Characterization of major peptides in Creutzfeldt-Jakob disease and scrapie.

In Creutzfeldt-Jakob disease three major peptides cosediment with the infectious agent. These distinct peptides are not present in identical fractions from uninfected brain, and bind to polyclonal antibodies raised against "prion protein" purified by protease treatment. Three similar distinct peptides are also found in scrapie-infected brain fractions purified without the use of proteases. To clarify the relationships between these distinct peptides and prion protein, peptides were analyzed on immunoblots after cleavage with various glycosidases. There are two different 34-kDa peptides. One binds to ricin and cannot be detected by nonequilibrium pH gradient electrophoresis, presumably due to its highly acidic or basic pI. A second basic 34-kDa glycopeptide (Gp34) contains multiple terminal sialic acid residues responsible for charge heterogeneity (pI values, 7.2-7.8) and is reduced to a single spot with a pI value of 7.8 after neuraminidase treatment. After (but not before) neuraminidase treatment, secondary D-galactose-like sugars are detectable on Gp34, and a small number of N-acetylglucosamine residues probably represent the third sugar residue in an N-linked chain. When virtually all sugar residues are removed with endoglycosidase H the molecular weight of Gp34 is reduced by only approximately equal to 2 kDa. The residual peptide strongly binds antibody. A third acidic 24- to 26-kDa species (p26) also binds polyclonal antibodies but, in contrast to Gp34, was unaffected by any glycosidase treatment. Protease-treated peptides showed a very broad array of pI spots, consistent with a heterogeneous protein origin. None of the nonproteolyzed peptides show a clear relationship to prion protein. The number of sugar residues on Gp34 is not consistent with those estimated for prion protein. Although p26 could be the source of the "prion sequence," p26 does not appear to be glycosylated. Regardless, it is likely that all the major peptides described thus far are accumulated or modified normal gene products and are not integral components of the infectious agent.     

p53 mutations increase resistance to ionizing radiation.

Mouse and human tumors of diverse origin frequently have somatically acquired mutations or rearrangements of the p53 gene, or they have lost one or both copies of the gene. Although wild-type p53 protein is believed to function as a tumor-suppressor gene, it is as yet unclear how p53 mutations lead to neoplastic development. Wild-type p53 has been postulated to play a role in DNA repair, suggesting that expression of mutant forms of p53 might alter cellular resistance to the DNA damage caused by gamma radiation. Moreover, p53 is thought to function as a cell cycle checkpoint after irradiation, also suggesting that mutant p53 might change the cellular proliferative response to radiation. We have used transgenic mice expressing one of two mutant alleles of p53 to test this prediction. Our results show that expression of both mutant variants of the mouse p53 gene significantly increases the cellular resistance of a variety of hematopoietic cell lineages to gamma radiation. These observations provide direct evidence that p53 mutations affect the cellular response to DNA damage, either by increasing DNA repair processes or, possibly, by increasing cellular tolerance to DNA damage. The association of p53 mutations with increased radioresistance suggests possible mechanisms through which alterations in the p53 gene might lead to oncogenic transformation.     

Extreme resistance of bdelloid rotifers to ionizing radiation

Rotifers of class Bdelloidea are common invertebrate animals with highly unusual characteristics, including apparently obligate asexuality, the ability to resume reproduction after desiccation at any life stage, and a paucity of transposable genetic elements of types not prone to horizontal transmission. We find that bdelloids are also extraordinarily resistant to ionizing radiation (IR). Reproduction of the bdelloids Adineta vaga and Philodina roseola is much more resistant to IR than that of Euchlanis dilatata, a rotifer belonging to the desiccation-intolerant and facultatively sexual class Monogononta, and all other animals for which we have found relevant data. By analogy with the desiccation- and radiation-resistant bacterium Deinococcus radiodurans, we suggest that the extraordinary radiation resistance of bdelloid rotifers is a consequence of their evolutionary adaptation to survive episodes of desiccation encountered in their characteristic habitats and that the damage incurred in such episodes includes DNA breakage that is repaired upon rehydration. Such breakage and repair may have maintained bdelloid chromosomes as colinear pairs and kept the load of transposable genetic elements low and may also have contributed to the success of bdelloid rotifers in avoiding the early extinction suffered by most asexuals.     

