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R Gabizon

Publications and source records attributed to R Gabizon.

At least 37 records · Page 2Linked to original sources
PubMed1996

Evidence for the conformation of the pathologic isoform of the prion protein enciphering and propagating prion diversity.

The fundamental event in prion diseases seems to be a conformational change in cellular prion protein (PrPC) whereby it is converted into the pathologic isoform PrPSc. In fatal familial insomnia (FFI), the protease-resistant fragment of PrPSc after deglycosylation has a size of 19 kilodaltons, whereas that from other inherited and sporadic prion diseases is 21 kilodaltons. Extracts from the brains of FFI patients transmitted disease to transgenic mice expressing a chimeric human-mouse PrP gene about 200 days after inoculation and induced formation of the 19-kilodalton PrPSc fragment, whereas extracts from the brains of familial and sporadic Creutzfeldt-Jakob disease patients produced the 21-kilodalton PrPSc fragment in these mice. The results presented indicate that the conformation of PrPSc functions as a template in directing the formation of nascent PrPSc and suggest a mechanism to explain strains of prions where diversity is encrypted in the conformation of PrPSc.

Animals↗
PubMed1996

Effect of scrapie infection on the activity of neuronal nitric-oxide synthase in brain and neuroblastoma cells.

Nitric-oxide synthase (NOS) is responsible for the synthesis of nitric oxide which serves as a neural messenger in the central nervous system. NOS activity was markedly inhibited in brains of mice and hamsters and neuroblastoma cells infected with scrapie (ScN2a). The decrease in activity was in accordance with decreased NADPH-diaphorase-positive cells and decreased staining of NOS-positive cells demonstrated by specific anti-NOS antibodies. However, the specific nNOS mRNA in ScN2a was elevated when compared with normal neuroblastoma cells (N2a). Immunoblotting of fractions from these cell lines with an anti-nNOS monoclonal antibody revealed a band of nNOS from N2a and two bands with a lower molecular weight in ScN2a cells. Furthermore, NOS in ScN2a cells was insoluble in nondenaturing detergents. This insolubility is one of the landmark properties of PrPSc. It is, therefore, possible that nNOS in scrapie-infected cells and brains is aberrantly folded, resulting in an insoluble and inactive enzyme.

Animals↗
PubMed1996

Insoluble wild-type and protease-resistant mutant prion protein in brains of patients with inherited prion disease.

We studied prion proteins (PrP) in skin and brains of Libyan Jews carrying the E200K mutation who died of familial Creutzfeldt-Jakob disease (CJD). Unexpectedly, studies with brain showed that PrP molecules encoded both by the wild-type (wt) and mutant alleles exhibit altered properties characteristic of the prion protein associated with prion diseases (PrPSc). Using monospecific antisera, we found that wtPrP was insoluble in the brains of three patients who were heterozygous for the E200K mutation, whereas mutant PrP was both insoluble and protease-resistant. Our results argue that both wild-type and mutant PrP undergo conformational changes and are particularly intriguing, because the normal isoform PrPc is soluble in nondenaturing detergents and is readily digested by proteases, whereas PrPSc is insoluble and resistant to proteolytic digestion. Our findings indicate that insoluble wtPrP represents a conformational intermediate, the first to be identified, within a pathway in which PrPc is converted to PrPSc.

Amino Acid Sequence↗
PubMed1995

Prion protein (PrP) synthetic peptides induce cellular PrP to acquire properties of the scrapie isoform.

Conversion of the cellular isoform of prion protein (PrPC) into the scrapie isoform (PrPSc) involves an increase in the beta-sheet content, diminished solubility, and resistance to proteolytic digestion. Transgenetic studies argue that PrPC and PrPSc form a complex during PrPSc formation; thus, synthetic PrP peptides, which mimic the conformational pluralism of PrP, were mixed with PrPC to determine whether its properties were altered. Peptides encompassing two alpha-helical domains of PrP when mixed with PrPC produced a complex that displayed many properties of PrPSc. The PrPC-peptide complex formed fibrous aggregates and up to 65% of complexed PrPC sedimented at 100,000 x g for 1 h, whereas PrPC alone did not. These complexes were resistant to proteolytic digestion and displayed a high beta-sheet content. Unexpectedly, the peptide in a beta-sheet conformation did not form the complex, whereas the random coil did. Addition of 2% Sarkosyl disrupted the complex and rendered PrPC sensitive to protease digestion. While the pathogenic A117V mutation increased the efficacy of complex formation, anti-PrP monoclonal antibody prevented interaction between PrPC and peptides. Our findings in concert with transgenetic investigations argue that PrPC interacts with PrPSc through a domain that contains the first two putative alpha-helices. Whether PrPC-peptide complexes possess prion infectivity as determined by bioassays remains to be established.

