PubMed Health⌕ Search

Biomedical subjects

C L Masters

Publications and source records attributed to C L Masters.

At least 181 records · Page 10Linked to original sources
PubMed1994

Blood buffy coat from Alzheimer's disease patients and their relatives does not transmit spongiform encephalopathy to hamsters.

There was a report of spongiform encephalopathy transmitted to Syrian hamsters by intracerebral inoculation with the blood buffy coat of patients with Alzheimer's disease (AD) and their unaffected first-degree relatives. We attempted to verify that report, taking measures to reduce the risk of contaminating samples with agents causing spongiform encephalopathies. We obtained blood from 50 subjects, including six patients with familial AD, 21 unaffected first-degree relatives (siblings and offspring) of patients with familial AD, and 20 control subjects. We inoculated the buffy coats intracerebrally into Syrian LVG hamsters, observed them for signs of neurologic disease, examined their brains for neuropathologic changes at time of death, and performed serial (blind) passages by inoculating suspensions of all recovered brains into fresh LVG hamsters. We discerned no clinical illness or histopathologic changes resembling experimental spongiform encephalopathy in any hamster inoculated with human buffy coat nor in blind-passage hamsters, nor were the life spans of those hamsters shortened. We conclude that AD is not caused by an agent that transmits spongiform encephalopathy to hamsters.

Alzheimer Disease↗
PubMed1994

A heparin-binding domain in the amyloid protein precursor of Alzheimer's disease is involved in the regulation of neurite outgrowth.

The amyloid protein precursor (APP) of Alzheimer's disease is synthesized as an integral transmembrane protein that is released from cells in culture following proteolytic cleavage. The function of released APP is not known, although there is evidence that the protein may bind to components of the extracellular matrix (ECM). In the present study, substratum-bound APP stimulated neurite outgrowth in cultures of chick sympathetic and mouse hippocampal neurons. This effect was dependent upon the presence of substratum-bound heparan sulfate proteoglycans (HSPG). The effect of APP on neurite outgrowth was comparable to that of laminin. A 14 K N-terminal fragment of APP was found to bind heparin and a region close to the N-terminus of APP (residues 96-110) identified as a potential heparin-binding domain based on secondary structure predictions and molecular modeling. Mutagenesis of three basic residues (lysine-99, arginine-100, and arginine-102) resulted in a recombinant protein (APPhep) with decreased heparin-binding capacity. A peptide homologous to the heparin-binding domain was synthesized and found to bind strongly to heparin and to inhibit binding of 125I-labeled APP to heparin (IC50 approximately 10(-7) M). The peptide blocked the effect of APP on neurite outgrowth (IC50 approximately 10(-7) M), whereas two other peptides homologous to other domains in APP had no effect. The results indicate that the binding of APP to HSPG in the ECM may stimulate the effects of APP on neurite outgrowth.

Amino Acid Sequence↗
PubMed1993

Amyloid-like properties of peptides flanking the epitope of amyloid precursor protein-specific monoclonal antibody 22C11.

Alzheimer's disease is one of the prevalent forms of human dementia. Its pathology is distinguished by proteinaceous deposits ("amyloid") in the brain. They contain a peptide (beta A4) that is proteolytically derived from a larger transmembrane protein. To follow the different metabolic pathways of this Amyloid Precursor Protein (APP) may thus lead to the elucidation of the molecular basis of Alzheimer's disease. Specific antibodies are necessary tools for this task. Using synthetic peptides, we have characterized the epitope of the APP-specific monoclonal antibody 22C11; it is localized between residues 66 and 81 of APP. Some of the peptides flanking this site exhibited properties generally associated with amyloid, i.e. low solubility, filament formation, and birefringence after Congo Red staining. Exploiting differences in the peptides' aggregational properties, we present evidence that the two dyes Eosin and Direct Red 254, in conjunction with classical amyloid staining by Congo Red, can be used to characterize aggregating, amyloid-like peptides in vitro.

Amino Acid Sequence↗
PubMed1993

Regulation and expression of the Alzheimer's beta/A4 amyloid protein precursor in health, disease, and Down's syndrome.