Specific proteins associated with Creutzfeldt-Jakob disease and scrapie share antigenic and carbohydrate determinants.

Small amounts of brain tissue (2 g) infected with Creutzfeldt-Jakob disease (CJD) can be fractionated by using a simple 1-day method that includes lysis with N-lauroylsarcosine. Unique fibrils have been identified previously in scrapie- and CJD-infected tissue. These fibrils were abundant in final fractions. Preparations from human CJD autopsy material and from experimental hamster and guinea pig CJD all displayed readily identifiable fibrils that were not seen in control preparations. Thus, these methods appear to be of value in biopsy diagnosis of suspected human cases of CJD. Lysis with N-lauroylsarcosine quantitatively solubilized infectivity from membrane-rich fractions. Significant infectivity was recovered in microfractionations. After proteinase K digestion, a diffuse band at 29 kDa was detectable on NaDodSO4/PAGE. This 29-kDa material was not present in uninfected control brain and was similar to that seen in scrapie. Protein blots of human, guinea pig, and hamster CJD fractions were tested with an antibody raised against a 29-kDa band from mouse scrapie; 29-kDa proteins were labeled in all CJD and scrapie fractions but not in controls. These results indicate that specific proteins in both these diseases share common antigenic determinants. Ricin and wheat germ agglutinin, but not concanavalin A, also labeled a portion of the 29-kDa band from hamster CJD and hamster scrapie fractions, but they did not label any bands in normal hamster fractions at the same gel protein loads. When proteinase K treatment was omitted, specific bands of approximately equal to 35 kDa were detected in CJD samples. These results are consistent with the idea that some CJD- and scrapie-specific proteins are glycoproteins or sialoglycoproteins that can reside in or possibly derive from cell membranes.     

Cell fusion induced by scrapie and Creutzfeldt-Jakob virus-infected brain preparations.

Cell fusion was induced by brain extracts containing the scrapie virus and the virus of Creutzfeldt-Jakob disease. The assay involved quantitation of colony-forming ability in a double selection system, strandardized against fusion induced by Sendai virus. Correlation between the logarithm of virus dilution and the hybrid colony number gave similar curves for scrapie virus and Sendai virus. Fusion induction may explain some aspects of pathogenesis in these diseases and provide a potential in vitro assay.     

The 200- and 150-kDa neurofilament proteins react with IgG autoantibodies from patients with kuru, Creutzfeldt-Jakob disease, and other neurologic diseases.

Sera from 65 patients with spongiform virus encephalopathies (29 with kuru, 36 with Creutzfeldt-Jakob disease), 79 with other neurologic diseases, and 65 control subjects were examined for reactivity in immunoblots of preparations of myelinated axons and neurofilaments from mouse brain. The sera reacted most frequently with the 200-kDa and 150-kDa neurofilament proteins and less frequently with the 70-kDa neurofilament protein and a 62-kDa neurofilament-associated protein. The sera reacted with the same proteins as those which reacted with rabbit and mouse polyclonal antibodies and mouse monoclonal antibody to neurofilament proteins. Serum reactions were also seen with Trixon X-100 extracts of chimpanzee brain and bovine spinal cord but not with Triton extracts of liver, kidney, and muscle.     

