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D Groth

Publications and source records attributed to D Groth.

At least 55 records · Page 3Linked to original sources
PubMed1993

Conversion of alpha-helices into beta-sheets features in the formation of the scrapie prion proteins.

Prions are composed largely, if not entirely, of prion protein (PrPSc in the case of scrapie). Although the formation of PrPSc from the cellular prion protein (PrPC) is a post-translational process, no candidate chemical modification was identified, suggesting that a conformational change features in PrPSc synthesis. To assess this possibility, we purified both PrPC and PrPSc by using nondenaturing procedures and determined the secondary structure of each. Fourier-transform infrared (FTIR) spectroscopy demonstrated that PrPC has a high alpha-helix content (42%) and no beta-sheet (3%), findings that were confirmed by circular dichroism measurements. In contrast, the beta-sheet content of PrPSc was 43% and the alpha-helix 30% as measured by FTIR. As determined in earlier studies, N-terminally truncated PrPSc derived by limited proteolysis, designated PrP 27-30, has an even higher beta-sheet content (54%) and a lower alpha-helix content (21%). Neither PrPC nor PrPSc formed aggregates detectable by electron microscopy, while PrP 27-30 polymerized into rod-shaped amyloids. While the foregoing findings argue that the conversion of alpha-helices into beta-sheets underlies the formation of PrPSc, we cannot eliminate the possibility that an undetected chemical modification of a small fraction of PrPSc initiates this process. Since PrPSc seems to be the only component of the "infectious" prion particle, it is likely that this conformational transition is a fundamental event in the propagation of prions.

Animals↗
PubMed1993

Ablation of the prion protein (PrP) gene in mice prevents scrapie and facilitates production of anti-PrP antibodies.

Mice, homozygous for prion protein (PrP) gene ablation (Prn-p0/0), develop normally and remain well > 500 days after inoculation with murine scrapie prions. In contrast, wild-type mice developed scrapie < 165 days after inoculation and most Prn-p0/+ mice, heterozygous for disruption of the PrP gene, exhibited signs of central nervous system dysfunction between 400 and 465 days after inoculation. In situ immunoblots showed widespread deposition of scrapie PrP (PrPSc) in the brains of both wild-type Prn-p+/+ and Prn-p0/+ mice, while neither cellular PrP (PrPC) nor PrPSc was detected in the brains of Prn-p0/0 mice. In contrast to Prn-p+/+ and Prn-p0/+ mice, Prn-p0/0 mice failed to propagate prion infectivity as measured by bioassays. Syrian hamster (SHa) PrP transgenes rendered Prn-p0/0 mice susceptible to prions containing SHaPrPSc. Immunization of Prn-p0/0 mice with purified, infectious mouse or SHa prions dispersed in Freund's adjuvant produced antisera that bound mouse, SHa, and human PrP on Western blots. Presumably, the lack of PrPC expression in Prn-p0/0 mice prevents them from becoming tolerant to the immunogen. The resistance of Prn-p0/0 mice to developing scrapie after inoculation with murine prions supports the hypothesis that PrPSc is essential for both transmission and pathogenesis of the prion diseases.

Animals↗
PubMed1993

Three scrapie prion isolates exhibit different accumulation patterns of the prion protein scrapie isoform.

To investigate the molecular basis of prion diversity, we inoculated transgenic mice expressing the Syrian hamster prion protein (PrP) with three distinct prion isolates. We compared the three isolates designated Sc237, 139H, and Me7H in Tg(SHaPrP)7 mice with clinical signs of scrapie because the incubation times with these mice are considerably shorter than the times found with hamsters. Each prion isolate produced a distinctive pattern of the scrapie isoform of PrP (PrPSc) accumulation, as determined by histoblotting, a technique developed for the regional mapping of PrPSc deposition. The PrPSc pattern with the Me7H isolate was particularly interesting because it appeared to be confined to the hypothalamus and related structures--including the interstitial nucleus of the stria terminalis, the paraventricular nucleus of the thalamus, and periaqueductal grey. Additionally, the regions of PrPSc accumulation remained highly restricted, even though the incubation time for Me7H scrapie was significantly longer than with Sc237 and 139H isolates. Neuropathological changes characterized by neuronal vacuolation and astrocytic gliosis were confined to those regions where PrPSc accumulated. These findings argue that the cell-specific propagation of prion isolates may be responsible for different properties exhibited by each of the isolates.