A four- to fivefold overexpression of the gene for the Alzheimer beta/A4 amyloid precursor protein (APP) in individuals with Down's Syndrome (DS) appears to be responsible for the fifty year earlier onset of Alzheimer's disease (AD) pathology in DS compared to the normal population. It is therefore likely that a deregulated overexpression of the APP gene is a risk factor for the beta/A4 amyloid formation. To test this hypothesis and to get a better understanding of how APP expression is regulated, we studied the 5' control region of the human APP gene, alternative splicing of the 19 APP exons, and APP biogenesis, metabolism and function. The analysis of the APP promoter revealed its similarity with those of housekeeping genes by the presence of a GC-rich region around the transcription start site and the lack of a TATA box. Gene transfer experiments showed this GC-rich region to contain overlapping binding sites for different transcription factors whose binding is mutually excluded. An imbalance between these factors may cause APP overexpression and predispose to AD pathology. Another putative risk factor for AD is regulation of splicing of exon 7 in APP mRNA's which changes in brain during aging. This is relevant for APP processing since exon 7 codes for a Kunitz protease inhibitory domain. Investigation of further splicing adjacent to the beta/A4 exons 16 and 17 which might also interfere with APP processing led to the identification of the leukocyte-derived (L-APP) splice forms which lack exon 15. In brain this splicing occurs in activated astrocytes and microglia. The localization of APP at synaptic sites in brain suggests that APP regulation and expression are critical determinants of a potential and early impairment of central synapses. This may be the case during pathological evolution of AD and DS when beta/A4 derived from synaptic APP is converted to beta/A4 amyloid by radical generation.

Aging↗
PubMed1993

The role of extracellular matrix in the processing of the amyloid protein precursor of Alzheimer's disease.

Alzheimer's disease (AD) is characterized by the presence of extracellular amyloid plaques, which contain a protein referred to as the amyloid or beta A4 protein. The beta A4 protein is derived from a larger precursor protein (APP). Studies of autosomal-dominant forms of AD have established the central role of APP in the pathogenesis of the disease. Despite considerable research, the function of APP is unknown. APP can be processed by at least two separate routes. The first route involves a protease known as "APP secretase," which cleaves within the amyloid sequence, thereby mitigating amyloid formation. The second route may result in the production of potentially amyloidogenic fragments. Our studies suggest that following release from the cell membrane, APP interacts with components of the extracellular matrix (ECM) such as the heparan sulfate proteoglycans (HSPG's). The interaction of APP with HSPG's may be important for the function of APP. Substratum-bound APP was found to dramatically increase neurite outgrowth and survival of chick sympathetic neurons in vitro. This effect was dependent upon the presence of substratum-bound HSPG. The results suggest that normally, when bound to the ECM, APP functions to promote neurite outgrowth and/or cell survival. Loss of this normal trophic function might occur in AD, when APP is proteolytically processed via the amyloidogenic pathway.

Alzheimer Disease↗
PubMed1993

The beta A4 amyloid protein precursor in human circulation.

beta A4, the principal constituent of the brain amyloid collections in Alzheimer's disease, is derived from a much larger precursor, the amyloid protein precursor (APP). APP exists in the blood as full-length, potentially amyloidogenic forms in platelets, and as an attenuated species in plasma and T-lymphocytes. Studies of circulating APP facilitate the elaboration of the function of this protein, as well as the elucidation of its processing in health and disease.

Alzheimer Disease↗
PubMed1993

Expression of L-APP mRNA in brain cells.