Cells infected with scrapie and Creutzfeldt-Jakob disease agents produce intracellular 25-nm virus-like particles

We had repeatedly found approximately 25-nm-diameter virus-like particles in highly infectious brain fractions with little prion protein (PrP), and therefore we searched for similar virus-like particles in situ in infected cell lines with high titers. Neuroblastoma cells infected with the 22L strain of scrapie as well as hypothalamic GT cells infected with the FU Creutzfeldt-Jakob disease agent, but not parallel mock controls, displayed dense 25-nm virus-like particles in orthogonal arrays. These particles had no relation to abnormal PrP amyloid in situ, nor were they labeled by PrP antibodies that faithfully recognized rough endoplasmic reticulum membranes and amyloid fibrils, the predicted sites of normal and pathological intracellular PrP. Additionally, phorbol ester stimulated the production of abnormal PrP gel bands by >5-fold in infected N2a + 22L cells, yet this did not increase either the number of virus-like arrays or the infectious titer of these cells. Thus, the 25-nm infection-associated particles could not be prions. Synaptic differentiation and neurodegeneration, as well as retroviruses that populate the rough endoplasmic reticulum of neuroblastoma cells, were not required for particle production. The 25-nm particle arrays in cultured cells strongly resembled those first described in 1968 in synaptic regions of scrapie-infected brain and subsequently identified in many natural and experimental TSEs. The high infectivity of comparable, isolated virus-like particles that show no intrinsic PrP by antibody labeling, combined with their loss of infectivity when nucleic acid-protein complexes are disrupted, make it likely that these 25-nm particles are the causal TSE virions that induce late-stage PrP brain pathology.     

The 200- and 150-kDa neurofilament proteins react with IgG autoantibodies from chimpanzees with kuru or Creutzfeldt-Jakob disease; a 62-kDa neurofilament-associated protein reacts with sera from sheep with natural scrapie.

Sera from 46 chimpanzees with spongiform encephalopathy (18 kuru, 28 Creutzfeldt-Jakob disease) and sera from 12 sheep with natural scrapie were tested for reactivity with immunoblots of neurofilament preparations obtained from mouse brain. The sera from the chimpanzees reacted mainly with the 200- and 150-kDa proteins of the neurofilament triplet and less frequently with the 70-kDa component of the triplet and with a 62-kDa neurofilament-associated protein. In contrast, the sera of sheep with natural scrapie reacted exclusively against the 62-kDa protein. The specificity of the reactions was established by comparison of sera reactivities with those of rabbit and mouse polyclonal antibodies and mouse monoclonal antibody to neurofilament proteins.     

The kuru infectious agent is a unique geographic isolate distinct from Creutzfeldt–Jakob disease and scrapie agents

Human sporadic Creutzfeldt–Jakob disease (sCJD), endemic sheep scrapie, and epidemic bovine spongiform encephalopathy (BSE) are caused by a related group of infectious agents. The new U.K. BSE agent spread to many species, including humans, and clarifying the origin, specificity, virulence, and diversity of these agents is critical, particularly because infected humans do not develop disease for many years. As with viruses, transmissible spongiform encephalopathy (TSE) agents can adapt to new species and become more virulent yet maintain fundamentally unique and stable identities. To make agent differences manifest, one must keep the host genotype constant. Many TSE agents have revealed their independent identities in normal mice. We transmitted primate kuru, a TSE once epidemic in New Guinea, to mice expressing normal and ≈8-fold higher levels of murine prion protein (PrP). High levels of murine PrP did not prevent infection but instead shortened incubation time, as would be expected for a viral receptor. Sporadic CJD and BSE agents and representative scrapie agents were clearly different from kuru in incubation time, brain neuropathology, and lymphoreticular involvement. Many TSE agents can infect monotypic cultured GT1 cells, and unlike sporadic CJD isolates, kuru rapidly and stably infected these cells. The geographic independence of the kuru agent provides additional reasons to explore causal environmental pathogens in these infectious neurodegenerative diseases.     

