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Biomedical subjects

R I Carp

Publications and source records attributed to R I Carp.

At least 91 records · Page 5Linked to original sources
PubMed1986

Chromatin structure in scrapie and Alzheimer's disease.

Scrapie affected brains exhibit a number of pathological features in common with the human neurodegenerative condition, Alzheimer's disease. The present report describes studies on chromatin structure seen in these two disease processes. Chromatin associated proteins influence transcriptional activity of DNA through an effect upon chromatin structure. We examined chromatin structure by: measuring the capacity of the enzyme micrococcal nuclease to release mono- and dinucleosomes from isolated nuclei and measuring DNA-histone interactions by examining the effect of ambient tonicity upon the release of chromatin proteins. In two strains of mice infected with two strains of scrapie agent there was reduced accessibility to micrococcal nuclease and an increased content on dinucleosomes of the histone H1 and H1(0) types. These changes precede clinical signs of scrapie and resemble those found in the human conditions of Alzheimer's and Pick's disease. Scrapie mouse brain differs from Alzheimer brain in that scrapie does not alter histone-DNA interactions as monitored by ionically induced histone release from chromatin. Despite similarities, the scrapie agent appears to operate upon different molecular mechanisms than those found in Alzheimer's disease.

Alzheimer Disease↗
PubMed1986

Isolation of a cDNA clone encoding the leader peptide of prion protein and expression of the homologous gene in various tissues.

We have isolated a hamster cDNA clone representing the coding sequences for the entire precursor of prion protein (PrP) 27-30. This clone encodes a protein of 254 residues and contains an in-frame ATG codon 42 bases upstream from the one previously reported. Analysis of the predicted amino acid sequence suggests that the PrP precursor protein contains an amino-terminal signal sequence, and a membrane-spanning domain in the carboxyl terminus. Cleavage of the signal peptide would produce a mature protein of 232 amino acids. Sequences homologous to the hamster PrP cDNA were detected in hamster, mouse, sheep, human, and rabbit genomes. A related 2.5-kilobase transcript was present in the brain of normal and scrapie-infected rodents. Two homologous transcripts, 2.5 and 1.1 kilobases, were detected in the lung and heart of normal animals. No PrP mRNA was detected in spleen stroma, a tissue known to contain high titers of scrapie. Antisera raised to the 27- to 30-kDa polypeptide detected the PrP in both normal and infected brains but failed to detect this protein in either normal or infected spleens. Homologous mRNA species were detected in human, sheep, and rabbit brain, even though the latter is resistant to scrapie infection. Our data suggest that PrP is not a necessary component of the infectious agent.

Amino Acid Sequence↗
PubMed1986

Detection of scrapie-associated fibril (SAF) proteins using anti-SAF antibody in non-purified tissue preparations.

Antisera raised to scrapie-associated fibril (SAF) proteins were used to detect scrapie-specific polypeptides in three different non-purified brain preparations: a synaptosomal-mitochondrial fraction, 20% brain homogenate and 20% brain homogenate extracted with Sarkosyl. The concentration of SAF proteins in the preparations was greater than the quantity of SAF as detected by negative stain electron microscopy. This suggests that not all of the protein exists in the form of SAF. An immunologically reactive 33K to 35K protein was detected in both normal and scrapie brain preparations. This protein was susceptible to complete proteinase K (PK) digestion in normal brain preparations and it is suggested that scrapie infection is responsible for post-translational modifications which confer PK resistance in scrapie preparations. These modifications may also play a role in the antigenic differences seen in a variety of scrapie agents. SAF-specific proteins were also detected in the spinal cords and spleens from scrapie-affected animals. Detergent extraction of material followed by PK treatment and Western blot analysis is a highly specific and sensitive method for the detection of SAF proteins. This procedure could be applied to human neurological diseases of unknown aetiology.

Animals↗
PubMed1986

Immunological comparison of scrapie-associated fibrils isolated from animals infected with four different scrapie strains.

Scrapie-associated fibrils (SAFs) are abnormal filamentous structures that are uniquely associated with unconventional slow virus diseases. The antigenic relationships of SAFs from animals infected with four biologically distinct scrapie strains were investigated by using antisera raised to purified SAF proteins. Rabbit antisera were raised to SAFs isolated from mice infected with the ME7 scrapie strain and to SAFs isolated from hamsters infected with the 263K scrapie strain. A strong antigenic relationship was shown among SAF proteins (PrPs) isolated from all scrapie-infected animals (ME7, 139A, and 87V in mice and 263K in hamsters), and this relationship was demonstrable regardless of which antiserum was used. SAF proteins were antigenically distinct from those of paired helical filaments or amyloid isolated from patients with Alzheimer disease. Distinct Western blot profiles were demonstrated for SAFs isolated from animals infected with each scrapie strain. Differences seen among SAFs were independent, at least in part, of host species or genotype, implying that certain specific structural and molecular properties of SAFs are mediated by the strain of scrapie agent.

