Biomedical subjects

D Ernst

Publications and source records attributed to D Ernst.

At least 37 records · Page 2Linked to original sources

PubMed1993

Foreign PrP expression and scrapie infection in tissue culture cell lines.

PrP expression and scrapie infectivity was studied in a group of tissue culture cell lines from mice, hamsters, rats and humans. Cell surface PrP protein was detected in seven of ten cell lines from a variety of tissues and showed both cell surface and cytoplasmic perinuclear distributions. Mouse N2a neuroblastoma cells could be persistently infected with scrapie in vitro, but OBL-21 mouse neuroma cells were never successfully infected. Foreign PrP molecules including hamster PrP, mutant hamster PrP with leucine at amino acid 102, and mutant mouse PrP expressing a hamster-encoded PrP antigen epitope were expressed in mouse N2a cells. However, none of these cell lines could be successfully infected with the 263K hamster scrapie agent. Furthermore, expression of these foreign PrP molecules did not lead to spontaneous generation of protease-resistant PrP or a transmissible scrapie-like agent in vitro.

Animals↗

PubMed1992

Ozone-induced changes of mRNA levels of beta-1,3-glucanase, chitinase and 'pathogenesis-related' protein 1b in tobacco plants.

Treatment of the ozone-sensitive tobacco cultivar Bel W3 with an ozone pulse (0.15 microliter/l, 5 h) markedly increased the mRNA level of basic beta-1,3-glucanase and to a lower degree that of basic chitinase. The increase of beta-1,3-glucanase mRNA level occurred within 1 h and showed a transient maximum. Seventeen hours after ozone treatment, the beta-1,3-glucanase mRNA level decreased to lower values. The increase of basic chitinase mRNA level was delayed and was less pronounced than that of beta-1,3-glucanase mRNA. Cultivar Bel B showed only a small increase of beta-1,3-glucanase mRNA level after the same ozone treatment, whereas its basic chitinase mRNA was more strongly induced. Prolonged ozone treatment for 2 days of tobacco Bel W3 led to a persistent level of beta-1,3-glucanase and basic chitinase mRNAs, as well as to an increase of acidic chitinase and 'pathogenesis-related' (PR) 1b mRNA levels. The results indicate that genes so far considered to code for PR proteins may also be involved in the plant response to oxidative stress.

Blotting, Northern↗

PubMed1992

Social networks as predictors of ischemic heart disease, cancer, stroke and hypertension: incidence, survival and mortality.

Three social network measures were obtained from a randomly sampled cohort of 2603 HMO members in 1970-71. Mortality and first incidence of ischemic heart disease, cancer, stroke, and hypertension were assessed over the next 15 years. Outcome data were adjusted for age, sex, smoking, SES, and baseline health status. Persons with histories of a given morbidity at or prior to baseline were excluded from the analyses of that morbidity. Social network measures, particularly network scope (a measure of the number of different domains in which a person has social contacts), were powerful predictors of 15-year mortality hazard, but weak predictors of incident disease. Only network scope predicted IHD incidence, and none of the other morbidities was predicted by the social network measures. However, social network measures were strong predictors of both cause-specific and all-cause mortality among persons who had incident cases of IHD, cancer, and stroke. These data suggest that social networks may be more effective in supporting recovery after illness has occurred than in preventing the incidence of new disease.

Adult↗

PubMed1992

Detection of proteinase K-resistant prion protein and infectivity in mouse spleen by 2 weeks after scrapie agent inoculation.

The sequential accumulation of the protease-resistant form of the endogenous prion protein (PrP-res) was compared to levels of scrapie infectivity in the spleen and brain of scrapie-infected mice at various times after inoculation. In mouse spleen PrP-res was detected 1 week after inoculation, and increased 65-fold between 1 and 3 weeks post-inoculation and an additional 15-fold during the next 17 weeks. Infectivity in spleen reached a maximum plateau level by 3 weeks. In contrast, in mouse brain PrP-res was not detected until 8 weeks after inoculation and then increased 200-fold during the next 12 weeks. During this same time, infectivity increased approximately 10,000-fold. Therefore, in both spleen and brain of scrapie-infected mice accumulation of PrP-res and infectivity appear to be associated. However, it was not possible to show quantitative correlations between PrP-res detection and infectivity, perhaps owing to the inaccuracy of the infectivity assay.

