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D Groth

Publications and source records attributed to D Groth.

At least 37 records · Page 2Linked to original sources
PubMed1997

Structure of the recombinant full-length hamster prion protein PrP(29-231): the N terminus is highly flexible.

The prion diseases seem to be caused by a conformational change of the prion protein (PrP) from the benign cellular form PrPC to the infectious scrapie form PrPSc; thus, detailed information about PrP structure may provide essential insights into the mechanism by which these diseases develop. In this study, the secondary structure of the recombinant Syrian hamster PrP of residues 29-231 [PrP(29-231)] is investigated by multidimensional heteronuclear NMR. Chemical shift index analysis and nuclear Overhauser effect data show that PrP(29-231) contains three helices and possibly one short beta-strand. Most striking is the random-coil nature of chemical shifts for residues 30-124 in the full-length PrP. Although the secondary structure elements are similar to those found in mouse PrP fragment PrP(121-231), the secondary structure boundaries of PrP(29-231) are different from those in mouse PrP(121-231) but similar to those found in the structure of Syrian hamster PrP(90-231). Comparison of resonance assignments of PrP(29-231) and PrP(90-231) indicates that there may be transient interactions between the additional residues and the structured core. Backbone dynamics studies done by using the heteronuclear [1H]-15N nuclear Overhauser effect indicate that almost half of PrP(29-231), residues 29-124, is highly flexible. This plastic region could feature in the conversion of PrPC to PrPSc by template-assisted formation of beta-structure.

Amino Acid Sequence↗
PubMed1997

Marked decrease of neuropeptide Y Y2 receptor binding sites in the hippocampus in murine prion disease.

Using autoradiographic binding methodology with monoiodinated peptide YY together with the agonists neuropeptide Y (NPY) and NPY (13-36), as well as in situ hybridization with oligonucleotide probes complementary to the NPY Y2 receptor (Y2-R) mRNA, we have studied whether or not intracerebral prion inoculation affects Y2-Rs in male CD-1 mice. Monoiodinated peptide YY binding, mainly representing Y2-Rs, was down-regulated by 85% in the CA1 strata oriens and radiatum and by 50-65% in the CA3 stratum oriens 110-140 days postinoculation. In the CA3 stratum radiatum, where the mossy fibers from the dentate granule cells project, there was a significant decrease in PYY binding at 110-120 days. Y2-R mRNA, moderately expressed both in the CA1 and CA3 pyramidal cell layers and the granule cell layer in the dentate gyrus, showed a slight, but not significant, decrease in CA3 neurons 130 days postinoculation. The results indicate that the accumulation of the scrapie prion protein in the CA1-3 region strongly inhibits NPY binding at the Y2-Rs, which, however, is only marginally due to reduced Y2-R mRNA expression. The loss of the ability of NPY to bind to inhibitory Y2-Rs may cause dysfunction of hippocampal circuits and may contribute to the clinical symptoms in mouse scrapie.

Animals↗
PubMed1997

Difference between lambs with chronic and mild dermatophilosis in frequency of alleles of CD3 gamma.

Southern blots prepared with DNA from 20 Merino lambs that previously had chronic dermatophilosis (chronic) and 20 lambs that previously had mild dermatophilosis lesions (resistant) were hybridised with DNA sequences of the genes for the T-cell receptor-beta (TCR beta), a TCR-associated peptide 'cluster designation 3 gamma chain' (CD3 gamma) and ovine Major Histocompatibility Complex class 1 (ov. MHC class 1). There was a significant difference in the incidence of an allele of CD3 gamma between the chronic and resistant lambs. No significant difference in the incidence of alleles of TCR beta or ov. MHC class 1 was detected.

Actinomycetales Infections↗
PubMed1997

Solution structure of a 142-residue recombinant prion protein corresponding to the infectious fragment of the scrapie isoform.

The scrapie prion protein (PrPSc) is the major, and possibly the only, component of the infectious prion; it is generated from the cellular isoform (PrPC) by a conformational change. N-terminal truncation of PrPSc by limited proteolysis produces a protein of approximately 142 residues designated PrP 27-30, which retains infectivity. A recombinant protein (rPrP) corresponding to Syrian hamster PrP 27-30 was expressed in Escherichia coli and purified. After refolding rPrP into an alpha-helical form resembling PrPC, the structure was solved by multidimensional heteronuclear NMR, revealing many structural features of rPrP that were not found in two shorter PrP fragments studied previously. Extensive side-chain interactions for residues 113-125 characterize a hydrophobic cluster, which packs against an irregular beta-sheet, whereas residues 90-112 exhibit little defined structure. Although identifiable secondary structure is largely lacking in the N terminus of rPrP, paradoxically this N terminus increases the amount of secondary structure in the remainder of rPrP. The surface of a long helix (residues 200-227) and a structured loop (residues 165-171) form a discontinuous epitope for binding of a protein that facilitates PrPSc formation. Polymorphic residues within this epitope seem to modulate susceptibility of sheep and humans to prion disease. Conformational heterogeneity of rPrP at the N terminus may be key to the transformation of PrPC into PrPSc, whereas the discontinuous epitope near the C terminus controls this transition.