Occupational exposure to ionizing radiation is associated with autoimmune thyroid disease

CONTEXT: The thyroid gland is a potential target organ for radiation-related damage. OBJECTIVE: The aim of the analysis was to investigate the association between occupational exposure to ionizing radiation and autoimmune thyroid disease (AITD). DESIGN: Our design was the cross-sectional Study of Health in Pomerania. SETTING: The setting was the general community. SUBJECTS: Analyses were performed in a population-based sample of 4299 subjects. Among them, 160 persons reported a history of occupational exposure to ionizing radiation. MAIN OUTCOME MEASURE: AITD was defined as the combined presence of hypoechogenicity in thyroid ultrasound and antithyroxiperoxidase antibodies greater than 200 IU/ml. RESULTS: Females with occupational exposure to ionizing radiation had more often AITD than nonexposed females (10.0 vs. 3.4%; P < 0.05). This association persisted after adjustment for relevant confounders (odds ratio, 3.46; 95% confidence interval, 1.16-10.31; P < 0.05). In males, there were too few subjects who fulfilled the criteria of AITD, but the association between the exposure to radiation and hypoechogenicity of the thyroid gland barely missed statistical significance (odds ratio, 2.20; 95% confidence interval, 0.92-5.26; P = 0.08). In both females and males, subjects who reported a length of exposure of more than 5 yr exhibited the highest risk of the endpoints. CONCLUSIONS: We conclude that occupational exposure to ionizing radiation is related to the risk of AITD. The usage of thyroid protection shields by radiation workers is strongly recommended.     

Exposure of the operator to ionizing radiation during intracoronary radiation therapy

This study examined the radiation exposure levels and safety of medical personnel during intracoronary radiotherapy procedures. The data of 34 stenosis patients from a total of 42 irradiated patients who participated in the Seoul National University Hospital Postangioplasty Rhenium (SPARE) trial were analyzed. Intracoronary radiotherapy was delivered to the patient immediately after angioplasty ballooning. The prescribed dose was 17 Gy to the media of the diseased artery and was delivered via a Re-188 filled balloon catheter. Dosimetry was carried out with a Geiger-Müller counter at eight different points. The selected maximum and whole-body exposed doses to the operator were 10 cm and 40 cm from the patient's heart. Median delivered activity was 111.6 MCi, with an average treatment time of 576 seconds. Average exposed dose rates at 10 cm and 40 cm from the patient's heart level were 47.2 mrem/hour and 29.6 mrem/hour, respectively, and average exposed doses per treatment were 7.0 mrem and 4.9 mrem, respectively. Exposure levels measured were much lower than the recommended limit of 50 mSv for radiation workers or 1 mSv for the general population, as recommended by the International Commission on Radiological Protection. This study proves that the method of intracoronary radiotherapy currently adopted and which is the basis of this trial is safe with respect to radiation protection.     

Predicted highly expressed and putative alien genes of Deinococcus radiodurans and implications for resistance to ionizing radiation damage

Predicted highly expressed (PHX) and putative alien genes determined by codon usages are characterized in the genome of Deinococcus radiodurans (strain R1). Deinococcus radiodurans (DEIRA) can survive very high doses of ionizing radiation that are lethal to virtually all other organisms. It has been argued that DEIRA is endowed with enhanced repair systems that provide protection and stability. However, predicted expression levels of DNA repair proteins with the exception of RecA tend to be low and do not distinguish DEIRA from other prokaryotes. In this paper, the capability of DEIRA to resist extreme doses of ionizing and UV radiation is attributed to an unusually high number of PHX chaperone/degradation, protease, and detoxification genes. Explicitly, compared with all current complete prokaryotic genomes, DEIRA contains the greatest number of PHX detoxification and protease proteins. Other sources of environmental protection against severe conditions of UV radiation, desiccation, and thermal effects for DEIRA are the several S-layer (surface structure) PHX proteins. The top PHX gene of DEIRA is the multifunctional tricarboxylic acid (TCA) gene aconitase, which, apart from its role in respiration, also alerts the cell to oxidative damage.