Several reports addressed the issue of how the alternative splicing of exon 7 and 8 in the APP pre-mRNA is regulated in different tissues. Of special interest here was the potential involvement of exon 7 containing APP splice isoforms, since this exon codes for a serine protease inhibitor and is therefore of putative relevance for amyloidogenic catabolism of the precursor protein. The recent identification of a third alternative splice site in close proximity to the beta A4-amyloid portion in the APP gene which may also increase APP amyloidogenicity, allowed us to investigate its regulation in cells of the central nervous system. With our assay, we were able to resolve six different APP isoforms of the eight potential isoforms which can be generated from the three alternatively spliced exons 7, 8, and 15. We demonstrate here that, in addition to rat brain microglia cells, astrocyte-enriched cultures also skip the novel alternative 3'-splice site in front of exon 15, generating L-APP mRNA. Neurons are the only cells in the central nervous system which seem to use the 3'-splice site of intron 14 nearly 100%. Interestingly, this very 3'-splice site is the only one present in the APP gene that completely matches the consensus sequence for the branchpoint sequence proposed for introns. We would therefore suggest that neurons lack a specific splicing factor which inhibits the use of the rather strong 3'-splice site in front of exon 15. It remains to be shown whether this is also the case for neurons in Alzheimer's disease.

Alternative Splicing↗
PubMed1993

A novel zinc(II) binding site modulates the function of the beta A4 amyloid protein precursor of Alzheimer's disease.

Abnormalities of zinc metabolism occur in Alzheimer's disease (AD), a condition where pathological catabolism of the amyloid protein precursor (APP) causes cerebral beta A4 amyloidosis. An association between zinc and APP metabolism was sought by studying the binding of 65Zn2+ to APP. 65Zn2+ bound in a rapid, saturable, and specific manner (KD = 764 nM). A novel zinc binding motif, strongly conserved between members of the APP family, was located between the cysteine-rich and negatively charged domains of the protein. Zinc increased binding of APP to heparin and has been shown to potentiate the inhibition of coagulation factor XIa by an APP isoform containing a Kunitz-type inhibitory domain (Komiyama, Y., Murakami, T., Egawa, H., Okubo, S., Yasunaga, K., and Murata, K. (1992) Thromb. Res. 66, 397-408) situated near the zinc binding region. Zinc is a factor that modulates the functional properties of the substrate for beta A4 amyloidogenesis.

Alzheimer Disease↗
PubMed1993

Amyloidogenicity of rodent and human beta A4 sequences.

Previously we have shown that aggregation of the C-terminal 100 residues (A4CT) of the beta A4 amyloid protein precursor (APP) and also of beta A4 itself depends on the presence of metal-catalyzed oxidation systems [T. Dyrks et al. (1988) EMBO J. 7, 949-957]. We showed that aggregation of the amyloidogenic peptides induced by radical generation systems requires amino acid oxidation and protein cross-linking. Here we report that aggregation of A4CT and beta A4 induced by radical generation systems involves oxidation of histidine, tyrosine and methionine residues. The rodent beta A4 sequence lacking the single tyrosine and one of the three histidine residues of human beta A4 and a beta A4 variant in which the tyrosine and the three histidine residues were replaced showed a reduced tendency for aggregation. Thus our results may explain why beta A4 amyloid deposits could so far not been detected in the rodent brain.

Amino Acid Sequence↗
PubMed1993

Amyloid precursor protein (APP) in the striatum in Alzheimer's disease: an immunohistochemical study.

Increasing recognition of diffuse plaques has raised questions about the differences between diffuse and neuritic plaques, particularly in regard to the role of amyloid precursor protein (APP) processing in their formation. To address this issue, corpus striatum (containing almost exclusively diffuse plaques) and cerebral cortex (containing an admixture of plaque types) from patients with Alzheimer's disease (AD) were examined immunohistochemically with antibodies to domain-specific sites of APP (N-terminal, C-terminal, beta A4-related, isoform-specific, and other epitopes). Striatal plaques labeled strongly with beta A4 antibodies as did cortical plaques in AD and the occasional diffuse plaques in cortex from nondemented elderly controls. Weak labeling of some cortical neuritic plaques but not diffuse plaques was observed with antibodies directed against other APP epitopes. Electron microscopy of diffuse plaque-rich striatum in AD cases revealed only rare degenerating neurites without apparent fibrillar amyloid; no changes were noted in the plaque-free striatum of controls. These results suggest that antibodies to beta A4 recognize not only fibrillar amyloid of neuritic plaques but also antigenic determinants of diffuse plaques which lack fibrillar amyloid. Furthermore, the finding that antibodies to non-A4 domains of APP labeled only cortical but not striatal plaques suggests that APP processing mechanisms in cortical and striatal tissues may differ.