Amino Acid Sequence↗
PubMed1997

Physical studies of conformational plasticity in a recombinant prion protein.

PrP(Sc) is known to be the major, if not the only, component of the infectious prion. Limited proteolysis of PrP(Sc) produces an N-terminally truncated polypeptide of about 142 residues, designated PrP 27-30. Recently, a recombinant protein (rPrP) of 142 residues corresponding to the Syrian hamster PrP 27-30 was expressed in Escherichia coli and purified (Mehlhorn et al., 1996). rPrP has been refolded into both alpha-helical and beta-sheet structures as well as various intermediates in aqueous buffers. The beta-sheet state and two pH-dependent alpha-helical states were characterized by CD and NMR. The alpha-helical conformation occurred only after the formation of an intramolecular disulfide bond, whereas the beta-sheet form was accessible either with or without the disulfide. Of the different alpha-helical forms studied, only those refolded in the pH range 5-8 were substantially soluble at physiological pH, exhibiting similar conformations and monomeric analytical sedimentation profiles throughout the above pH range. Furthermore, refolded alpha-rPrP showed NMR chemical shift dispersion typical of proteins with native conformations, although 2D NMR indicated large segments of conformational flexibility. It displayed a cooperative thermal denaturation transition; at elevated temperatures, it converted rapidly and irreversibly to the thermodynamically more stable beta-sheet form. Unfolding of alpha-rPrP by GdnHCl revealed a two-phase transition with a relatively stable folding intermediate at 2 M GdnHCl. The deltaG values were estimated to be 1.9 +/- 0.4 kcal/mol for the first phase and 6.5 +/- 1.2 kcal/mol for the second, consistent with a folding core surrounded by significant segments of flexible conformation. By NMR, alpha-rPrP(acid) isolated at pH 2 without refolding exhibited heterogeneous line widths, consistent with an acid-denatured molten globular state. We conclude that to the extent that rPrP constitutes a relevant folding domain of PrP(C), the various conformations exhibited by rPrP suggest that the PrP sequence may be intrinsically plastic in its conformations; indeed, portions of PrP(C) may possess a relatively open conformation which makes it susceptible to conversion into PrP(Sc) under appropriate conditions.

Animals↗
PubMed1997

Genetic analysis of the freshwater crayfish Cherax tenuimanus.

The marron (Cherax tenuimanus) is one of the few species of freshwater crayfish native to Australia that is suitable for aquaculture and occurs only in the southwest of Western Australia. This study describes polymorphic microsatellite markers which differentiate marron populations from several geographically distinct regions (including rivers and streams, dams, and commercial marron farms) throughout Western Australia. Twenty microsatellite loci, primarily of the (CA)n. (GT)n type, were isolated and sequenced from a marron cosmid library. Three of these loci were characterised further. Two loci exhibited extensive polymorphism and one was monomorphic. The polymorphic loci exhibited Mendelian codominant inheritance in the family group comprising two individual parents and approximately 100 offspring bred for this study. These loci permitted differentiation between the five geographically distinct populations studied and thus provide a basis for genetic characterisation of marron stock in Western Australia.

Animals↗
PubMed1997

Selective neuronal targeting in prion disease.

The pattern of scrapie prion protein (PrP(Sc)) accumulation in the brain is different for each prion strain. We tested whether the PrP(Sc) deposition pattern is influenced by the Asn-linked oligosaccharides of PrP(C) in transgenic mice. Deletion of the first oligosaccharide altered PrP(C) trafficking and prevented infection with two prion strains. Deletion of the second did not alter PrP(C) trafficking, permitted infection with one prion strain, and had a profound effect on the PrP(Sc) deposition pattern. Our data raise the possibility that glycosylation can modify the conformation of PrP(C). Glycosylation could affect the affinity of PrP(C) for a particular conformer of PrP(Sc), thereby determining the rate of nascent PrP(Sc) formation and the specific patterns of PrP(Sc) deposition.

