Biomedical subjects

B Caughey

Publications and source records attributed to B Caughey.

At least 73 records · Page 4Linked to original sources

PubMed1992

Secondary structure of the pentraxin female protein in water determined by infrared spectroscopy: effects of calcium and phosphorylcholine.

The secondary structure of hamster female protein in aqueous solutions in the presence or absence of calcium and phosphorylcholine has been investigated using Fourier transform infrared spectroscopy. Our present studies provide the first evaluation of the secondary structure of FP and its calcium- and phosphorylcholine-dependent conformational changes. Quantitative analysis indicated that FP is composed of 50% beta-sheet, 11% alpha-helix, 29% beta-turn, and 10% random structures. Calcium- and phosphorylcholine-dependent infrared spectral changes were observed in regions assigned to beta-sheet, alpha-helix, turn, and random structures. The infrared-based secondary structure compositions were used as constraints to compute theoretical locations for the different secondary structures along the amino acid sequence of the FP protein. Two putative calcium-binding sites were proposed for FP (residues 93-109 and 150-168) as well as other members of the pentraxin family on the basis of the theoretical secondary structure predictions and the similarity in sequence between the pentraxins and EF-hand calcium-binding proteins. The changes in protein conformation detected upon binding of calcium and phosphorylcholine provide a mechanism for the effects of these ligands on physiologically important properties of the protein, e.g., activation of complement and association with amyloids.

Alpha-Globulins↗

PubMed1992

Potent inhibition of scrapie-associated PrP accumulation by congo red.

Transmissible spongiform encephalopathies (prion diseases), Alzheimer's disease, and other amyloidoses result in the accumulation of certain abnormally stable proteins that are thought by many to play central roles in disease pathogenesis. Using scrapie-infected neuroblastoma cells as a model system, we found that Congo red, an amyloid-binding dye, potently inhibits the accumulation of the scrapie-associated, protease-resistant isoform of protein PrP without affecting the metabolism of the normal isoform. Growth of the cells with submicromolar concentrations of Congo red for 5 days reduced the amount of protease-resistant PrP detected in the cultures by greater than 90%. This activity of Congo red suggests that it selectively disrupts the conversion of PrP to the protease-resistant isoform or destabilizes this isoform once it is made. Potential therapeutic applications of Congo red are discussed.

Animals↗

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

The scrapie-associated form of PrP is made from a cell surface precursor that is both protease- and phospholipase-sensitive.

A common feature of scrapie and related transmissible spongiform encephalopathies is the accumulation of an abnormal protease-resistant form of PrP which may be the major component of the infectious agent. While it is known that both the normal (protease-sensitive) PrP and protease-resistant PrP are encoded by the same endogenous gene, the nature of the disease-associated modification of PrP is not understood. To study the cellular events leading to the formation of protease-resistant PrP, we have compared its biosynthesis to that of its normal isoform in scrapie-infected mouse neuroblastoma cells. In pulse-chase labeling experiments, the protease-resistant PrP was synthesized and degraded much more slowly than the normal PrP, suggesting that protease-resistant PrP is made from a protease-sensitive precursor. More significantly, we found that the precursor of protease-resistant PrP was eliminated from intact cells by treatments with phosphatidylinositol-specific phospholipase C and trypsin. This demonstrated that, unlike the protease-resistant PrP itself, the precursor is phospholipase- and protease-sensitive and at least transiently found on the cell surface. By these criteria, the precursor of protease-resistant PrP is indistinguishable from the normal PrP isoform. These results indicate that the conversion of PrP to the protease- and phospholipase-resistant state is a post-translational event that occurs after the precursor reaches the cell surface.

Animals↗

PubMed1991

Electron microscopic characterization of neonatal platelet ultrastructure: effects of sampling techniques.

Detailed ultrastructure of 720 neonatal platelets was systematically examined in 200-300 microliters cord blood samples from four normal term infants. There were no differences in platelet organelles between samples collected into standard sodium citrate anticoagulant and minimally heparinized samples collected into sodium citrate. Samples collected into EDTA showed significant differences from the other two solutions. There were also differences in organelle characteristics within a solution group, as time from sample collection to EM fixation varied. These results verify utilization of small minimally heparinized blood samples for examination of platelet ultrastructure. This system can now be expanded to the premature infant, where small blood sample requirements are essential, and minimally heparinized blood may be more readily available.

Anticoagulants↗

PubMed1991

In vitro expression and biosynthesis of prion protein.

