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P H Jensen

Publications and source records attributed to P H Jensen.

At least 19 recordsLinked to original sources
PubMed2001

Ca2+ binding to alpha-synuclein regulates ligand binding and oligomerization.

alpha-Synuclein is a protein normally involved in presynaptic vesicle homeostasis. It participates in the development of Parkinson's disease, in which the nerve cell lesions, Lewy bodies, accumulate alpha-synuclein filaments. The synaptic neurotransmitter release is primarily dependent on Ca(2+)-regulated processes. A microdialysis technique was applied showing that alpha-synuclein binds Ca(2+) with an IC(50) of about 2-300 microm and in a reaction uninhibited by a 50-fold excess of Mg(2+). The Ca(2+)-binding site consists of a novel C-terminally localized acidic 32-amino acid domain also present in the homologue beta-synuclein, as shown by Ca(2+) binding to truncated recombinant and synthetic alpha-synuclein peptides. Ca(2+) binding affects the functional properties of alpha-synuclein. First, the ligand binding of (125)I-labeled bovine microtubule-associated protein 1A is stimulated by Ca(2+) ions in the 1-500 microm range and is dependent on an intact Ca(2+) binding site in alpha-synuclein. Second, the Ca(2+) binding stimulates the proportion of (125)I-alpha-synuclein-containing oligomers. This suggests that Ca(2+) ions may both participate in normal alpha-synuclein functions in the nerve terminal and exercise pathological effects involved in the formation of Lewy bodies.

Biopolymers↗
PubMed2001

Evaluation and ranking of restoration strategies for radioactively contaminated sites.

An international project, whose aim was the development of a transparent and robust method for evaluating and ranking restoration strategies for radioactively contaminated sites (RESTRAT), was carried out under the Fourth Framework of the Nuclear Fission Safety Programme of the EU. The evaluation and ranking procedure used was based on the principles of justification and optimisation for radiation protection. A multi-attribute utility analysis was applied to allow for the inclusion of radiological health effects, economic costs and social factors. Values of these attributes were converted into utility values by applying linear utility functions and weighting factors, derived from scaling constants and expert judgement. The uncertainties and variabilities associated with these utility functions and weighting factors were dealt with by a probabilistic approach which utilised a Latin Hypercube Sampling technique. Potentially relevant restoration techniques were identified and their characteristics determined through a literature review. The methodology developed by this project has been illustrated by application to representative examples of different categories of contaminated sites; a waste disposal site, a uranium tailing site and a contaminated freshwater river.

Costs and Cost Analysis↗
PubMed2000

Microtubule-associated protein 1B is a component of cortical Lewy bodies and binds alpha-synuclein filaments.

Lewy bodies, neuropathological hallmarks of Parkinson's disease and dementia with Lewy bodies, comprise alpha-synuclein filaments and other less defined proteins. Characterization of Lewy body proteins that interact with alpha-synuclein may provide insight into the mechanism of Lewy body formation. Double immunofluorescence labeling and confocal microscopy revealed approximately 80% of cortical Lewy bodies contained microtubule-associated protein 1B (MAP-1B) that overlapped with alpha-synuclein. Lewy bodies were isolated using an immunomagnetic technique from brain tissue of patients dying with dementia with Lewy bodies. Lewy body proteins were resolved by polyacrylamide gel electrophoresis. Immunoblotting confirmed the presence of MAP-1B and alpha-synuclein in purified Lewy bodies. Direct binding studies revealed a high affinity interaction (IC(50) approximately 20 nm) between MAP-1B and alpha-synuclein. The MAP-1B-binding sites were mapped to the last 45 amino acids of the alpha-synuclein C terminus. MAP-1B also bound in vitro assembled alpha-synuclein fibrils. Thus, MAP-1B may be involved in the pathogenesis of Lewy bodies via its interaction with monomeric and fibrillar alpha-synuclein.

Aged↗
PubMed2000

In situ and in vitro study of colocalization and segregation of alpha-synuclein, ubiquitin, and lipids in Lewy bodies.

alpha-Synuclein and ubiquitin are two Lewy body protein components that may play antagonistic roles in the pathogenesis of Lewy bodies. We examined the relationship between alpha-synuclein, ubiquitin, and lipids in Lewy bodies of fixed brain sections or isolated from cortical tissues of dementia with Lewy bodies. Lewy bodies exhibited a range of labeling patterns for alpha-synuclein and ubiquitin, from a homogeneous pattern in which alpha-synuclein and ubiquitin were evenly distributed and overlapped across the inclusion body to a concentric pattern in which alpha-synuclein and ubiquitin were partially segregated, with alpha-synuclein labeling concentrated in the peripheral domain and ubiquitin in the central domain of the Lewy body. Lipids represented a significant component in both homogeneous and concentric Lewy bodies. These results suggest that Lewy bodies are heterogeneous in their subregional composition. The segregation of alpha-synuclein to Lewy body peripheral domain is consistent with the hypothesis that alpha-synuclein is continually deposited onto Lewy bodies.