Animals↗

PubMed1992

Biochemical Plant Responses to Ozone : III. Activation of the Defense-Related Proteins beta-1,3-Glucanase and Chitinase in Tobacco Leaves.

A single pulse of O(3) (0.15 microliter per liter, 5 hours) induced beta-1,3-glucanase and chitinase activities in O(3)-sensitive and -tolerant tobacco (Nicotiana tabacum L.) cultivars. In the O(3)-sensitive cultivar Bel W3, the response was rapid (maximum after 5 to 10 hours) and was far more pronounced for beta-1,3-glucanase (40- to 75-fold) than for chitinase (4-fold). In the O(3)-tolerant cultivar Bel B, beta-1,3-glucanase was induced up to 30-fold and chitinase up to 3-fold under O(3) concentrations that did not lead to visible damage. Northern blot hybridization showed a marked increase in beta-1,3-glucanase mRNA in cultivar Bel W3 between 3 and 24 hours following O(3) treatment, a transient induction in cultivar Bel B, and no change in control plants. The induction of beta-1,3-glucanase and chitinase activities following O(3) treatment occurred within the leaf cells and was not found in the intercellular wash fluids. In addition, O(3) treatment increased the amount of the beta-1,3-glucan callose, which accumulated predominantly around the necrotic spots in cultivar Bel W3. The results demonstrate that near-ambient O(3) levels can induce pathogenesis-related proteins and may thereby alter the disposition of plants toward pathogen attack.

Journal Article↗

PubMed1992

Diagnostic implications of detection of proteinase K-resistant protein in spleen, lymph nodes, and brain of sheep.

Brain, spleen, and selected lymph nodes from sheep with clinical signs of scrapie were analyzed for presence of proteinase K-resistant protein (PrP-res). Diagnosis of scrapie on the basis of detection of PrP-res was compared with diagnosis on the basis of histologic evaluation of the brain from clinically affected or exposed sheep. Proteinase K-resistant protein was found in every brain that was histologically positive for scrapie, and in addition, was found in the brain of several clinically positive sheep that were not diagnosed as scrapie-positive by histologic evaluation. Proteinase K-resistant protein was also found in 87% of the spleens and lymph nodes from sheep that had PrP-res detected in brain homogenates. Therefore, analysis of sheep brain, spleen, or lymph nodes for PrP-res provided a diagnostic approach that was superior to histologic examination alone for detection of naturally scrapie agent-infected sheep.

Animals↗

PubMed1991

Isolation of functional RNA from plant tissues rich in phenolic compounds.

A method for the isolation of RNA from different tissues of trees (seedlings, saplings, and adult trees) is described. Using this procedure it is possible to remove large amounts of disturbing polyphenolic compounds from nucleic acids. The method involves an acetone treatment of the freeze-dried and powdered plant material, the use of high salt concentrations in the extraction buffer and an aqueous two-phase system. These steps were combined with the conventional phenol/chloroform extraction and CsCl centrifugation. The method has been successfully applied to the isolation and purification of RNA from pine (Pinus sylvestris L. and Pinus mugo Turr.), Norway spruce (Picea abies L.), and beech (Fagus sylvatica L.). The functional quality of RNA extracted by this procedure has been characterized by its uv spectrum, by agarose gel electrophoresis with ethidium bromide staining, Northern blot hybridization, and in vitro translation.

Acetone↗

PubMed1991

Secondary structure analysis of the scrapie-associated protein PrP 27-30 in water by infrared spectroscopy.

