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Biomedical subjects

C L Masters

Publications and source records attributed to C L Masters.

At least 145 records · Page 8Linked to original sources
PubMed1996

Iatrogenic and zoonotic Creutzfeldt-Jakob disease: the Australian perspective.

The transmissible brain diseases of humans and animals, the spongiform encephalopathies, continue to stimulate interest, and the announcement that exposure to "mad cow disease" (bovine spongiform encephalopathy [BSE]) is a possible explanation for more than 10 cases of a variant Creutzfeldt-Jakob disease in humans in the United Kingdom is a recent example. Cases of iatrogenic Creutzfeldt-Jakob disease (from previous use of human cadaveric tissues for pituitary hormone therapy and neurosurgical grafts) are still being identified, and the unique nosological status of this group of disorders-that they are both transmissible and inherited and that the only known component of their infectious agent is protein-alone makes these diseases remarkable.

Animals↗
PubMed1996

The amyloid precursor protein of Alzheimer's disease in the reduction of copper(II) to copper(I)

The transition metal ion copper(II) has a critical role in chronic neurologic diseases. The amyloid precursor protein (APP) of Alzheimer's disease or a synthetic peptide representing its copper-binding site reduced bound copper(II) to copper(I). This copper ion-mediated redox reaction led to disulfide bond formation in APP, which indicated that free sulfhydryl groups of APP were involved. Neither superoxide nor hydrogen peroxide had an effect on the kinetics of copper(II) reduction. The reduction of copper(II) to copper(I) by APP involves an electron-transfer reaction and could enhance the production of hydroxyl radicals, which could then attack nearby sites. Thus, copper-mediated toxicity may contribute to neurodegeneration in Alzheimer's disease.

Alzheimer Disease↗
PubMed1996

Regulation of amyloid protein precursor (APP) binding to collagen and mapping of the binding sites on APP and collagen type I.

The specific binding of the amyloid precursor protein (APP) to extracellular matrix molecules suggests that APP regulates cell interactions and has a function as a cell adhesion molecule and/or substrate adhesion molecule. On the molecular level APP has binding sites for collagen, laminin, and glycosaminoglycans which is a characteristic feature of cell adhesion molecules. We have examined the interactions between the APP and collagen types I and IV and identified the corresponding binding sites on APP and collagen type I. We show that APP bound most efficiently to collagen type I in a concentration-dependent and specific manner in the native and heat-denatured states, suggesting an involvement of a contiguous binding site on collagen. This binding site was identified on the cyanogen bromide fragment alpha 1(I)CB6 of collagen type I, which also binds heparin. APP did not bind to collagen type I-heparin complexes, which suggests that there are overlapping binding sites for heparin and APP on collagen. We localized the site of APP that mediates collagen binding within residues 448-465 of APP695, which are encoded by the ubiquitously expressed APP exon 12, whereas the high affinity heparin binding site of APP is located in exon 9. Since a peptide encompassing this region binds to collagen type I and inhibits APP-collagen type I binding in nanomolar concentrations, this region may comprise the major part of the collagen type I binding site of APP. Moreover, our data also indicate that the collagen binding site is involved in APP-APP interaction that can be modulated by Zn(II) and heparin. Taken together, the data suggest that the regulation of APP binding to collagen type I by heparin occurs through the competitive binding of heparin and APP to collagen.

Amino Acid Sequence↗
PubMed1996

APP gene family. Alternative splicing generates functionally related isoforms.