Alzheimer Disease↗
PubMed1986

Partial copurification of scrapie-associated fibrils and scrapie infectivity.

The association between scrapie infectivity and scrapie-associated fibrils (SAF) during a partial purification procedure for infectivity was investigated. Scrapie infectivity and SAF can be separated from most membrane components by subcellular fractionation of infected mouse brain to obtain a synaptosomal fraction, followed by detergent treatment and density gradient centrifugation. After different detergent treatments, with either octyl glucoside or sodium N-lauroyl sarcosinate, SAF showed differing sedimentation characteristics but nevertheless cosedimented with scrapie infectivity in both cases. Copurification under two different conditions provides more evidence that SAF may be a form of the infectious agent of scrapie.

Animals↗
PubMed1986

Scrapie incubation periods and end-point titers in mouse strains differing at the H-2D locus.

In extending findings on the influence of the mouse H-2D locus on the scrapie incubation period, we showed that with the intracerebral (i.c.) route of injection, SJL and NZW mice (s and z alleles, respectively) had shorter incubation periods than C57BL mice (b allele) at several concentrations of two scrapie strains, ME7 and 139A. The three mouse strains have the same Sinc genotype, s7s7. Incubation period data among the three mouse strains after intraperitoneal (i.p.) injection revealed a different rank order of incubation periods from that seen after i.c. injection. End-point titers in the three mouse strains were similar after i.c. injection, but both scrapie strains yielded very low titers in NZW mice after i.p. injection. There were marked differences between 139A and ME7 incubation periods after both i.c. and i.p. injections in the three mouse genotypes, providing an additional parameter that distinguishes these two scrapie strains.

Animals↗
PubMed1985

Biochemical differences among scrapie-associated fibrils support the biological diversity of scrapie agents.

Scrapie-associated fibrils (SAF) were isolated and purified from animals infected with three different scrapie agents: ME7 and 139A in mice, and 263K in hamsters. Mouse ME7 and 139A SAF differed from hamster 263K SAF in morphology, sedimentation rate and protein composition. SAF from the three scrapie agents were distinguishable from each other by their sensitivity to proteinase K digestion. SAF copurified with infectivity in both the hamster and mouse systems. SAF appear to be a unique class of structures which are related but specific for each individual scrapie agent. These properties may correlate with the biological and pathological differences seen among these agents.

Animals↗
PubMed1984

In vitro replication of scrapie agent in a neuronal model: infection of PC12 cells.

A rat phaeochromocytoma cell line, termed PC12, was used to study scrapie replication. These cells, in response to the addition of nerve growth factor (NGF), exhibit a number of neuronal properties including morphological differentiation, electrophysiological responsiveness, and neurotransmitter synthesis. Cultures were exposed to scrapie brain homogenate (strain 139A), harvested every week for up to 6 weeks, and assayed for scrapie infectivity. Scrapie replication in vitro was monitored by injecting scrapie agent-exposed NGF-treated PC12 cells into mice and measuring time intervals from injection to onset of clinical symptoms. Mouse incubation periods vary inversely with the amount of scrapie infectivity present. Cells harvested at 7 and 14 days after exposure to scrapie agent showed a decrease in the level of infectivity followed by an increase at subsequent time points. The increase in scrapie infectivity from early to late time intervals after agent exposure clearly indicated replication in vitro. A fusion agent was not necessary to establish infection, and the addition of mouse peritoneal macrophages caused a reduction in the yield of infectivity per culture. Examination of cells by phase-contrast microscopy failed to reveal any cytopathology.

Animals↗
PubMed1984

The effects of detergents on the composition of postsynaptic densities.

A method of purifying postsynaptic densities (PSD) of Cohen et al. (1977) has been modified, primarily by the substitution of octyl glucoside as the detergent used to solubilize synaptosomal fractions. Subsequent extraction with other detergents resulted in the selective removal of specific polypeptides. In particular sulphobetaine 3-14 removed most of the beta-tubulin but not alpha-tubulin. Sodium N-lauroyl sarcosinate completely destroyed the structural integrity of the PSD when the in vitro formation of intermolecular disulphide bonds was minimized. These results suggest that the structure of PSDs is more labile than previously thought and demonstrate a technique for further examining their composition.

Animals↗
PubMed1984

Senile dementia of the Alzheimer type: possibility of infectious etiology in genetically susceptible individuals.

The concept that SDAT is caused by an infectious agent acting in a genetically susceptible host was approached from a number of standpoints. The similarities between SDAT and the transmissible encephalopathies are discussed. One of the areas of similarity is the influence of genetic background on the development and expression of both conditions. Evidence is presented showing that genetics plays a role in many cases of SDAT and that there are known genetically controlled phenomena, the incidence of which is positively correlated with SDAT. For human encephalopathies the genetics of CJD and GSS are detailed. In experimental systems with scrapie, the influence of genetic control, operating through both host and agent, on the outcome of infection with scrapie is described. The events controlled include length of incubation period, type of lesions and their distribution and intensity. In the context of the human diseases, scrapie provides a model for the known human encephalopathies and for SDAT.