Animals↗
PubMed1995

Prion propagation in mice expressing human and chimeric PrP transgenes implicates the interaction of cellular PrP with another protein.

Transgenic (Tg) mice expressing human (Hu) and chimeric prion protein (PrP) genes were inoculated with brain extracts from humans with inherited or sporadic prion disease to investigate the mechanism by which PrPC is transformed into PrPSc. Although Tg(HuPrP) mice expressed high levels of HuPrPC, they were resistant to human prions. They became susceptible to human prions upon ablation of the mouse (Mo) PrP gene. In contrast, mice expressing low levels of the chimeric transgene were susceptible to human prions and registered only a modest decrease in incubation times upon MoPrP gene disruption. These and other findings argue that a species-specific macromolecule, provisionally designated protein X, participates in prion formation. While the results demonstrate that PrPSc binds to PrPC in a region delimited by codons 96 to 167, they also suggest that PrPC binds protein X through residues near the C-terminus. Protein X might function as a molecular chaperone in the formation of PrPSc.

Animals↗
PubMed1995

Complete penetrance of Creutzfeldt-Jakob disease in Libyan Jews carrying the E200K mutation in the prion protein gene.

BACKGROUND: Creutzfeldt-Jakob disease (CJD) is a prion disease which is manifest as a sporadic, inherited, and transmissible neurodegenerative disorder. The mean age at onset of CJD is approximately 60 years, and as such, many people destined to succumb undoubtedly die of other illnesses first. The delayed onset of CJD has complicated the analysis of inherited forms of the illness and led to the suggestion that mutations in the prion protein (PrP) gene are necessary but not sufficient for prion disease despite genetic linkage; indeed, an environmental factor such as a ubiquitous virus has been proposed as a second necessary factor. MATERIALS AND METHODS: To examine what appeared to be incomplete penetrance, we applied a life-table analysis to clinical and pedigree data from a cluster population of Libyan Jews in which the E200K mutation is prevalent. The study population included 42 affected and 44 unaffected members of 13 Libyan Jewish families, all of whom possessed the E200K mutation. RESULTS: The calculated value using life table analysis is 0.77 at age 70 which increases to 0.89 if a mutation carrier survives to age 80 and 0.96 if age 80 is surpassed. CONCLUSIONS: These data argue that the E200K mutation alone is sufficient to cause prion disease and does so in an age-dependent manner.

Adolescent↗
PubMed1994

Mutation in codon 200 and polymorphism in codon 129 of the prion protein gene in Libyan Jews with Creutzfeldt-Jakob disease.

Various mutations in the prion protein (PrP) gene are associated with Creutzfeldt-Jakob disease (CJD), a transmissible fatal neurodegenerative disorder. Among Libyan Jews, CJD is a familial disease with an incidence about 100 times higher than the worldwide population. CJD in this community segregates with a point mutation at codon 200 of the PrP gene which causes the substitution of lysine for glutamate. This mutation was found in all definitely affected individuals and yields a maximum lod score of 4.85. Some healthy elderly mutation carries above 65 years of age were identified, suggesting partial penetrance. Homozygous patients have the same disease pattern and age of onset as heterozygous patients, which argues that CJD associated with the codon 200 lysine mutation is a true dominant disorder. In the caucasian population, Palmer et al. (1991) reported an association between homozygosity in a polymorphic site at codon 129 of the PrP gene, coding for either valine or methionine, with a tendency to acquire the sporadic or iatrogenic forms of CJD, as well as with disease age of appearance in the genetic type. The incidence of the polymorphism at codon 129 in the control Libyan population is similar to the one found in the caucasian population. In the Libyan CJD patients, the codon 200 mutation is within a Met129-encoding allele. The incidence of the Met allele is significantly higher in the affected pedigrees than in the control Libyan population; however, no difference was detected between CJD patients, codon 200 healthy carriers, and their normal family members.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Genetics and biochemistry of Creutzfeldt-Jakob disease in Libyan Jews.

