tubulin became stronger along with the number of the octapeptide repeats increased. Microtubule assembly assays, sedimental tests and transmission electron microscopy demonstrated that the full-length PrP (aa 23-231) obviously inhibited the microtubule polymerization processes in vitro, whereas the N- (aa 23-91) and C- (aa 91-231) terminal peptides of PrP did not affect microtubule polymerization. Moreover, the familial Cruetzfeldt Jacob disease (fCJD) related PrP mutants with inserted or deleted octapeptide repeats showed much stronger inhibitive capacities on the microtubule dynamics in vitro than wild-type PrP. Our data highlight a potential role of PrP in regulating the microtubule dynamics in neurons.

A patient with Creutzfeldt-Jakob disease with an insertion of 7 octa-repeats in the PRNP gene: molecular characteristics and clinical features.

BACKGROUND: We evaluated the features of neuropathology, abnormal prion protein (PrP) molecules, and clinical data of a Chinese woman diagnosed with familiar Creutzfeldt-Jakob disease (CJD), having 7 octa-repeats inserted with codon 129 methionine homozygote in the PRNP gene. METHODS: Neuropathologic characteristics of the brain were analyzed by hemotoxylin-eosin stain and electronic microscopy. The presence of abnormal PrP in brains was detected by proteinase K and PrP molecules were evaluated by deglycosylation assay. RESULTS: Spongiform degeneration, with diffuse neuronal loss and mild astrocytic gliosis, as well as with profound degeneration of neurons and astrocytes was observed. Proteinase K-resistant PrP was deposited widely in various regions of the brain. Calculation of the glycosylation ratios of proteinase K-resistant PrP molecules identified that the monoglycosyl isomer was predominant. PrP deglycosylation tests allowed for the identification of a predominant 19-kDa PrP signal that represents a partially proteolytic C-terminal segment, a 27-kDa band that represents the full-length wild-type PrP molecule, and a 30-kDa band that probably corresponds to the full-length mutant PrP molecule. CONCLUSION: : Sporadic CJD-like neuropathologic changes and deposits of proteinase K-resistant PrP have been identified in this familiar CJD case with a 168 base pair nucleotide insertion. The clinical features differ from previously reported cases that had 7 octa-repeat insertion, but bear similarities to sporadic CJD.