Animals↗
PubMed1993

Propagation of prions with artificial properties in transgenic mice expressing chimeric PrP genes.

Transgenic mice expressing chimeric prion protein (PrP) genes derived from Syrian hamster (SHa) and mouse (Mo) PrP genes were constructed. One SHa/MoPrP gene, designated MH2M PrP, contains five amino acid substitutions encoded by SHaPrP, while another construct, designated MHM2 PrP, has two substitutions. Transgenic (Tg) (MH2M PrP) mice were susceptible to both Syrian hamster and mouse prions, whereas three lines expressing MHM2 PrP were resistant to Syrian hamster prions. The brains of Tg(MH2M PrP) mice dying of scrapie contained chimeric PrPSc and prions with an artificial host range favoring propagation in mice that express the corresponding chimeric PrP and were also transmissible, at reduced efficiency, to nontransgenic mice and hamsters. Our findings provide genetic evidence for homophilic interactions between PrPSc in the inoculum and PrPc synthesized by the host.

Animals↗
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↗
PubMed1991

Search for a putative scrapie genome in purified prion fractions reveals a paucity of nucleic acids.

Scrapie can be transmitted by novel infectious pathogens termed prions. No evidence for a scrapie-specific nucleic acid has been detected to date. To investigate amounts, types and sizes of nucleic acid molecules associated with prions in purified preparations, aliquots were deproteinized, and the nucleic acids analysed by PAGE and silver staining. Digestion with nucleases and exposure to Zn2+ prior to analysis substantially diminished the content of nucleic acids, but did not alter the prion titre indicating that those nucleic acids which were removed are not essential for infectivity. Since a single species of scrapie-specific nucleic acid could not be identified, we explored the unprecedented possibility of scrapie-specific nucleic acids of variable length which are biologically active. If such molecules of variable length exist then they might be hidden within the background smear on silver-stained gels after PAGE. A new procedure designated return refocusing gel electrophoresis (RRGE) was developed to identify heterogeneous nucleic acids in purified prion fractions. The content of variable length nucleic acids was reduced by a factor of 10 by exhaustive Bal 31 exonuclease digestion after dispersion of purified prions into detergent-lipid-protein complexes. For example, a typical sample after Bal 31 digestion contained approximately 4 ng of nucleic acid of variable length and 10(8.7) ID50 units of scrapie prion infectivity. Consideration of different models for a hypothetical scrapie-specific nucleic acid suggests that such a molecule would have to be: (i) quite small (less than 100 nucleotides), (ii) possess a particle-to-infectivity ratio near unity or (iii) heterogeneous in size. Although our results do not eliminate the possibility that prions possess a scrapie-specific nucleic acid of variable length, they narrow considerably the spectrum of features specifying such a candidate molecule.

Animals↗
PubMed1991

Spontaneous neurodegeneration in transgenic mice with prion protein codon 101 proline----leucine substitution.

Gerstmann-Sträussler-Scheinker syndrome (GSS) is an autosomal, dominantly inherited, human neurodegenerative disease that can sometimes be transmitted to non-human primates and rodents through intracerebral inoculation of brain homogenates from patients. Recent studies of GSS demonstrated significant genetic linkage between GSS and a leucine substitution at codon 102 of the human prion protein (PrP) gene. Transgenic mice were created to test the biologic activity of this mutation. Spontaneous neurologic disease with spongiform degeneration developed in one of three lines of transgenic mice containing murine PrP genes with a leucine substitution at codon 101 (homologous to codon 102 in humans). Transmission studies of brain homogenates from affected mice are in progress. These results indicate that some of the clinical and pathologic features of GSS can be reproduced in a transgenic mouse paradigm; this represents the first time a dominantly inherited, neurodegenerative process similar to a human disease has been genetically modeled in an experimental animal (Hsiao and Prusiner 1990).