A focus of Creutzfeldt-Jakob disease (CJD) among Jews from Libyan origin was identified in Israel 20 years ago. The incidence of the disease in this ethnic group is about 100 times more than in the worldwide population. The consumption of lightly cooked sheep brain has been invoked to explain the high incidence of CJD in this community. The discovery of mutations in the PrP gene which segregates with other familial prion diseases such as Gerstmann-Straussler syndrome (GSS) lead us to perform a molecular genetic study and compare it to an epidemiological survey among the Libyan community. The epidemiological data suggests a very high familial incidence of CJD in this population and a molecular genetic research elucidated that CJD segregates with a point mutation at codon 200 of the PrP gene resulting in the substitution of Lysine for Glutamate. This mutation was found in some 40 CJD patients of Libyan origin and was not found in one Moroccan Jew suffering from CJD. It was also absent in almost 100 healthy Libyan controls above the age of 60. This result strongly supports a genetic etiology for CJD pathogenesis in the Libyan Jewish community and disregards the previous culinary hypothesis. The disease is vertically transmitted in autosomal dominant inheritance with unknown penetrance. All our patients were heterozygote for the mutation except one homozygote patient. The course of the disease in this patient was identical to the heterozygote patients, strongly arguing that inherited CJD displays complete phenotypic dominance.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Northern↗
PubMed1993

Attempts to restore scrapie prion infectivity after exposure to protein denaturants.

A wealth of experimental evidence argues that infectious prions are composed largely, if not entirely, of the scrapie isoform of the prion protein. We attempted to restore scrapie infectivity after exposure to protein denaturants including urea, chaotropic salts, and SDS. None of the procedures restored infectivity. Dialysis to remove slowly chaotropic ions and urea failed to restore scrapie infectivity. Attempts to create monomers of the scrapie isoform of the prion protein under nondenaturing conditions using a wide variety of detergents have been unsuccessful, to date, except for one report claiming that scrapie infectivity could be recovered from 12% polyacrylamide gels after SDS/PAGE [Brown, P., Liberski, P. P., Wolff, A. & Gajdusek, D. C. (1990) Proc. Natl. Acad. Sci. USA 87, 7240-7244]. We found that < 0.001% of the infectious prion titer could be recovered from the region of a polyacrylamide gel where the denatured proteinase K-resistant core of the scrapie isoform of the prion protein and other 30-kDa proteins migrate. We conclude that under the denaturing conditions used for SDS/PAGE, the scrapie isoform of the prion protein is denatured and little or no renaturation occurs upon injection of fractions eluted from gels into animals for bioassays.

Animals↗
PubMed1993

Heparin-like molecules bind differentially to prion-proteins and change their intracellular metabolic fate.

PrPSc is the only known component of the scrapie prion. The difference between PrPSc and its normal isoform PrPc is probably conformational, since no difference has been found in the amino acid sequence or postranslational modifications between both proteins. Heparan sulfate (HS) has been shown to be a component of amyloid plaques in a number of diseases including the prion diseases. We now present evidence that PrP can specifically bind to heparin-like compounds and that this interaction might have a physiological significance. HS can increase the concentration of PrP in normal neuroblastoma cells, whereas low molecular weight heparin (LMWH) does not. In contrast, LMWH and other heparin-like molecules, excluding HS, can inhibit the synthesis of PrPSc in scrapie infected cells and reverse their phenotype back to normal as judged by measurement of PrPSc by immunoblotting and by infectivity experiments. Whether an interaction between PrP and glycosaminoglycans plays a direct role in the conversion of PrPc into PrPSc remains to be established.