A protease-resistant form of the protein PrP (PrP-res) accumulates in tissues of mammals infected with scrapie, Creutzfeldt-Jakob disease, and related transmissible neurodegenerative diseases. This abnormal form of PrP can aggregate into insoluble amyloid-like fibrils and plaques and has been identified as the major component of brain fractions enriched for scrapie infectivity. Using a recently developed technique in Fourier transform infrared spectroscopy which allows protein conformational analysis in aqueous media, we have studied the secondary structure of the proteinase K resistant core of PrP-res (PrP-res 27-30) as it exists in highly infectious fibril preparations. Second-derivative analysis of the infrared spectra has enabled us to quantitate the relative amounts of different secondary structures in the PrP-res aggregates. The analysis indicated that PrP-res 27-30 is predominantly composed of beta-sheet (47%), which is consistent with its amyloid-like properties. In addition, significant amounts of turn (31%) and alpha-helix (17%) were identified, indicating that amyloid-like fibrils need not be exclusively beta-sheet. The infrared-based secondary structure compositions were then used as constraints to improve the theoretical localization of the secondary structures within PrP-res 27-30.

Acetylglucosamine↗

PubMed1991

Empirical status of cognitive theory of depression.

Studies testing cognitive theory of depression (Beck, 1963, 1987) and defining depression as a clinical syndrome are reviewed. Many aspects of the theory's descriptive claims about depressive thinking have been substantiated empirically, including (a) increased negativity of cognitions about the self, (b) increased hopelessness, (c) specificity of themes of loss to depressive syndromes rather than psychopathology in general, and (d) mood-congruent recall. Evidence that depressive thinking is especially inaccurate or illogical, however, is weak. Fewer studies have tested the theory's causal (diathesis-stress) hypotheses, and there is no strong evidence supporting them.

Attitude↗

PubMed1991

N-terminal truncation of the scrapie-associated form of PrP by lysosomal protease(s): implications regarding the site of conversion of PrP to the protease-resistant state.

Scrapie and related transmissible spongiform encephalopathies result in the accumulation of a protease-resistant form of an endogenous brain protein called PrP. As an approach to understanding the scrapie-associated modification of PrP, we have studied the processing and sedimentation properties of protease-resistant PrP (PrP-res) in scrapie-infected mouse neuroblastoma cells. Like brain-derived PrP-res, the neuroblastoma cell PrP-res aggregated in detergent lysates, providing evidence that the tendency to aggregate is an intrinsic property of PrP-res and not merely a secondary consequence of degenerative brain pathology. The PrP-res species had lower apparent molecular masses than the normal, protease-sensitive PrP species and were not affected by moderate treatments with proteinase K. This suggested that the PrP-res species were partially proteolyzed by the neuroblastoma cells. Immunoblot analysis of PrP-res with a panel of monospecific anti-PrP peptide sera confirmed that the PrP-res species were quantitatively truncated at the N terminus. The metabolic labeling of PrP-res in serum-free medium did not prevent the proteolysis of PrP-res, showing that the protease(s) involved was cellular rather than serum-derived. The PrP-res truncation was inhibited in intact cells by leupeptin and NH4Cl. This provided evidence that a lysosomal protease(s) was involved, and therefore, that PrP-res was translocated to lysosomes. When considered with other studies, these results imply that the conversion of PrP to the protease-resistant state occurs in the plasma membrane or along an endocytic pathway before PrP-res is exposed to endosomal and lysosomal proteases.

Animals↗

PubMed1991

Protease sensitivity and nuclease resistance of the scrapie agent propagated in vitro in neuroblastoma cells.

The scrapie agent has been propagated in vitro in mouse neuroblastoma cells. To further characterize the tissue culture-derived scrapie agent, we studied the effects of protease and nuclease digestion on the agent derived from these cells. The scrapie agent in these cells was found to be resistant to protease digestions for short times but was inactivated by prolonged digestion at high protease concentrations. In contrast, digestion with a variety of nucleases did not alter the agent titer. These results demonstrate that the agent requires an essential protein or proteins for infectivity. If the agent also contains a nucleic acid genome, it must be more nuclease resistant than the majority of cellular DNA and RNA. These properties of the tissue culture-derived scrapie agent were identical to those of brain-derived scrapie agent and thus cannot be attributed to secondary effects of tissue pathology, since the infected cell cultures show no cytopathic effects as a result of infection.