Animals↗
PubMed1997

Propagation of prion strains through specific conformers of the prion protein.

Two prion strains with identical incubation periods in mice exhibited distinct incubation periods and different neuropathological profiles upon serial transmission to transgenic mice expressing chimeric Syrian hamster/mouse (MH2M) prion protein (PrP) genes [Tg(MH2M) mice] and subsequent transmission to Syrian hamsters. After transmission to Syrian hamsters, the Me7 strain was indistinguishable from the previously established Syrian hamster strain Sc237, despite having been derived from an independent ancestral source. This apparent convergence suggests that prion diversity may be limited. The Me7 mouse strain could also be transmitted directly to Syrian hamsters, but when derived in this way, its properties were distinct from those of Me7 passaged through Tg(MH2M) mice. The Me7 strain did not appear permanently altered in either case, since the original incubation period could be restored by effectively reversing the series of passages. Prion diversity enciphered in the conformation of the scrapie isoform of PrP (PrP(Sc)) (G. C. Telling et al., Science 274:2079-2082, 1996) seems to be limited by the sequence of the PrP substrates serially converted into PrP(Sc), while prions are propagated through interactions between the cellular and scrapie isoforms of PrP.

Animals↗
PubMed1997

Failure to transmit disease from gray tremor mutant mice.

Mice homozygous for mutant alleles at the gray tremor (gt) locus develop a marked non-intention tremor beginning at 8 days of age. Most homozygous mice die by 3 months. Homozygotes exhibit intense vacuolation of the central nervous system gray matter and vacuolation and hypomyelination of some white matter tracts. Based on neuropathological similarities with scrapie, other investigators inoculated wild-type mice with gray tremor brain homogenates to test the hypothesis of transmissibility. Published reports indicated that spongiform encephalopathy (R. L. Sidman, H. C. Kinney, and H. O. Sweet, Proc. Natl. Acad. Sci. USA 82:253-257, 1985) and disease, including hind limb paralysis in NFS mice (P. M. Hoffman, R. G. Rohwer, C. MacAuley, J. A. Bilello, J. W. Hartley, and H. C. Morse III, Proc. Natl. Acad. Sci. USA 84:3866-3870, 1987), were transmitted by inoculation of gt/gt brain homogenates. In our hands, however, no NFS/NCr animals inoculated intracerebrally with gt/gt or +/+ brain preparations showed any signs of disease or pathological changes in the brain. Positive transmission by other investigators may reflect the microbiological status of their donor or recipient mice.

Animals↗
PubMed1996

Aberrant induction of neuropeptide Y mRNA in hippocampal CA3 pyramidal neurones in scrapie-infected mice.

The neurochemical alterations preceding neurological dysfunction and neuronal death in prion diseases are not well characterized. Here we examined, using in situ hybridization histochemistry, the expression of neuropeptide Y (NPY), an inducible and abundant neuropeptide in mammalian brain with known neuroregulatory functions, and glial fibrillary acidic protein (GFAP), a marker for astroglial activation, in the hippocampus at different time points following intracerebral prion inoculation in male CD-1 mice. Between 110 and 140 days postinoculation NPY mRNA expression was specifically up-regulated in CA3 pyramidal neurones, whereas expression of NPY in hilar neurones remained unaltered. Up-regulation of GFAP mRNA was observed in the CA1 stratum radiatum at 60 days, and spread throughout the hippocampus, cortex and thalamus between 110 and 140 days, suggesting early accumulation of scrapie prion protein in these regions. The clinical symptoms were first manifested 120 days postinoculation. Aberrant induction of NPY mRNA in the hippocampal CA3 pyramidal neurones preceded the onset of neurological symptoms, and may be involved in the regulation of glutamate release at the Schaffer collateral-CA1 synapses in scrapie-infected mice.

Animals↗
PubMed1996

High-level expression and characterization of a purified 142-residue polypeptide of the prion protein.