Aged↗
PubMed2000

Are public dose limits necessary?

The distinction between practices and interventions in the System of Radiation Protection has created a lot of confusion in the population and amongst decision-makers, especially with regards to the concepts of dose limits and intervention levels. The experience gained after the Chernobyl accident indicated that many actions taken led to an unnecessarily large expenditure of national resources and many instances occurred of contradictory national responses. A major reason was the mixture of dose limits for the population, which apply only to exposures from practices, and intervention levels, which apply only to protective measures in de-facto exposure situations. The existing System of Radiation Protection is revisited and it is suggested that the System can be revised with no dose limits for the public without causing a lower degree of protection of the population. With the widespread use of source-related dose constraints and practical restrictions on the sources of public exposure from practices, generally applicable dose limits are rarely limiting in any practical situation, even if dose constraints might, at least in principle, fail to take adequate account of the exposures from other practices. Constraints can be expressed as operational protection quantities, e.g., nuclide-specific release rates, dose rate at the fence of a facility, or nuclide-specific surface contamination density in the environment. A revised System of Radiation Protection without public dose limits would not cause any reduced protection of the public compared to the existing System, and it has a potential for removing much of the confusion with regards to application of intervention/action levels. It would also have the potential for improving public perception of radiation protection and radiation risks as well as for saving vast resources in intervention situations for better application in general health care of the public.

Humans↗
PubMed1999

alpha-synuclein binds to Tau and stimulates the protein kinase A-catalyzed tau phosphorylation of serine residues 262 and 356.

alpha-Synuclein has been implicated in the pathogenesis of several neurodegenerative disorders based on the direct linking of missense mutations in alpha-synuclein to autosomal dominant Parkinson's disease and its presence in Lewy-like lesions. To gain insight into alpha-synuclein functions, we have investigated whether it binds neuronal proteins and modulates their functional state. The microtubule-associated protein tau was identified as a ligand by alpha-synuclein affinity chromatography of human brain cytosol. Direct binding assays using (125)I-labeled human tau40 demonstrated a reversible binding with a IC(50) about 50 pM. The interacting domains were localized to the C terminus of alpha-synuclein and the microtubule binding region of tau as determined by protein fragmentation and the use of recombinant peptides. High concentrations of tubulin inhibited the binding between tau and alpha-synuclein. Functionally, alpha-synuclein stimulated the protein kinase A-catalyzed phosphorylation of tau serine residues 262 and 356 as determined using a phospho-epitope-specific antibody. We propose that alpha-synuclein modulates the phosphorylation of soluble axonal tau and thereby indirectly affects the stability of axonal microtubules.

Axons↗
PubMed1999

Localization of a single transglutaminase-reactive glutamine in the third domain of RAP, the alpha2-macroglobulin receptor-associated protein.

The 39-kDa receptor-associated protein (RAP) is an intracellular glycoprotein that interacts with hitherto unknown sites in several members of the low-density-lipoprotein receptor gene family. Upon binding to these receptors, RAP inhibits all ligand interactions with the receptors. In the present study, the transglutaminase-catalyzed incorporation of radioactively labeled putrescine and a dansylated glutamine-containing peptide into human RAP has been studied. The results indicate the presence of both glutamine and lysine residues in RAP, accessible for transglutaminase cross-linking. Moreover, enzymatic digestion followed by sequence analysis of radiolabeled fractions demonstrated that Gln261 acts as the amine acceptor site. This residue is located in the third domain of RAP and is conserved among the RAP interspecies homologues. Insertion of a reporter group into the protein could prove useful to assess ligand/receptor interactions.

Animals↗
PubMed1999

Identification of a neuritogenic ligand of the neural cell adhesion molecule using a combinatorial library of synthetic peptides.

The neural cell adhesion molecule (NCAM) plays a key role in neural development, regeneration, and learning. In this study, we identified a synthetic peptide-ligand of the NCAM Ig1 module by combinatorial chemistry and showed it could modulate NCAM-mediated cell adhesion and signal transduction with high potency. In cultures of dissociated neurons, this peptide, termed C3, stimulated neurite outgrowth by activating a signaling pathway identical to that activated by homophilic NCAM binding. A similar effect was shown for the NCAM Ig2 module, the endogenous ligand of NCAM Ig1. By nuclear magnetic resonance spectroscopy, the C3 binding site in the NCAM Ig1 module was mapped and shown to be different from the binding site of the NCAM Ig2 module. The C3 peptide may prove useful as a lead in development of therapies for neurodegenerative disorders, and the C3 binding site of NCAM Ig1 may represent a target for discovery of nonpeptide drugs.

Amino Acid Sequence↗
PubMed1999

Structure and interactions of NCAM modules 1 and 2, basic elements in neural cell adhesion.