Amino Acid Sequence↗
PubMed1990

Transgenetic studies implicate interactions between homologous PrP isoforms in scrapie prion replication.

Transgenic (Tg) mice expressing both Syrian hamster (Ha) and mouse (Mo) prion protein (PrP) genes were used to probe the mechanism of scrapie prion replication. Four Tg lines expressing HaPrP exhibited distinct incubation times ranging from 48 to 277 days, which correlated inversely with HaPrP mRNA and HaPrPC. Bioassays of Tg brain extracts showed that the prion inoculum dictates which prions are synthesized de novo. Tg mice inoculated with Ha prions had approximately 10(9) ID50 units of Ha prions per gram of brain and less than 10 units of Mo prions. Conversely, Tg mice inoculated with Mo prions synthesized Mo prions but not Ha prions. Similarly, Tg mice inoculated with Ha prions exhibited neuropathologic changes characteristic of hamsters with scrapie, while Mo prions produced changes similar to those in non-Tg mice. Our results argue that species specificity of scrapie prions resides in the PrP sequence and prion synthesis is initiated by a species-specific interaction between PrPSc in the inoculum and homologous PrPC.

Animals↗
PubMed1990

Intracellular accumulation of the cellular prion protein after mutagenesis of its Asn-linked glycosylation sites.

The cellular isoform of the prion protein (PrPC) is a sialoglycoprotein bound almost exclusively on the external surface of the plasma membrane by a glycosyl phosphatidylinositol anchor. The deduced amino acid sequence of Syrian hamster PrPC identifies two potential sites for the addition of Asn-linked carbohydrates at amino acids 181-183 (Asn-Ile-Thr) and 197-199 (Asn-Phe-Thr). We have altered these sites by replacing the threonine residues with alanine and expressed the mutant proteins transiently in CV1 cells utilizing a mutagenesis vector with the T7 promoter located upstream from the PrP gene. The T7 RNA polymerase was supplied by infection with a recombinant vaccinia virus. The 3 mutant proteins (PrPAla183, PrPAla199 and PrPAla183/199) have a reduced relative molecular weight compared to wild-type (wt) PrP. Deglycosylation as well as synthesis in the presence of tunicamycin reduced the relative molecular weight of all the PrP species to that of the double mutant PrPAla183/199. Our results indicate that both single-site mutant prion proteins are glycosylated at non-mutated sites and they suggest that both potential sites for Asn-linked glycosylation are utilized in wt PrPC. Immunofluorescence studies demonstrate that while wt PrPC localizes to the cell surface, all the mutant PrP molecules accumulate intracellularly. The site of accumulation of PrPAla183 is probably prior to the mid-Golgi stack since this protein does not acquire resistance to endoglycosidase H. Whether the intracellular locations of the mutant PrPC species are the same as those identified for the scrapie isoform of the prion protein (PrPSc) remains to be established.

Amino Acid Sequence↗
PubMed1989

Transgenic mice expressing hamster prion protein produce species-specific scrapie infectivity and amyloid plaques.

Three transgenic mouse lines designated Tg 69, 71, and 81 were produced harboring a Syrian hamster (Ha) prion protein (PrP) gene; all expressed the cellular HaPrP isoform in their brains. Inoculation of Tg 81 mice or hamsters with Ha prions caused scrapie in integral of 75 days; nontransgenic control mice failed to develop scrapie after greater than 500 days. Tg 71 mice inoculated with Ha prions developed scrapie in integral of 170 days. Both Tg 71 and Tg 81 mice exhibited spongiform degeneration and reactive astrocytic gliosis, and they produced the scrapie HaPrP isoform in their brains. Tg 81 brains also showed HaPrP amyloid plaques characteristic of Ha scrapie and contained integral of 10(9) ID50 units of Ha prions based on Ha bioassays. Our findings argue that the PrP gene modulates scrapie susceptibility, incubation times, and neuropathology; furthermore, they demonstrate synthesis of infectious scrapie prions programmed by a recombinant DNA molecule.