Amino Acids↗
PubMed1993

Mutation and polymorphism of the prion protein gene in Libyan Jews with Creutzfeldt-Jakob disease (CJD).

The inherited prion diseases are neurodegenerative disorders which are not only genetic but also transmissible. More than a dozen mutations in the prion protein gene that result in nonconservative amino acid substitutions segregate with the inherited prion diseases including familial Creutzfeldt-Jakob disease (CJD). In Israel, the incidence of CJD is about 1 case/10(4) Libyan Jews. A Lys200 substitution segregates with CJD and is reported here to be genetically linked to CJD with a lod score of > 4.8. Some healthy elderly Lys200 carriers > age 65 years were identified, suggesting the possibility of incomplete penetrance. In contrast, no linkage was found between the development of familial CJD and a polymorphism encoding either Met129 or Val129. All Libyan Jewish CJD patients with the Lys200 mutation encode a Met129 on the mutant allele. Homozygosity for Met129 did not correlate with age at disease onset or the duration of illness. The frequency of the Met129 allele was higher in the affected pedigrees than in a control population of Libyan Jews. The frequency of the Met129 and Val129 alleles in the control Libyan population was similar to that found in the general Caucasian population. The identification of three Libyan Jews homozygous for the Lys200 mutation suggests frequent intrafamilial marriages, a custom documented by genealogical investigations.

Age of Onset↗
PubMed1992

Presence of prion protein in peripheral tissues of Libyan Jews with Creutzfeldt-Jakob disease.

The prion protein (PrP) gene on chromosome 20 encodes a protein designated PrPC. An abnormal, protease-resistant isoform of PrPC, denoted PrPCJD or PrPSc, is present in the brains of patients with Creutzfeldt-Jakob disease (CJD). In Libyan Jews, CJD segregates with a point mutation at codon 200 of the PrP gene, resulting in the substitution of lysine for glutamate. In the present study, we examined the presence of PrP in fibroblasts and leukocytes derived from eight CJD patients with the codon 200 mutation. In cultured fibroblasts as well as in leukocytes, there was a significant increase in PrP as judged by immunocytochemistry in addition to immunoblotting. Most of the PrP in fibroblasts and leukocytes could be released from the external surface by phosphatidylinositol-specific phospholipase C, a property characteristic of PrPC. In leukocytes only, part of the protein was protease resistant, resembling PrPCJD. The concentration of PrP mRNA was similar in fibroblast lines derived from controls and CJD patients. These results suggest that in CJD patients carrying a mutation at codon 200 of the PrP gene, the metabolism of PrP, rather than PrP synthesis, is abnormal.

Animals↗
PubMed1991

Mutation of the prion protein in Libyan Jews with Creutzfeldt-Jakob disease.

BACKGROUND: Creutzfeldt-Jakob disease is a transmissible neurodegenerative disorder that occurs more than 100 times more frequently among Libyan Jews than in the worldwide population. We examined 11 patients with the disease--10 Libyan Jews from Israel and 1 Libyan Jew from Italy--to determine whether abnormalities of the prion protein could be detected in them. Abnormal forms of this host-encoded protein are the predominant if not sole components of the transmissible agent that causes the disease. METHODS: The prion-protein open-reading frame in peripheral-leukocyte DNA from the Italian patient was amplified with the polymerase chain reaction and sequenced. Allele-specific oligonucleotide hybridization was used to assess a prion-protein codon 200 lysine mutation in the 10 Israeli patients and 37 control subjects. RESULTS: The prion-protein sequence in DNA from the Italian patient revealed a single nucleotide change (G----A) at the first position of codon 200 that resulted in a substitution of lysine for glutamate. This substitution was detected in all 10 Israeli patients, 8 of whom had a positive family history of Creutzfeldt-Jakob disease. One patient was homozygous for the lysine mutation, and her clinical course did not differ from that of the patients heterozygous for the mutation. The lysine mutation was not found in one Moroccan Jew from Israel with Creutzfeldt-Jakob disease. CONCLUSIONS: The codon 200 lysine mutation of the prion-protein gene is consistently present among Libyan Jews with Creutzfeldt-Jakob disease, strongly supporting a genetic pathogenesis of their illness. The similarity of the clinical courses of the patient homozygous for this mutation and the patients heterozygous for it argues that familial Creutzfeldt-Jakob disease is a true dominant disorder.