In addition to whatever function PrP may have normally, its involvement in scrapie-like neurodegenerative diseases has become clearer in recent years. In vitro studies have made important contributions to the understanding of normal PrP biosynthesis and turnover and how they can be influenced by scrapie infection. Cell-free transcription and translation experiments have indicated that PrP gene translation products are capable of assuming two different topologies, one spanning microsomal membranes and the other completely translocated into the microsomal lumen (Hay et al. 1987a, b). A novel stop transfer signal in the polypeptide is critical to the formation of the transmembrane topology (Yost et al. 1990). Expression of recombinant PrP genes has been accomplished in mouse (Caughey et al. 1988b), monkey (Scott et al. 1988), frog (Hay et al. 1987a), and insect (Scott et al. 1988) tissue culture cells. PrP products encoded by PrP cDNAs cloned from scrapie-infected brain tissues are not infectious and do not have the protease-resistance characteristic of the scrapie-associated form of PrP isolated from diseased tissue (Caughey et al. 1988b; Scott et al. 1988). Studies of PrP encoded by the endogenous gene of mouse neuroblastoma cells have identified the precursors (Caughey et al. 1989) and products (Race et al. 1988; Caughey et al. 1989) of normal PrP biosynthesis and shown that most of the PrP of normal cells is linked to the cell surface by phosphatidylinositol (Stahl et al. 1987; Caughey et al. 1989, 1990; Borchelt et al. 1990). In scrapie-infected clones, and additional pool of PrP is present which, unlike the normal PrP, aggregates (B. Caughey, unpublished observations) and is partially protease resistant (Butler et al. 1988; Caughey et al. 1990; Borchelt et al. 1990; Stahl et al. 1990). This scrapie-associated pool of PrP differs from the normal PrP in that it is primarily intracellular (Caughey et al. 1990; Borchelt et al. 1990; Taraboulos et al. 1990) and resistant to removal from cells by phospholipase or protease (Caughey et al. 1990; Borchelt et al. 1990; Stahl et al. 1990) treatments. Kinetic studies have shown that while PrP-sen is synthesized and degraded relatively rapidly (Caughey et al. Borchelt et al. 1990), PrP-res is synthesized slowly and has a very long half-life (Borchelt et al. 1990). Further studies with the scrapie-infected mouse neuroblastoma cells should lead toward the elucidation of the molecular details of the scrapie-associated modification of PrP and whether the modification is directly related to scrapie agent replication.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

Production of monoclonal antibodies reactive with a denatured form of the Friend murine leukemia virus gp70 envelope protein: use in a focal infectivity assay, immunohistochemical studies, electron microscopy and western blotting.

Four monoclonal antibodies were selected for their ability to recognize the envelope protein of Friend murine leukemia virus (F-MuLV) in methanol-fixed tissue culture cells. Each of these monoclonal antibodies was found to react only with F-MuLV. By using recombinant retroviruses, it was determined that each of the monoclonal antibodies recognized the C-terminal one-third of the F-MuLV gp70 envelope protein. The monoclonal antibodies were effective in radioimmunoprecipitation of F-MuLV proteins, and one of the antibodies, 720, was also effective in Western blotting. The ability of antibody 720 to react with F-MuLV in methanol-fixed cells facilitated the use of a sensitive immunoperoxidase method with a focal virus infectivity assay. In immunohistochemical studies using light microscopy, antibody 720 could specifically label F-MuLV-infected cells in acetone-fixed tissue sections from F-MuLV-infected animals. Finally, in immuno-gold labelling studies using electron microscopy, antibody 720 could be used to distinguish F-MuLV from amphotropic MuLV.

3T3 Cells↗

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

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↗

PubMed1989

Comparative sequence analysis, in vitro expression and biosynthesis of mouse PrP.

A number of studies have indicated that an endogenous brain protein, PrP, is associated with transmissible agents causing spongiform encephalopathies such as scrapie, kuru, and Creutzfeldt-Jakob disease. It has been proposed that PrP derived from scrapie brain is the scrapie agent itself. To test directly whether the PrP mRNA in scrapie brain tissue can encode the scrapie agent, we expressed PrP cDNA cloned from scrapie-infected mouse brain in vitro. The expressed PrP did not transmit scrapie to susceptible mice. Thus either PrP is not the scrapie agent, or the expressed PrP requires additional modification to be infectious. The normal function of PrP is unknown, however, comparison of the amino acid sequences of PrP from mouse, hamster, and human revealed that many structural features of potential functional significance have been conserved during evolution. To learn about normal PrP and whether it is altered by scrapie infection in vitro, we have performed studies of PrP biosynthesis in normal and scrapie-infected mouse neuroblastoma tissue culture cells. The major PrP species were glycoproteins anchored at the cell surface by covalent linkage to phosphatidylinositol. No scrapie-associated modifications of PrP biosynthesis were observed, and, none of the metabolically labeled PrP observed in either scrapie-infected normal cells was resistant to proteinase K.