Amyloid↗
PubMed1989

Diversity of oligosaccharide structures linked to asparagines of the scrapie prion protein.

Prion proteins from humans and rodents contain two consensus sites for asparagine-linked glycosylation near their C-termini. The asparagine-linked oligosaccharides of the scrapie isoform of the hamster prion protein (PrP 27-30) were released quantitatively from the purified molecule by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. The radioactive oligosaccharides were fractionated into one neutral and three acidic oligosaccharide fractions by anion-exchange column chromatography. All oligosaccharides in the acidic fractions could be converted to neutral oligosaccharides by sialidase digestion. Structural studies on these oligosaccharides including sequential exoglycosidase digestion in combination with methylation analysis revealed that PrP 27-30 contains a mixture of bi-, tri-, and tetraantennary complex-type sugar chains with Man alpha 1----6(GlcNAc beta 1----4)(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4-(Fuc alpha 1----6)GlcNAc as their core. Variation is produced by the different combination of the oligosaccharides Gal beta 1----4GlcNAc beta 1----, Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----, GlcNAc beta 1----, Sia alpha 2----3Gal beta 1----4GlcNAc beta 1----, and Sia alpha 2----6Gal beta 1----4GlcNAc beta 1---- in their outer chain moieties. When both asparagine-linked consensus sites are glycosylated, the diversity of oligosaccharide structures yields over 400 different forms of the scrapie prion protein. Whether these diverse asparagine-linked oligosaccharides participate in scrapie prion infectivity or modify the function of the cellular prion protein remains to be established.

Acetylation↗
PubMed1989

Asparagine-linked glycosylation of the scrapie and cellular prion proteins.

Post-translational modification of the scrapie prion protein (PrP) is thought to account for the unusual features of this protein. Molecular cloning of a PrP cDNA identified two potential Asn-linked glycosylation sites. Both the scrapie (PrPSc) and cellular (PrPC) isoforms were susceptible to digestion by peptide N-glycosidase F (PNGase F) but resistant to endoglycosidase H as measured by migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PNGase F digestion of PrPC yielded two proteins of Mr26K and 28K; however, the 26-k species was only a minor component. In contrast, PNGase F digestion of PrPSc yielded equimolar amounts of two proteins of Mr26K and 28K. The significance of this altered stoichiometry between the 26- and 28-kDa deglycosylated forms of PrP during scrapie infection remains to be established. Both isoforms as well as PrP 27-30, which is produced by limited proteolysis of PrPSc, exhibited a reduced number of charge isomers after PNGase F digestion. The molecular weight of PrP 27-30 was reduced from 27K-30K by PNGase F digestion to 20K-22K while anhydrous hydrogen fluoride or trifluoromethanesulfonic acid treatment reduced the molecular weight to 19K-21K and 20K-22K, respectively. Denatured PrP 27-30 was radioiodinated and then assessed for its binding to lectin columns. PrP 27-30 was bound to wheat germ agglutinin (WGA) or lentil lectins and eluted with N-acetylglucosamine or alpha-methyl-mannoside, respectively. Digestion of PrP 27-30 with sialidase prevented its binding to WGA but enhanced its binding to Ricinus communis lectin. These findings argue that PrP 27-30 probably possesses Asn-linked, complex oligosaccharides with terminal sialic acids, penultimate galactoses, and fucose residues attached to the innermost N-acetyl-glucosamine. Whether differences in Asn-linked oligosaccharide structure between PrPC and PrPSc exist and are responsible for the distinct properties displayed by these two isoforms remain to be established.

