Biomedical subjects

K Kellings

Publications and source records attributed to K Kellings.

5 recordsLinked to original sources

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

Nucleic acids in prion preparations: unspecific background or essential component?

As recently published (Kellings et al. J. gen Vir. 73, 1025-1029 (1992)), the analysis of purified scrapie prions by return refocusing gel electrophoresis revealed remaining nucleic acids in the size range up to 1100 nucleotides. The results defined the possible characteristics of a hypothetical scrapie-specific nucleic acid. If homogeneous in size, such a molecule would be less than 80 nucleotides in length at a particle-to-infectivity ratio (P:I) near unity; if heterogeneous, scrapie-specific nucleic acids would have to include molecules smaller than 240 nucleotides. To decrease the amount of nucleic acids, several modifications of the PrPSc purification scheme were introduced. Instead of sucrose gradient, ultrafiltration was applied as a purification step and nucleic acids were degraded by Benzonase after ultrafiltration, but significant reduction of the P:I ratio could not be achieved. To prevent trapping of nucleic acids in prion rods, nuclease (Benzonase) was added into the tissue homogenate and incubated at 37 degrees C, overnight. The Benzonase treatment revealed no loss of infectivity, but the whole procedure of nucleic acid analysis did not lead to a reduction of the P:I ratio. In another approach the number of nucleic acid degradations steps was reduced to essentially two steps: Zn2+ hydrolysis and Benzonase digestion. Higher Zn2+ concentrations and prolonged incubation times resulted in a more efficient nucleic acid degradation. The bioassays yielded complete recovery of infectivity. Large-scale preparations for determining the P:I ratio are still underway.

Animals↗

PubMed1993

Analysis of nucleic acids in purified scrapie prion preparations.

Amount, type, and size of nucleic acid molecules associated with purified prion preparations were analyzed. Return refocusing gel electrophoresis (RRGE) was developed to detect homogeneous and heterogeneous nucleic acids extracted from highly purified scrapie prion preparations. With this method all types of nucleic acids in the size range from 13 to several thousand nucleotides could be analyzed. The recovery of all nucleic acids, after deproteinization and two-phase extraction was higher than 90%. Despite extensive nuclease digestions some small polynucleotides remained. Although a scrapie-specific nucleic acid cannot be excluded, the results further define the possible characteristics for such a hypothetical molecule. If it was homogeneous in size, then it would be < 80 nt in length at a particle-to-infectivity ratio (P/I) near unity; if the other extreme, i.e. totally heterogeneous scrapie-specific nucleic acids were assumed, then scrapie-specific nucleic acids would have to include molecules smaller than 240 nt. In order to exclude the possibility that unspecific background nucleic acid is entrapped in prion-rods, infectious material has to be prepared without a proteolysis and rod formation, and the analysis of nucleic acids performed with those preparations.

Animals↗

PubMed1993

Prions and nucleic acids: search for "residual" nucleic acids and screening for mutations in the PrP-gene.

Studies on prions involve nucleic acid chemistry under two headings: i) do infectious prion particles contain nucleic acids? ii) is it possible by a simple procedure to screen the prion protein (PrP)-gene for mutations? The return refocusing gel electrophoresis technique was developed to detect by sensitive silver staining homogeneous and heterogeneous nucleic acids extracted from highly purified scrapie prion preparations. With this method all kinds of nucleic acids from a length of 13 nucleotides up to several thousand could be recovered and detected with a yield over 90%. Despite extensive nuclease digestion some small polynucleotides remained. The results define clear restrictions for a hypothetical scrapie-specific nucleic acid. If homogeneous in size, such a molecule would be smaller than 80 nucleotides in length at a particle-to-infectivity ratio near unity; if heterogeneous, scrapie-specific nucleic acids would have to include molecules smaller than 240 nucleotides. To detect mutations in the PrP-gene, either known mutations from human prion diseases or artificial ones in transgenic animals, or to screen for not yet identified mutations in patients, a method is required which guarantees detection of mutations which might occur in every single position of the whole PrP-ORF. It will be shown that a combination of PCR and temperature-gradient gel electrophoresis fulfils these requirements. By thermodynamic calculations the shift in the gel electrophoresis due to a mutation can be calculated depending on the position of the mutation. The theoretical results were tested with the mutations known so far.

Animals↗

PubMed1992

Further analysis of nucleic acids in purified scrapie prion preparations by improved return refocusing gel electrophoresis.

Although increasingly unlikely, the possibility of a scrapie-specific nucleic acid carried by infectious prion particles is still unresolved. Return refocusing gel electrophoresis was developed to detect homogeneous and heterogeneous nucleic acids extracted from highly purified scrapie prion preparations. This method was improved with respect to the size range from 13 to 1100 nucleotides (nt) over which analyses could be performed. The yield of nucleic acid, particularly of small DNA oligonucleotides and polyadenylated RNA, was determined after deproteinization and two-phase extraction. Despite extensive nuclease digestions some small polynucleotides remained. Although a scrapie-specific nucleic acid cannot be excluded, the results further define the possible characteristics of a hypothetical molecule. If homogeneous in size, such a molecule would be less than 80 nt in length at a particle-to-infectivity ratio near unity, if heterogeneous, scrapie-specific nucleic acids would have to include molecules smaller than 240 nt.

Animals↗