The Alzheimer's beta A4-amyloid protein precursor (APP) and the APP-like proteins (APLPs) are transmembrane glycoproteins with a similar modular domain structure. APP exists in 8 isoforms generated by alternative splicing of exons 7, 8, and 15, of which the L-APP mRNAs lacking exon 15 are ubiquitously expressed in rat tissues but not in neurons. Rat APLP2, the nearest relative of APP, is similarly expressed in 4 different isoforms due to alternative splicing of inserts encoding a Kunitz protease inhibitor domain (KPI, homologous to exon 7 of APP) and a divergent region of 12 amino acids on the NH2-terminal side of the transmembrane domain (12 aa exon). KPI-APLP2 transcripts are highly expressed in neurons, in contrast to KPI-APPs, while L-APLP2 mRNA isoforms lacking the 12 aa exon are predominantly expressed in non-neuronal rat tissues, similar to L-APPs. Further examination of the divergent domains in APP and APLP2 harboring the similarly alternatively spliced APP exon 15 and the 12 aa exon of APLP2 revealed some structural similarities of the amino acid sequences and the predicted secondary structures. In both L-APLP2 and L-APP, a putative xylosyl-transferase recognition site for chondroitin sulfate glycosaminoglycan attachment is present that is interrupted in APP and APLP2 isoforms expressing APP exon 15 or the 12 aa exon of APLP2. Thus, a related function of the divergent domains and the corresponding alternatively spliced APP and APLP2 isoforms in regulation of the binding properties of the ectodomain is suggested. Additionally, beta-secretase cleavage of APP might be sterically hindered selectively in proteoglycan L-APP but not in APP lacking the proteoglycan attachment site. Neurons which have a uniquely low portion of L-APP and high content of APP might therefore be especially susceptible to beta A4-protein liberation. This could explain the selective vulnerability of neurons that is observed in Alzheimer's disease.

Alternative Splicing↗
PubMed1996

The role of heparan sulfate proteoglycans in the pathogenesis of Alzheimer's disease.

The hallmark of Alzheimer's disease (AD) is the deposition of amyloid plaques and neurofibrillary tangles in the brain. The relationship between amyloid deposition and the cognitive deficit is still unclear. The amyloid beta A4 protein is produced by proteolytic cleavage of the amyloid protein precursor (APP). Very little is known about the normal function of APP and the role the protein may play in pathogenesis. Several studies have shown that APP is important for the regulation of neurite outgrowth. Our studies support these findings and indicate that the neurite outgrowth-promoting effects of APP are stimulated by an interaction between APP and specific proteoglycans. Using site-directed mutagenesis, a heparan sulfate binding site which mediates this effect has been mapped to the N-terminus of APP (residues 96-110, HBD-1). A peptide homologous to HBD-1 blocks the trophic effects of APP in cell culture. To purify specific proteoglycans which stimulate the action of APP, an affinity column was constructed using a biotinylated peptide homologous to HBD-1 coupled to streptavidin-agarose. Two proteoglycans were isolated from a crude brain cell conditioned medium by affinity chromatography. The purified proteoglycans bound APP saturably with high affinity and stimulated the action of APP on neurite outgrowth from chick sympathetic neurons. Digestion of the proteoglycan fraction with heparitinase I or chondroitinase ABC demonstrated the presence of two major proteins, a heparan sulfate proteoglycan with a core protein of 63-67 kD molecular mass and a chondroitin sulfate proteoglycan with a core protein of 100-110 kD molecular mass. The results demonstrate that APP binds to at least two proteoglycans and that this interaction may regulate the trophic effects of the protein. The interaction of specific APP-binding proteoglycans with amyloid plaques may disturb the normal function of APP and contribute to the neuritic degeneration that is commonly seen around the amyloid plaque cores.

Alzheimer Disease↗
PubMed1996

Alzheimer's disease: genesis of amyloid.

Much of the present knowledge on the genes and genetic processes involved in the genesis of amyloid formation in Alzheimer's disease (AD) has come directly or indirectly from the retrospective molecular and genetic analysis of amyloid beta-protein (A beta or beta A4) deposits and from the identification of genes involved in inherited susceptibility to the disease. This analysis shows that the release and aggregation of the A beta fragment from the amyloid precursor protein (APP) is involved in APP (AD1), chromosome 14 (AD3), 1 (AD4) and 19(AD2) families as well as in the sporadic forms of AD, suggesting that AD is a single disease with a common APP/A beta amyloid pathogenesis. Synthetic A beta protein readily forms beta sheets, filaments and amyloid at micromolar concentrations. The principle to inhibit this process has been worked out by our groups with A beta variants. The N-terminal and C-terminal A beta sequences, oxidative radicals, membrane integrity and metal ions also affect the aggregation of A beta. Amino acid substitutions within the A beta sequence, as occur in rodents, alter A beta release and change the degree to which oxidation of the peptides occurs. Transgenic approaches resulting in overexpression of human APP have confirmed that A beta sequence and concentration are critical prerequisites to amyloid deposition in vivo.