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

Scrapie prion liposomes and rods exhibit target sizes of 55,000 Da.

Scrapie is a degenerative neurologic disease in sheep and goats which can be experimentally transmitted to laboratory rodents. Considerable evidence suggests that the scrapie agent is composed largely, if not entirely, of an abnormal isoform of the prion protein (PrPSc). Inactivation of scrapie prions by ionizing radiation exhibited single-hit kinetics and gave a target size of 55,000 +/- 9000 mol wt. The inactivation profile was independent of the form of the prion. Scrapie agent infectivity in brain homogenates, microsomal fractions, detergent-extracted microsomes, purified amyloid rods, and liposomes exhibited the same inactivation profile. Our data are consistent with the hypothesis that the infectious particle causing scrapie contains approximately 2 PrPSc molecules.

Animals↗
PubMed1988

Immunoaffinity purification and neutralization of scrapie prion infectivity.

Prions are unusual infectious pathogens causing scrapie of sheep and goats as well as Creutzfeldt-Jakob disease of humans. Biochemical and genetic studies contend that the scrapie isoform of the prion protein (PrPSc) is a major component of the prion. Limited proteinase K digestion of PrPSc produced a protein of 27-30 kDa. After dispersion of brain microsomes isolated from scrapie-infected hamsters into detergent-lipid-protein complexes, copurification of PrPSc and scrapie infectivity was obtained with scrapie prion protein of 27-30 kDa monoclonal antibody-affinity columns. PrPSc was enriched approximately equal to 5700-fold with respect to total brain protein, whereas scrapie prion infectivity was enriched approximately equal to 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 prion protein antiserum raised against NaDodSO4/PAGE-purified scrapie prion protein of 27-30 kDa reduced scrapie infectivity dispersed into detergent-lipid-protein complexes by a factor of 100. These results represent 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. That PrPSc is a host-encoded protein is an important feature distinguishing prions from viruses.

Animals↗
PubMed1986

Efficacy of urinary monitoring for 4,4'-methylenebis (2-chloroaniline).

The aromatic amine 4,4'-methylenebis (2-chloroaniline) (MBOCA), an animal carcinogen, is used commercially as a curing agent for isocyanate-containing polymers. It is structurally similar to other aromatic amines that cause bladder cancer in occupationally exposed workers. Since the late 1970s, MBOCA users have relied on urinary monitoring as the primary method of assessing MBOCA exposure in the workplace. This paper (1) outlines uncertainties about MBOCa's metabolism in humans that complicate interpretation of urinary MBOCA results; (2) describes alternative laboratory techniques for measuring MBOCA in urine; and (3) discusses observations from site visits concerning the practical application of urinary monitoring. Recommendations to improve the efficacy of monitoring programs for urinary MBOCA are outlined.

Animals↗
PubMed1984

Subchronic inhalation toxicity of dimethylformamide in rats and mice.

Fisher F344 rats and B6C3F1 mice were exposed to concentrations of 0, 150, 300, 600 and 1200 ppm of dimethylformamide (DMF) for 6 hours a day, 5 days a week for 12 weeks. Detailed clinical observations were obtained weekly and body weights biweekly on all animals. Clinical chemistry and hematology evaluations were made on all rats and approximately half the mice at terminal sacrifice. Gross necropsy examinations were made on all animals. Histopathologic evaluations were conducted on selected tissues of animals of both species at all dose levels. Few overt signs of toxicity were seen in either rats or mice. There was a dose related depression in body weight gain in rats that was significant at the 1200 ppm level from the second week of study onwards. A total of 11 mice died or were sacrificed moribund during the study, 8 from the high dose and 2 from the 600 ppm dose level. Both clinical chemistry (in rats only) and gross necropsy observations, and histopathology of tissues indicate the possibility that liver may be the target in specific organ toxicity. The no-effect DMF dose was below the 150 ppm level for both rats and mice and the maximum tolerated dose was below the 600 ppm level.

Air Pollutants↗