Animals↗

PubMed1990

Analysis of linkage between scrapie incubation period and the prion protein gene in mice.

A single gene is known to have a predominant influence on scrapie incubation period in mice. In crosses between strains that give a short incubation period, such as NZW mice, and those which give a long incubation period, such as I/LnJ mice, long incubation period was dominant using a Chandler scrapie agent isolate. Recently a close linkage was found between the incubation period gene and the prion protein (PrP) structural gene in I/LnJ mice crossed to NZW mice. Because this linkage suggested an important role for PrP in the pathogenesis of scrapie we sought to verify the linkage between these genes and extended the analysis to three additional mouse strains. All four of the mouse strains that we evaluated, I/LnJ, P/J, MA/MyJ, and RIIIS/J, had incubation periods longer than those of the NZW mice to which they were crossed. In addition, all four strains shared an XbaI restriction enzyme polymorphism, which suggested that all four strains might also exhibit linkage between the incubation period and the PrP structural gene. Very strong linkage between PrP and incubation period was found in I/LnJ and P/J mice crossed to NZW mice, whereas less obvious linkage was demonstrated for MA/MyJ mice crossed to NZW mice. In MA/MyJ mice genes other than PrP also had an obvious influence on incubation period. In RIIIS/J mice no linkage was shown. Although linkage between PrP and incubation period was very significant in I/LnJ and P/J mice, a few animals were identified in both crosses that represented potential recombinants in which PrP and incubation period did not segregate together. Therefore, although these phenotypes are certainly linked in I/LnJ and P/J mice, it is possible that PrP and incubation period are controlled by separate genes.

Animals↗

PubMed1990

Rhythmic regulation of the light-harvesting chlorophyll a/b protein and the small subunit of ribulose-1,5-bisphosphate carboxylase mRNA in rye seedlings.

In etiolated rye seedlings transferred to light the expression of chlorophyll a/b binding protein mRNA varies when the seedlings are grown in a day/night cycle. The fluctuation pattern follows a circadian rhythm. Exposure of 4-day old etiolated seedlings to continuous white light revealed two maxima within the first 24 h before the 24 h cycle period appeared. These first two maxima are also observable after a pulse of white light or after a pulse of red light. These results indicate a possible involvement of phytochrome in the endogenous regulation of the rhythm.

Chlorophyll↗

PubMed1990

Normal and scrapie-associated forms of prion protein differ in their sensitivities to phospholipase and proteases in intact neuroblastoma cells.

Previous studies have indicated that scrapie infection results in the accumulation of a proteinase K-resistant form of an endogenous brain protein generally referred to as prion protein (PrP). The molecular nature of the scrapie-associated modification of PrP accounting for proteinase K resistance is not known. As an approach to understanding the cellular events associated with the PrP modification in brain tissue, we sought to identify proteinase K-resistant PrP (PrP-res) in scrapie-infected neuroblastoma cells in vitro and to compare properties of PrP-res with those of its normal proteinase K-sensitive homolog, PrP-sen. PrP-res was detected by immunoblot in scrapie-infected but not uninfected neuroblastoma clones. Densitometry of immunoblots indicated that there was two- to threefold more PrP-res than PrP-sen in one infected clone. Metabolic labeling and membrane immunofluorescence experiments indicated that PrP-sen was located on the cell surface and could be removed from intact cells by phosphatidylinositol-specific phospholipase C and proteases. In contrast, PrP-res was not removed after reaction with these enzymes. Thus, either the scrapie-associated PrP-res was not on the cell surface or it was there in a form that is resistant to these hydrolytic enzymes. Attempts to detect intracellular PrP-res by immunofluorescent staining of fixed and permeabilized cells revealed that PrP was present in discrete perinuclear Golgi-like structures. However, the staining pattern was similar in both scrapie-infected and uninfected clones, and thus the intracellular staining may have represented only PrP-sen. Analysis of scrapie infectivity in cells treated with extracellular phospholipase, proteinase K, and trypsin indicated that, like PrP-res, the scrapie agent was not removed from the infected cells by any of these enzymes.