Aging↗
PubMed1984

Preclinical changes in weight of scrapie-infected mice as a function of scrapie agent-mouse strain combination.

Several inbred strains of mice were injected with different scrapie agents and their total body weight was monitored throughout the incubation period. As a control, mice were injected with normal mouse brain homogenate. For most combinations of scrapie agent and mouse strain, weights during the preclinical phase were similar to or lower than the average weight of controls. For some combinations there was a significant increase in weight (compared to controls) during the latter part of the preclinical phase of disease. The effect was dependent on both agent and mouse strain, i.e., in some cases a mouse strain showed the increase with one scrapie agent but not another and some scrapie agents caused the increase in one inbred strain of mouse but not in another strain. The increase in weight was due to accumulations of fat rather than a generalized increase in weight of various organs. With one mouse strain (SJL), there was increased vacuolation seen in the hypothalamus of mice injected with scrapie agents that showed the increase in weight compared to the lesion intensity with an agent which did not cause the weight increase.

Adipose Tissue↗
PubMed1983

Increased blood-brain barrier permeability in scrapie-infected mice.

The present investigation was designed to study the ultrastructural integrity of the blood-brain barrier (BBB) in the cerebral microvasculature of scrapie-infected mice showing clinical illness. Cerebral microvessels from either IM, VM, or C57BL/6J mice, terminally affected with various strains of scrapie agent showed a focal leakage of horseradish peroxidase (HRP) in all agent-strain and mouse-strain combinations. This leakage was most pronounced in and near the primary site of agent inoculation, but was also observed in microvessels scattered throughout the brain. Cytochemical studies also revealed a redistribution of plasmalemma-bound alkaline phosphatase in the endothelial cells. In control mice, the enzymatic activity was mainly concentrated in the luminal plasmalemma, while in the scrapie-infected mice the activity also appeared in the abluminal side in the majority of microvessels. Our observations are evidence that the BBB of the mouse is altered in some way by the scrapie agent. Such an alteration may have important implications for human disease, since the scrapie agent is related to the group of "slow" viral infections, including kuru and Creutzfeldt-Jakob disease. Scrapie may also serve as an important model for the study of senile dementia of the Alzheimer type (SDAT).

Animals↗
PubMed1983

Altered scrapie infectivity estimates by titration and incubation period in the presence of detergents.

During experiments on the purification of scrapie infectivity, changes were found in the dynamics of scrapie titration. After exposure to detergent, infectivity estimates by both endpoint titration and incubation period were altered. The addition of detergent to the diluent used in titration resulted in at least a 100-fold increase in the infectivity estimate. This suggests that the amount of scrapie in a sample, as measured by serial dilution and titration, may be underestimated to different extents, depending on the biochemical milieu of the inoculum. Membrane fractions treated with detergents before dilution exhibited longer incubation periods than untreated fractions for the same number of infectious units of scrapie. This demonstrates that detergent treatments and possibly other biochemical manipulations can cause changes in the response of the host to the inoculum that are not detectable if incubation periods alone are used to estimate scrapie 'titre'.

Animals↗
PubMed1983

Kinetics of replication of lactate dehydrogenase-elevating virus in age-dependent polioencephalomyelitis.

The kinetics of replication of lactate dehydrogenase-elevating virus (LDV) in age-dependent polioencephalomyelitis was studied in genetically susceptible (C58/J) and resistant (C57BL/6J) mice. The peripheral replication pattern (plasma concentration) for LDV was similar in both strains. However, the concentration of virus within the central nervous system was strikingly different. In nonsusceptible C57BL/6J mice, little or no virus was found within the central nervous system. In the lumbar cord of susceptible C58/J mice, an increase in the concentration of LDV began 5 days postinfection and continued during the preclinical stages of disease. A direct correlation was shown between the concentration of LDV in spinal cord and the appearance of motor neuron disease but not the degree of inflammatory reaction.

Aging↗
PubMed1982

Virological studies in amyotrophic lateral sclerosis.

Complement-fixing antibody response to eleven different viruses were measured in amyotrophic lateral sclerosis (ALS) patients, contacts of ALS patients, neurological controls, and normal controls. The normal controls showed a decreased response to adeno-associated virus and an increased response to adenovirus when compared to the other groups. Levels of interferon-like substances also were investigated in sera and cerebrospinal fluids of ALS patients and neurological controls. Responses were of a low titer and were not increased in the ALS group. Explant cultures were established from tissues of 24 ALS autopsy cases. Cultures were investigated directly or following fusion to various indicator cell lines for viral-like agents. Techniques such as interference assays, 5-bromodeoxyuridine (BudR) induction, hemadsorption, and fluorescent antibody staining failed to detect virus. By addition of helper adenovirus to primary explant cultures, adeno-associated virus was isolated from 2 of 11 ALS cases.

Adenoviruses, Human↗