The major, and possible only, component of the infectious prion is the scrapie prion protein (PrPSc); the protease resistant core of PrPSc is PrP 27-30, a protein of approximately 142 amino acids. PrPSc is derived from the cellular PrP isoform (PrPC) by a post-transliatonal process in which a profound conformational change occurs. Syrian hamster (SHa) PrP genes of varying length ranging from the N- and C- terminally truncated 90-228 up to the full-length mature protein 23-231 were inserted into various secretion and intracellular expression vectors that were transformed into Escherichia coli deficient for proteases. Maximum expression was obtained for a truncated SHaPrP containing residues 90-231, which correspond to the sequence of PrP 27-30; disruption of the bacteria using a microfluidizer produced the highest yields of this protein designated rPrP. After solubilization of rPrP in 8 M GdnHC1, it was purified by size exclusion chromatography and reversed phase chromatography. During purification the recovery was approximately 50%, and from each liter of E. coli culture, approximately 50 mg of purified rPrP was obtained. Expression of the longer species containing the basic N-terminal region was less successful and was not pursued further. The primary structure of rPrP was verified by Edman sequencing and mass spectrometry, and secondary structure determined by circular dichroism and Fourier transform infrared spectroscopy. When rPrP was purified under reducing conditions, it had a high beta-sheet content and relatively low solubility similar to PrPSc, particularly at pH values > 7. Refolding of rPrP by oxidation to form a disulfide bond between the two Cys residues of this polypeptide produced a soluble protein with a high alpha-helical content similar to PrPC. These multiple conformations of rPrP are reminiscent of the structural plurality that characterizes the naturally occurring PrP isoforms. The high levels of purified rPrP which can now be obtained should facilitate determination of the multiple tertiary structures that Prp can adopt.

Amino Acid Sequence↗
PubMed1996

Disruption of prion rods generates 10-nm spherical particles having high alpha-helical content and lacking scrapie infectivity.

An abnormal isoform of the prion protein (PrP) designated PrPSc is the major, or possibly the only, component of infectious prions. Structural studies of PrPSc have been impeded by its lack of solubility under conditions in which infectivity is retained. Among the many detergents examined, only treatment with the ionic detergent sodium dodecyl sulfate (SDS) or Sarkosyl followed by sonication dispersed prion rods which are composed of PrP 27-30, an N-terminally truncated form of PrPSc. After ultracentrifugation at 100,000 x g for 1 h, approximately 30% of the PrP 27-30 and scrapie infectivity were found in the supernatant, which was fractionated by sedimentation through 5 to 20% sucrose gradients. Near the top of the gradient, spherical particles with an observed sedimentation coefficient of approximately 6S, approximately 10 mm in diameter and composed of four to six PrP 27-30 molecules, were found. The spheres could be digested with proteinase K and exhibited little, if any, scrapie infectivity. When the prion rods were disrupted in SDS and the entire sample was fractionated by sucrose gradient centrifugation, a lipid-rich fraction at the meniscus composed of fragments of rods and heterogeneous particles containing high levels of prion infectivity was found. Fractions adjacent to the meniscus also contained spherical particles. Circular dichroism of the spheres revealed 60% alpha-helical content; addition of 25% acetonitrile induced aggregates high in beta sheet but remaining devoid of infectivity. Although the highly purified spherical oligomers of PrP 27-30 lack infectivity, they may provide an excellent substrate for determining conditions of renaturation under which prion particles regain infectivity.

Animals↗
PubMed1994

Serial transmission in rodents of neurodegeneration from transgenic mice expressing mutant prion protein.

Two lines of transgenic (Tg) mice expressing high (H) levels of the mutant P101L prion protein (PrP) developed a neurologic illness and central nervous system pathology indistinguishable from experimental murine scrapie; these mice were designated Tg(MoPrP-P101L)H. Brain homogenates from Tg(MoPrP-P101L)H mice were inoculated intracerebrally into CD-1 Swiss mice, Syrian hamsters, and Tg196 mice, Tg mice expressing the MoPrP-P101L transgene at low levels. None of the CD-1 mice developed central nervous system dysfunction, whereas approximately 10% of hamsters and approximately 40% of the Tg196 mice manifested neurologic signs between 117 and 639 days after inoculation. Serial transmission of neurodegeneration in Tg196 mice and Syrian hamsters was initiated with brain extracts, producing incubation times of approximately 400 and approximately 75 days, respectively. Although the Tg(MoPrP-P101L)H mice appear to accumulate only low levels of infections prions in their brains, the serial transmission of disease to inoculated recipients argues that prion formation occurs de novo in the brains of these uninoculated animals. These Tg mouse studies, taken together with similar findings in humans dying of inherited prion diseases, provide additional evidence that prions lack a foreign nucleic acid.

Animals↗
PubMed1994

Prion isolate specified allotypic interactions between the cellular and scrapie prion proteins in congenic and transgenic mice.