Alzheimer Disease↗
PubMed1996

Genes contributing to Alzheimer's disease.

We propose that Alzheimer's disease (AD) is a single disease with a common metabolic APP-beta A4-amyloid pathway. The multiple genetic and other factors already identified to induce this pathway are reviewed. The molecular genetics of AD has been successfully studied within the last years, and we now can account for the genetic and molecular alterations underlying the majority of familial AD cases inherited with an autosomal dominant pattern of complete penetrance. AD in these pedigrees can be caused by missense mutations within the recently identified PS1 (S182) gene on chromosome 14 (AD3 locus) and the PS2 (STM2/E5-1) gene on chromosome 1, in addition to previously described point mutations of the beta A4-amyloid protein precursor (APP) gene on chromosome 21 (AD1 locus). The majority of AD cases, however, appears to be sporadic or 'familial' in terms of an increased family-associated AD-probability. Genetic risk factors contributing to AD in these cases have also been identified. On chromosome 19, allelic segregation of the APOE gene with both late onset 'familial' (AD2) and sporadic AD has been demonstrated, with the APOE epsilon 4 allele conferring a relatively higher risk of developing AD at an earlier age. Several other risk factors have also been proposed, including the alpha 1-antichymotrypsin allele A (ACT-A), the 5-repeat allele of the VLDL-receptor (VLDL-R) gene, the A2 allele of the HLA-A locus, and possibly yet unknown mitochondrial mutations. All these findings are discussed against the background of what is known about APP metabolism leading to beta A4 amyloid formation, a process that is also modified by APP expression level, alternative splicing of APP exon 15, extracellular signalling and intracellular sorting.

Alzheimer Disease↗
PubMed1995

Gelatinase A possesses a beta-secretase-like activity in cleaving the amyloid protein precursor of Alzheimer's disease.

The ability of the 72 kDa gelatinase A to cleave the amyloid protein precursor (APP) was investigated. HeLa cells were transfected with an APP695 plasmid. The cells were incubated with gelatinase A, which cleaved the 110 kDa cell-surface APP, releasing a 100 kDa form of the protein. A peptide homologous to the beta-secretase site was cleaved by gelatinase A adjacent to a glutamate residue at position -3 (beta A4 numbering system). A peptide homologous to the alpha-secretase site was not cleaved. The results demonstrate that 72 kDa gelatinase A is not an alpha-secretase, but that it may have a beta-secretase activity.

Aggrecans↗
PubMed1995

Candidate gamma-secretases in the generation of the carboxyl terminus of the Alzheimer's disease beta A4 amyloid: possible involvement of cathepsin D.

beta A4 amyloid peptide, the main constituent of amyloid plaques and cerebrovascular amyloid deposits associated with Alzheimer's disease, derives from a large precursor protein (APP) by the action of beta- and gamma-secretases, the unidentified endoproteases which release the amino and carboxyl termini of beta A4, respectively. Several gamma-secretase cleavage sites exist which yield the more soluble (1-39/40) forms of beta A4 and the longer forms (1-42/43) which have a greater tendency to aggregate into amyloid plaques. gamma-Secretase activity may therefore be critical in amyloid formation. In this study, a synthetic peptide which encompasses the various gamma-secretase cleavage sites was used as a substrate to probe proteases of various classes and specificities. Elastase, collagenase, and cathepsin D cleaved at the amyloidogenic sites (after Ala42 or after Thr43) to release the carboxyl termini of the aggregating forms. In addition, collagenase and pepsin released the carboxyl terminus of the more soluble forms. Human brain fractions enriched in lysosomes contained a proteolytic activity that cleaved the substrate at the amyloidogenic site(s). This activity was more active at acidic pH and was inhibited by pepstatin, two characteristics of the lysosomal aspartyl proteinase cathepsin D. The same lysosomal fractions were found to contain APP carboxyl-terminal fragments which are potentially amyloidogenic. These were degraded, only in acidic conditions, by an endogenous protease activity inhibited by pepstatin. Thus, a cathepsin D-like activity from human brain is a candidate for APP gamma-secretase(s).