Aged↗
PubMed1993

APP+ T lymphocytes selectively sorted to endomysial tubes in polymyositis displace NCAM-expressing muscle fibers.

The characteristic pathogenic feature of polymyositis (PM) is muscle invasion by T lymphocytes penetrating the basal lamina and displacing the sarcolemma of normal muscle fibers (T cell invasion of endomysial tubes). Active forward movement of these T cells is indicated by cell extensions interdigitating with the muscle fiber surface. Here we describe for the first time high abundance of Alzheimer amyloid protein precursor (APP) in invasive T cells contacting the border of muscle fibers in PM. These are the sites of muscle fiber displacement. The percentage of APP+ T cells at these sites is significantly higher than in other neuromuscular disorders with inflammatory infiltrates suggesting a specific pathogenic function of these cells in PM. By using a new multiparameter immunofluorescence imaging procedure and confocal laser scanning microscopy, we show that APP+ T cells in PM are invasive front cells that penetrate the basal lamina of the endomysial tube and displace the muscle fiber. Mononuclear cells behind the invasive front are negative for APP or show much lower APP levels. Front T cells either express the CD8-CD4+APP+ or CD8+CD4-APP+ phenotypes, or are CD4+CD8+APP+ T cell chimeras. The highest APP concentration is found at the tip of T cell extensions interdigitating with the fiber surface. Although normal by morphological criteria, the same fibers show intense staining for the regeneration marker NCAM. This reactivity is highest at sites contacted by the APP+ T cells. The findings indicate that APP is specifically upregulated in T cells displacing muscle fibers in PM and suggest that NCAM, which may be abnormally regulated in these fibers, is a candidate molecule for interaction with APP.

Amyloid beta-Protein Precursor↗
PubMed1992

Alzheimer beta A4-amyloid protein precursor in immunocompetent cells.

The mechanism of proteolytic breakdown of the beta A4-amyloid protein precursor (APP) has attracted much attention because of its relevance for Alzheimer's disease. Apart from the pathological role of APP in the amyloidogenesis, many efforts have been made to identify the functional significance of this widely expressed protein in various biological processes. Employing biochemical techniques, we demonstrate that APP is involved in the initiation of the immune response. Upon stimulation, it is expressed by the major functional types of T-lymphocytes, i.e. CD4+ and CD8+ cells. As was demonstrated for the CD4+ lymphoid cell line H9, APP is predominantly secreted. The remaining COOH-terminal fragments generated upon secretion were highly unstable. Of the APP produced by immunocompetent cells, considerable amounts were shown to be leukocyte-derived APP (L-APP). In addition, we were able to identify the KPI-containing L-APP isoform, L-APP733, as the major expressed L-APP isoform in immunocompetent cells, including rat microglial cells and astrocytes. The L-APP expression pattern of these cells showed high similarity. These findings seem to be indicative of an important function of APP within the immune system. Therefore, APP may be involved in various immunopathogenic conditions of the periphery and in the central nervous system.

Amyloid beta-Peptides↗
PubMed1992

Substitutions of hydrophobic amino acids reduce the amyloidogenicity of Alzheimer's disease beta A4 peptides.

The deposition of amyloid protein aggregates in brain is the main pathological feature of Alzheimer's disease. Their principal constituent is a peptide termed beta A4, which comprises up to 43 amino acid residues. It is highly insoluble under physiological conditions and aggregates into filaments that form very dense clusters in vivo and in vitro. Based on a beta A4 prototype sequence spanning residues 10 to 42 or 43, we have designed analogues in which hydrophobic amino acid residues in position 17 to 20 were substituted by more hydrophilic residues. Depending on the kind of newly introduced amino acids and their position within the sequence, the substitution of only two residues led to variants exhibiting a broad spectrum of different properties. Common to them was a reduced beta-sheet content after solubilization in water and in the solid state. Some of the variants showed significantly reduced amyloidogenicity: although still forming filaments, they did not aggregate into the highly condensed depositions that are typical for amyloid. In addition, they could be solubilized in 200 mM-NaCl and KCl. When mixed with beta A4 peptides bearing the natural sequence, two of the analogues could inhibit the formation of filaments in vitro. These results demonstrate that a well-preserved hydrophobic core around residues 17 to 20 of beta A4 is crucial for the formation of beta-sheet structure and the amyloid properties of beta A4. The introduction of structural alterations within this region may guide the development of reagents for the therapy of Alzheimer's disease.