Different prion isolates, often referred to as "strains," present an enigma because considerable evidence argues that prions are devoid of nucleic acid. To investigate prion diversity, we inoculated three "strains" of prions into congenic and transgenic mice harboring variable numbers of two different alleles, designated a and b, of the prion protein (PrP) structural gene, Prn-p. The length of the incubation time was inversely related to the number of Prn-p(a) genes in mice inoculated with the Rocky Mountain Laboratory (RML) prion strain. Results with mice lacking this locus (Prn-p0/0) and transgenic mice argue that long incubation times are not a dominant trait as thought for many years, but rather they are due to reduced levels of the substrate PrPC-A (cellular isoform of PrP, allotype A) in (Prn-p(a) x Prn-pb)F1 mice. In contrast, the Prn-p(a) gene extended incubation times in mice inoculated with the 87V and 22A prion strains, whereas the Prn-pb gene was permissive. Experiments with the 87V isolate suggest that a genetic locus distinct from Prn-p controls deposition of the scrapie isoform of PrP (PrPSc) and attendant neuropathology. Each prion isolate produced distinguishable patterns of PrPSc accumulation in brain; of note, the patterns in Prn-p(a) and Prn-pb congenic mice inoculated with RML prions were more different than those in congenic Prn-pb mice with RML or 22A prions. Our results suggest that scrapie "strain-specific" incubation times can be explained by differences in the relative efficiency of allotypic interactions that lead to conversion of PrPC into PrPSc.

Alleles↗
PubMed1994

The neuropathological phenotype in transgenic mice expressing different prion protein constructs.

Neuropathologic examination of transgenic (Tg) mice which express different prion protein (PrP) constructs is essential because spongiform (vacuolar) degeneration of neurons, the distribution of PrPSc and whether PrP amyloid plaques form are the phenotypes of prion diseases. In Tg models of experimental scrapie, it was found that all of the parameters that define prion isolates ('strains') can be manipulated by changing the structure of PrP. In those studies, further evidence that PrPSc causes scrapie neuropathology and determines scrapie incubation time was obtained. In addition, the distribution of PrPSc in the brain was unique for each prion isolate. The implications of these findings are first, that prion isolates target different neuron populations for synthesis of nascent pathogenic PrPSc and, secondly, that prion isolate diversity is determined by neurons. In Tg mice which express mutated PrP mimicking human prion protein gene mutations linked to familial prion diseases, the neuropathological changes have been faithfully reproduced. A new age-related, neuromuscular disorder has also been identified in uninfected Tg mice which overexpress wild-type PrPc. All of the findings with different PrP constructs plus the absence of scrapie pathology in PrP null mice are the strongest argument that the prion protein is the main etiologic and pathogenic factor of prion disorders.

Age Factors↗
PubMed1994

Spectroscopic characterization of conformational differences between PrPC and PrPSc: an alpha-helix to beta-sheet transition.

Although no chemical modifications have been found to distinguish the cellular prion protein PrPC from its infectious analogue PrPSc, spectroscopic methods such as Fourier transform infrared (FTIR) spectroscopy reveal a major conformational difference. PrPC is rich in alpha-helix but is devoid of beta-sheet, whereas PrPSc is high in beta-sheet. N-terminal truncation of PrPSc by limited proteolysis does not destroy infectivity but it increases the beta-sheet content and shifts the FTIR absorption to lower frequencies, typical of the cross beta-pleated sheets of amyloids. Thus the formation of PrPSc from PrPC involves a conformational transition in which one or more alpha-helical regions of the protein is converted to beta-sheet. This transition is mimicked by synthetic peptides, allowing predictions of domains of PrP involved in prion diseases.

Amino Acid Sequence↗
PubMed1994

Degeneration of skeletal muscle, peripheral nerves, and the central nervous system in transgenic mice overexpressing wild-type prion proteins.

Prion diseases of humans and animals are known to be caused by infection with prions containing PrPSc or mutation of the prion protein (PrP) gene. During transgenetic studies, we discovered that uninoculated older mice harboring high copy numbers of wild-type (wt) PrP transgenes derived from Syrian hamsters (SHa), sheep (She), and PrP-B mice developed truncal ataxia, hindlimb paralysis, and tremors. These transgenic (Tg) mice exhibited a profound necrotizing myopathy involving skeletal muscle, a demyelinating polyneuropathy, and focal vacuolation of the central nervous system. Development of disease was dependent on transgene dosage. For example, half of all Tg(SHaPrP+/+)7 mice homozygous for the SHaPrP transgene array developed disease by approximately 460 days of age, while no hemizygous Tg(SHaPrP+/o)7 mice became ill before 650 days. The novel neurologic syndrome found in older Tg(wtPrP) mice implies that overexpression of wtPrPC is pathogenic and widens the spectrum of prion diseases.

Animals↗