Animals↗

PubMed1989

Prion protein biosynthesis in scrapie-infected and uninfected neuroblastoma cells.

Numerous studies have indicated that a modified proteinase K-resistant form of an endogenous brain protein, prion protein (PrP), is associated with scrapie infection in animals. This scrapie-associated PrP modification appears to occur posttranslationally in brain, but its molecular nature is not known. To learn about the normal PrP biosynthesis and whether it is altered by scrapie infection in vitro, we did metabolic labeling experiments with uninfected and scrapie-infected mouse neuroblastoma tissue culture cells. Pulse-chase labeling experiments indicated that, in both cell types, two major PrP precursors of 28 and 33 kilodaltons (kDa) were processed to mature 30- and 35- to 41-kDa forms. Endoglycosidase H, tunicamycin, and phospholipase treatments revealed that the 28- and 33-kDa precursors resulted from the addition of high-mannose glycans to a 25-kDa polypeptide containing a phosphatidylinositol moiety and that maturation of the precursors involved the conversion of the high-mannose glycans to hybrid or complex glycans. Treatments of the live cells with trypsin and phosphatidylinositol-specific phospholipase C indicated that the mature PrP species were expressed solely on the cell surface, where they were anchored by covalent linkage to phosphatidylinositol. Once on the cell surface, the major PrP forms had half-lives of 3 to 6 h. No differences in PrP biosynthesis were observed between the scrapie-infected versus uninfected neuroblastoma cells.

Acetylglucosaminidase↗

PubMed1988

Photooxidation of Plastids Inhibits Transcription of Nuclear Encoded Genes in Rye (Secale cereale).

Rye (Secale cereale cv Halo) seedlings treated with the herbicide Norflurazon SAN 9789 showed a reduced concentration of mRNA for the small subunit of ribulose-1,5-bisphosphate carboxylase and for the light-harvesting chlorophyll a/b protein. The inhibition of mRNA accumulation by Norflurazon occurred only in the presence of high light intensities and only after a period of days. The primary effect was an inhibition of the transcription rate that occurred within 1 day after exposure of the seedlings to light.

Journal Article↗

PubMed1988

Analyses of frequency of infection, specific infectivity, and prion protein biosynthesis in scrapie-infected neuroblastoma cell clones.

Scrapie, a spongiform encephalopathy of sheep and goats, is caused by a poorly understood transmissible agent in which no nucleic acid has been conclusively identified. Biochemical characterization of agent derived from animal tissues has not been precise because of the tenacious association of the agent with tissue components. As an approach toward obtaining homogeneous preparations of agent generated in vitro, we cloned scrapie-infected neuroblastoma cells. By frequency analysis, nearly every cell in expanded cultures contained scrapie agent. We also analyzed cell-dose infectivity relationships and developed a standard curve which allowed various cultures to be compared. Since a proteinase K (PK)-resistant form of a protein designated prion protein (PrP) has been found in partially purified preparations of scrapie agent from infected animal spleens and brains, we sought to identify this protein in cell cultures. No PK-resistant PrP was found in infected or uninfected cultures, although the PK-sensitive PrP was readily detected. These results suggested that PK-resistant PrP may not be an essential component of the infectious scrapie agent.

Animals↗

PubMed1984

Purified phytochrome influences in vitro transcription in rye nuclei.

Nuclei isolated from dark-grown seedlings of rye by Percoll density gradient centrifugation incorporate radioactive UTP into RNA. Transcription is reduced to 50% by the addition of alpha-amanitin (5 mug/ml) and correspondingly a decrease of the label in the mRNA fraction is seen on agarose gels. Purified 124-kd phytochrome in its far-red absorbing form (P(fr)) increases the incorporation of labeled UTP by 40-70% above untreated controls, whereas pre-illumination with far-red light or addition of alpha-amanitin prevents this effect. Nuclei isolated from light-grown seedlings show only an increase of 5% upon addition of P(fr). Other proteins such as bovine serum albumin or cytochrome c do not enhance the rate of transcription. We conclude that the accumulation of mRNA species is influenced by phytochrome in its P(fr) form.

Journal Article↗