Alzheimer Disease↗
PubMed1995

Amyloid, aluminium and the aetiology of Alzheimer's disease.

Several lines of evidence suggest that neurotoxic beta A4 amyloid deposits are of prime importance in the pathogenesis of Alzheimer's disease. Epidemiologically determined risk factors such as Down's syndrome, head injury and apoE allelic status can be explained on the basis of this hypothesis. However, there are difficulties with the hypothesis--amyloid accumulation may be necessary, but is not sufficient to produce the neuronal damage seen in Alzheimer's disease. The association between aluminum exposure and Alzheimer's disease remains unproven and is considered to be increasingly peripheral to recent developments in our understanding of the disease.

Aluminum↗
PubMed1995

Expression of the amyloid protein precursor of Alzheimer's disease in the developing rat olfactory system.

The expression of the amyloid protein precursor (APP) of Alzheimer's disease (AD) was examined in the olfactory system of the developing rat. Two monoclonal antibodies were used to detect APP: Alz-90, which specifically recognizes APP, and 22C11 which recognizes both APP and the structurally related protein APLP-2. Very similar patterns of immunoreactivity were observed with both antibodies. APP immunoreactivity was first detected in a subpopulation of olfactory epithelial cells at embryonic day 16 (E16), at a time when primary sensory olfactory axons are first beginning to pierce the glia limitans of the olfactory bulb. At E16, there were more olfactory receptor neurons which expressed APP than the olfactory marker protein (OMP), indicating that some APP-containing neurons were not fully mature. Between E16 and postnatal day 8 (P8), there was a marked increase in the number of primary sensory olfactory neurons expressing APP. In the olfactory bulb, APP was first detected in the mitral cell layer at E18, at a time when synapses are first beginning to form between the dendrites of these cells and primary sensory axons. The level of APP detected within mitral cell perikarya decreased after birth and could no longer be detected between P3 and P8. This indicated that once synaptic connections had been initiated within olfactory glomeruli, the expression of APP within the mitral cells was down-regulated. High levels of APP were, however, detected within the olfactory nerve fiber layer and glomeruli between P3 and P8. The results demonstrate that APP expression in the olfactory system is coordinately regulated with the major periods of synaptogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Alzheimer Disease↗
PubMed1995

Intracellular production of beta A4 amyloid of Alzheimer's disease: modulation by phosphoramidon and lack of coupling to the secretion of the amyloid precursor protein.

The amyloid precursor protein (APP) undergoes abnormal metabolism in Alzheimer's disease, resulting in the accumulation of beta A4 amyloid in the brain. Normal APP metabolism includes the release of a truncated form (sAPP) which has been cleaved at the alpha-secretase site within the beta A4 amyloidogenic domain. However, intact forms of beta A4 protein may also be generated by the beta- and gamma-secretases. Soluble forms of beta A4 have been detected in various cell lines and in cerebrospinal fluid. Previous studies of protein kinase C activation have suggested a reciprocal relationship between sAPP secretion and beta A4 production and release. We find that phorbol ester activation of protein kinase C in untransfected SH-SY5Y neuroblastoma cells increases the release of sAPP without affecting beta A4 secretion. We provide further evidence for intracellular beta A4 production. Treatment of SY5Y cells with the protease inhibitor phosphoramidon results in a 2-fold increase in beta A4 secretion and an increase in the amount of beta A4 recovered from cell lysates, yet it does not affect sAPP secretion. The protease inhibitors thiorphan and N-[(RS)-2-carboxy-3-phenylpropanoyl]-L-leucine had no effect on beta A4 or sAPP secretion. The lysosomotropic agents chloroquine and NH4Cl decreased beta A4 secretion, providing additional evidence for the involvement of intracellular acidic compartments in the production of beta A4. Our results therefore demonstrate a double dissociation between the secretion of sAPP and beta A4 in the SH-SY5Y cell line. The effect of phosphoramidon supports previous studies which show that metalloproteases are involved in the biogenesis of beta A4.