Adult↗
PubMed1990

Prion liposomes.

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Animals↗
PubMed1989

Immunoaffinity purification and neutralization of scrapie prions.

The scrapie agent causes a degenerative neurologic disease and can be transmitted to laboratory rodents. The unusual properties of the scrapie agent prompted introduction of the term prion in order to distinguish this class of novel pathogens from viruses and viroids. The scrapie prion protein (PrPSc) is the only component of the infectious scrapie prion identified, to date. Although many biochemical and genetic lines of evidence argue that PrPSc is a major component of the infectious particle, the most convincing data is derived from immunoaffinity purification studies. Limited proteinase K digestion of hamster brain PrPSc produced PrP 27-30. After dispersion of brain microsomes isolated from scrapie-infected hamsters into detergent-lipid-protein complexes (DLPC), copurification of PrPSc and scrapie infectivity was obtained with PrP 27-30 monoclonal antibody affinity columns. PrPSc was enriched approximately 5700-fold with respect to total brain protein while scrapie prion infectivity was enriched approximately -4000-fold. The ratio of prion titer to PrPSc remained constant throughout purification. Heterologous monoclonal antibody columns failed to bind either PrpSc or scrapie infectivity. Polyclonal rabbit PrP antiserum raised against sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-purified PrP 27-30 reduced scrapie infectivity dispersed into DLPC by a factor of 100. Our findings represent the first direct immunologic and chromatographic demonstrations of a relationship between PrPSc and prion infectivity as well as providing additional support for the contention that PrPSc is a major component of the infectious scrapie particle. While these results and those of other studies establish that PrPSc is a component of the infectious prion, the possibility of a second component such as a small nucleic acid which might be required for infection must still be considered. PrPSc is encoded by a single copy cellular gene and not by a hypothetical-nucleic acid within purified prion preparations. Normal, uninfected cells express the cellular prion protein (PrPC). Both PrPSc and PrPC appear to be translated from the same 2.1-kb mRNA. The N-terminal amino acid sequences of hamster PrPC and PrPSc are identical; both correspond to that predicted by the translated prion protein (PrP) gene sequence. While the chemical difference between PrPC and PrPSc remains unknown, the organization of the PrP gene argues that it results from a posttranslational event. The mouse PrP gene is on chromosome 2 and is linked to a gene controlling the scrapie incubation time (Prn-i).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Properties of scrapie prion protein liposomes.

Purified scrapie prions contain one identifiable macromolecule, PrP 27-30, which polymerizes into rod-shaped amyloids. The rods can be dissociated with retention of scrapie infectivity upon incorporation of PrP 27-30 into detergent-lipid-protein complexes (DLPC) as well as liposomes. As measured by end-point titration, scrapie infectivity was increased greater than 100-fold upon dissociating the rods into liposomes. The incorporation of PrP 27-30 into liposomes was demonstrated by immunoelectron microscopy using colloidal gold. Detergent extraction of prion liposomes followed by chloroform/methanol extraction resulted in the reappearance of rods, indicating that this process is reversible. Scrapie prion infectivity in rods and liposomes was equally resistant to inactivation by irradiation at 254 nm and was unaltered by exposure to nucleases. A variety of lipids used for producing DLPC and liposomes did not alter infectivity. Fluorescently labeled PrP 27-30 in liposomes was used to study its entry into cultured cells. Unlike the rods which remained as large fluorescent extracellular masses, the PrP 27-30 in liposomes rapidly entered the cells and was seen widely distributed within the interior of the cell. PrP 27-30 is derived by limited proteolysis from a larger protein designated PrP(Sc) which is membrane bound. PrP(Sc) in membrane fractions was solubilized by incorporation in DLPC, thus preventing its aggregation into amyloid rods. The functional solubilization of scrapie prion proteins in DLPC and liposomes offers new approaches to the study of prion structure and the mechanism by which they cause brain degeneration.

Animals↗