Amino Acid Sequence↗

PubMed1988

In vitro expression of cloned PrP cDNA derived from scrapie-infected mouse brain: lack of transmission of scrapie infectivity.

A cDNA for the prion protein (PrP) derived from scrapie-infected mouse brain was expressed in C127 mouse cells in vitro under the control of the mouse metallothionein promoter. PrP synthesis was detected by immunoprecipitation using a rabbit antibody specific for a 15 amino acid PrP peptide. Homogenates of cells expressing the cloned PrP cDNA inoculated into weanling mice failed to induce clinical scrapie during 190 days of observation. We conclude that either PrP is not the transmissible agent of scrapie or the PrP is not processed appropriately in this cell system to create the infectious agent.

Animals↗

PubMed1988

In vitro expression in eukaryotic cells of a prion protein gene cloned from scrapie-infected mouse brain.

It has been proposed that the causative agent of scrapie represents a class of infectious particle that is devoid of nucleic acid and that an altered form of the endogenous prion protein (PrP) is the agent. However, it has been difficult to exclude the possibility that PrP purified from scrapie tissues might be contaminated with a more conventional viral agent. To obtain PrP uncontaminated by scrapie-infected tissues, PrP cDNA cloned from a scrapie-infected mouse brain was expressed in mouse C127 cells in vitro. mRNA and protein encoded by the cloned PrP gene were identified. The expressed PrP polypeptides appeared to be glycosylated and were released from the cell surface into the medium. Homogenates of the cells expressing the cloned PrP gene were inoculated into susceptible mice but failed to induce clinical signs of scrapie. Thus, either PrP is not the transmissible agent of scrapie or the expressed PrP requires additional modification to be infectious.

Animals↗

PubMed1988

Detection of prion protein mRNA in normal and scrapie-infected tissues and cell lines.

Prion protein (PrP) forms the fibrils or prion rods isolated from scrapie-infected brain and has been proposed as the major component of the infectious agent of this slowly progressive spongiform encephalopathy. In previous Northern blot analyses PrP-specific mRNAs have been found in both normal and scrapie-infected brains but not in spleen, an organ which harbours large titres of infectivity. In the present study, mouse PrP DNA was used to probe for PrP mRNA in assorted tissues and cells. A reexamination of mouse and hamster spleens revealed that they contained low levels of PrP mRNA (approx. 0.8% of that in brain mRNA). No consistent differences were observed between normal and scrapie-infected tissues. Also positive for PrP mRNA under stringent hybridization conditions were mouse epithelial, neuroblastoma, erythroid, B-lymphocytic and embryo fibroblast tissue culture cell lines, a hamster ovary cell line, a rat glioma cell line, and human T lymphocytic and neuroblastoma cell lines. In contrast, no PrP mRNA was detected in two mouse myeloid cell lines and one T cell lymphoma. These results provide evidence that PrP is a protein common to numerous, but not all, cell types besides those of the brain.

Animals↗

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↗

PubMed1987

Effects of reserpine and tetrabenazine on catecholamine and ATP storage in cultured bovine adrenal medullary chromaffin cells.

The in vivo storage relationship between catecholamines and ATP in chromaffin vesicles of cultured bovine adrenal medulla cells was investigated using drugs that block vesicular catecholamine uptake. Three-day treatments with reserpine and tetrabenazine causing 85-90% depletion of catecholamines resulted in 41-46% reductions in cellular ATP content. Subcellular fractionation of reserpine-treated cells indicated that the ATP is lost from the chromaffin vesicle pool. This was confirmed in experiments using metabolic inhibitors to differentiate the vesicular and extravesicular ATP pools. The vesicular ATP loss was not proportional to that of catecholamines, resulting in a reduction by 50% in the chromaffin vesicle mole ratio of catecholamines to ATP after 48 h of treatment. In metabolic labeling studies, it was found that reserpine treatment reduced the incorporation of [3H]adenosine into vesicular ATP selectively, but it reduced the incorporation of 32Pi into both the vesicular and extravesicular pools. The reduction of the [3H]adenosine incorporation was not due to diminished vesicular nucleotide uptake resulting from low catecholamine levels, because when the catecholamines were depleted by tetrabenazine pretreatment followed by removal of the drug before labeling, no reduction in [3H]adenosine incorporation was observed. When present during the labeling, tetrabenazine was found to be a reversible inhibitor of plasma membrane adenosine uptake. The observed loss of adenine nucleotides from catecholamine-depleted chromaffin vesicles in vivo provides evidence that interactions between ATP and catecholamines are important in the vesicular storage of high concentration of these compounds.

Adenosine↗