Alzheimer Disease↗
PubMed1995

Proteolytic processing of Alzheimer's disease beta A4 amyloid precursor protein in human platelets.

The processing of amyloid precursor protein (APP) and production of beta A4 amyloid are events likely to influence the development and progression of Alzheimer's disease, since beta A4 is the major constituent of amyloid deposited in this disorder. Our previous studies showed that human platelets contain full-length APP (APPFL) and are a suitable substrate to study normal APP processing. In the present study, we show that a 22-kDa beta A4-containing carboxyl-terminal fragment (22-CTF) of APP is present in unstimulated platelets. Both APPFL and 22-CTF are proteolytically degraded when platelets are activated with thrombin, collagen, or calcium ionophore A23187. Complete cleavage of APPFL and 22-CTF require the presence of extracellular calcium. Following stimulation in the presence of calcium, a new CTF of 17 kDa is generated, and the NH2-terminal epitope of beta A4 amyloid is lost. Preincubation of platelets with the cell-permeable cysteine protease inhibitors calpeptin, (2S,3S)-trans-epoxysuccinyl-L-leucyl-amido-3-methylbutane ethyl ester (E64d), Na alpha-p-tosyl-L-lysine chloromethyl ketone, or calcium chelator EGTA before platelet stimulation inhibits the degradation of both APPFL and 22-CTF. Divalent metal ions including zinc, copper, and cobalt inhibit the degradation of APPFL and 22-CTF. This study suggests that a calcium-dependent neutral cysteine protease is involved in the proteolytic processing of an amyloidogenic species of APP in human platelets.

Amyloid beta-Protein Precursor↗
PubMed1995

Extracellular matrix influences the biogenesis of amyloid precursor protein in microglial cells.

During axotomy studies, we discovered that the beta A4-amyloid precursor protein (APP) participates in immune responses of the central nervous system. Since microglia constitute the main immune effector cell population of this response, we used the murine microglial cell line BV-2 to analyze immune response-related APP expression. We show that interaction of microglia with the extracellular environment, particularly components of the extracellular matrix, affects APP secretion as well as intracellular APP biogenesis and catabolism. Fibronectin enhanced APP secretion and decreased the level of cellular mature transmembrane APP, whereas laminin and collagen caused a decrease in secretion and an accumulation of cellular mature APP and APP fragments. Our results demonstrate that APP plays a fundamental role in the regulation of microglial mobility, i.e. migration, initial target recognition, and binding. The decrease in APP secretion and the concomitant increase in cellular mature APP were accompanied by an accumulation of C-terminal APP fragments. Enrichment of APP and APP fragments is assumedly based on inhibition of catabolic processes that is caused by a disorganization of the actin microfilament network. These observations provide evidence that microglia, which are closely associated with certain amyloid deposits in the brain of Alzheimer patients, can play a key role in initial events of amyloidogenesis by initiating accumulation of APP and also of amyloidogenic APP fragments in response to physiological changes upon brain injury.

Alzheimer Disease↗
PubMed1995

Expression of the amyloid precursor protein of Alzheimer's disease on the surface of transfected HeLa cells.

The principal component of the amyloid which accumulates in Alzheimer's Disease brain is a 4-kDa beta A4 fragment of the amyloid precursor protein (APP). Although APP has the structural features of an integral transmembrane receptor, there has been limited evidence for expression of APP at the plasma membrane. The function of APP and related molecules is unknown. Using rabbit antisera to purified human brain APP, surface labeling of APP is demonstrable in HeLa cells transfected with the APP695 isoform. Indirect immunofluorescence indicates the presence of APP at the surface of unfixed or aldehyde-fixed cells; preembedding immunoelectron microscopy using 5- or 1-nm gold particles and silver enhancement confirms plasma membrane labeling as well as labeling within intracellular membrane vesicles. Immunolabeling of unfixed cells at 4 degrees C followed by incubation at 37 degrees C shows APP within endocytic vesicles. Transfected HeLa cells with prominent surface APP were larger with more extensive microvilli than nonimmunoreactive HeLa cells. This is consistent with the postulated role of APP as a mediator of cell surface adhesion and membrane-matrix stabilization.

Alzheimer Disease↗