Alzheimer Disease↗
PubMed1992

Identification and differential expression of a novel alternative splice isoform of the beta A4 amyloid precursor protein (APP) mRNA in leukocytes and brain microglial cells.

The gene for the beta A4-amyloid precursor protein (APP) consists of 19 exons which code for a typical N- and O-glycosylated transmembrane protein with four extracellular domains followed by the transmembrane domain and a short cytoplasmic domain. The beta A4-amyloid sequence is part of exons 16 and 17. Several APP isoforms can be generated by alternative splicing of exons 7 and 8, encoding domains with homologies to Kunitz-type protease inhibitors and the MRC OX-2 antigen, respectively. The mechanism by which the pathological beta A4 is generated is unknown, it is however a critical event in Alzheimer's disease and is distinct from the normally occurring cleavage and secretion of APPs within the beta A4 sequence. We report here for the first time considerable APP mRNA expression by rat brain microglial cells. In addition we showed by S1 nuclease protection and polymerase chain reaction analysis of reverse transcribed RNA (RT-PCR) that T-lymphocytes, macrophages, and microglial cells expressed a new APP isoform by selection of a novel alternative splice site and exclusion of exon 15 of the APP gene. This leads to a transmembrane, beta A4 sequence containing APP variant, lacking 18 amino acid residues close to the amyloidogenic region. The use of this novel alternative splice site alters the structure of APP in close proximity to the beta A4 region and thus may determine a variant, potentially pathogenic processing of leukocyte-derived APP in brain.

Amyloid beta-Protein Precursor↗
PubMed1992

The expression of the amyloid precursor protein (APP) is regulated by two GC-elements in the promoter.

The structure of the promoter of the human APP gene resembles that of housekeeping genes, with the presence of a GC-rich region and the lack of a canonical TATA box. Since analysis of the expression of the APP gene, especially at the transcriptional level, might reveal factors or elements, which influence amyloid formation in Alzheimer's disease, a 5' deletion analysis of the APP promoter was performed, leading to the identification of an activating DNA fragment (Ac), acting also on a heterologous promoter. DNaseI-footprint analysis revealed three protected regions on the Ac fragment. Further gene transfer experiments showed, that at least two elements, designated A and C, confer transcriptional activity in HeLa cells. Whereas the proximal element A is a 19bp long GC-rich DNA sequence, the distal element C is a GC-palindrome with the sequence 5'GCGGCGCCGC.

Amyloid beta-Protein Precursor↗
PubMed1992

Beta A4 amyloid protein and its precursor in Alzheimer's disease.

The beta A4 amyloid protein is now understood to play a pivotal role in the development of Alzheimer's disease. This protein is generated by the abnormal processing of the amyloid protein precursor, a large membrane glycoprotein. Insights into the mechanisms of this abnormal processing will give information relevant to the design of new therapeutic strategies for Alzheimer's disease.

Alzheimer Disease↗
PubMed1992

The amyloid protein precursor of Alzheimer's disease is a mediator of the effects of nerve growth factor on neurite outgrowth.

The beta A4 protein, the major component of the amyloid deposition characterizing Alzheimer's disease, derives from the amyloid protein precursor (APP), an integral membrane protein with soluble derivatives. The function of APP is unknown. Both soluble and membrane-associated human brain APP (10(-10) M) significantly increased (P less than 0.025) neurite length and branching in pheochromocytoma PC12 cells, but did not affect the number of neurites per cell. At higher concentrations, APP was cytotoxic, with a half-maximal concentration of 5 x 10(-9) M. Nerve growth factor (NGF) is known to affect APP expression in vivo and in vitro. Antibodies to APP specifically diminished the effects of NGF on neurite length and branching. Thus APP may act to mediate neurite outgrowth promotion by NGF.

Alzheimer Disease↗