The structure in solution of the second Ig-module fragment of residues 117-208 of NCAM has been determined. Like the first Ig-module of residues 20-116, it belongs to the I set of the immunogloblin superfamily. Module 1 and module 2 interact weakly, and the binding sites of this interaction have been identified. The two-module fragment NCAM(20-208) is a stable dimer. Removal of the charged residues in these sites in NCAM(20-208) abolishes the dimerization. Modeling the dimer of NCAM(20-208) to fit the interactions of these charges produces one coherent binding site for the formation of two antiparallel strands of the first two NCAM modules. This mode of binding could be a major element in trans-cellular interactions in neural cell adhesion.

Amino Acid Substitution↗
PubMed1999

Caspase I-related protease inhibition retards the execution of okadaic acid- and camptothecin-induced apoptosis and PAI-2 cleavage, but not commitment to cell death in HL-60 cells.

We have previously reported that the putative cytoprotective protease inhibitor, plasminogen activator inhibitor type 2 (PAI-2), is specifically cleaved during okadaic acid-induced apoptosis in a myeloid leukaemic cell line (Br J Cancer (1994) 70: 834-840). HL-60 cells exposed to okadaic acid and camptothecin underwent morphological and biochemical changes typical of apoptosis, including internucleosomal DNA fragmentation and PAI-2 cleavage. Significant endogenous PAI-2 cleavage was observed 9 h after exposure to okadaic acid; thus correlating with other signs of macromolecular degradation, like internucleosomal DNA fragmentation. In camptothecin-treated cells, PAI-2 cleavage was an early event, detectable after 2 h of treatment, and preceding internucleosomal DNA fragmentation. The caspase I selective protease inhibitor, YVAD-cmk, inhibited internucleosomal DNA fragmentation and PAI-2 cleavage of okadaic acid and camptothecin-induced apoptotic cells. YVAD-cmk rather sensitively and non-toxically inhibited camptothecin-induced morphology, but not okadaic acid-induced morphology. In in vitro experiments recombinant PAI-2 was not found to be a substrate for caspase I. The results suggest that caspase I selective protease inhibition could antagonize parameters coupled to the execution phase of okadaic acid- and camptothecin-induced apoptosis, but not the commitment to cell death.

Amino Acid Chloromethyl Ketones↗
PubMed1999

Axonal transport of synucleins is mediated by all rate components.

Synucleins are abundant nerve terminal proteins of hitherto unknown function. In diseases with Lewy bodies, human alpha-synuclein concentrates in these lesions in the cell body and mutations in alpha-synuclein lead to heritable Parkinson's disease with Lewy bodies. This indicates that changes in the normal metabolism and axonal transport of alpha-synuclein is perturbed in these diseases. To investigate the normal axonal transport of synucleins we studied the rat visual system by nerve crush operations and metabolic labelling of the retinal ganglion cells followed by immunoprecipitation of nerve segments. We found by immunofluorescence microscopy of the crush-operated nerves that synucleins are transported by fast antero- and retrograde transport and colocalize with synaptophysin and SNAP-25 around the lesion. The metabolic labelling studies demonstrated that synucleins were moved through the nerve with all the rate components, the fast component and the slow components a and b, with component b predominating. Two-dimensional gel electrophoresis revealed that both alpha- and beta-synuclein migrate through the nerve by slow component b in a ratio of 2:1.

Animals↗
PubMed1999

Alpha-synuclein immunoisolation of glial inclusions from multiple system atrophy brain tissue reveals multiprotein components.

Immunohistochemical studies have shown that oligodendroglial inclusions in multiple system atrophy contain alpha-synuclein, a synaptic protein also found in Lewy bodies in Parkinson's disease. We have now used density gradient enrichment and an anti-alpha-synuclein immunomagnetic technique to isolate pure and morphologically intact oligodendroglial inclusions from brain white matter of patients dying with multiple system atrophy. Filamentous inclusion structures were obtained only from multiple system atrophy tissue, but not from normal brain tissues, or from multiple system atrophy tissue processed without anti-alpha-synuclein antibody. We confirmed the purity and morphology of isolated inclusions by electron microscopy. The inclusions comprised multiple protein bands after separation by polyacrylamide gel electrophoresis. Immunoblotting demonstrated that these proteins included alpha-synuclein, alphaB-crystallin, tubulins, ubiquitin, and prominent, possibly truncated alpha-synuclein species as high-molecular-weight aggregates. Our study provides the first biochemical evidence that oligodendroglial inclusion filaments consist of multiple protein components, suggesting that these inclusions may form as a result of multiprotein interactions with alpha-synuclein.

Aged↗
PubMed1998

Binding of alpha-synuclein to brain vesicles is abolished by familial Parkinson's disease mutation.

The presynaptic protein alpha-synuclein has been implicated in the pathogenesis of Parkinson's disease. First, two missense mutations A30P and A53T cause inheritable early onset Parkinson's disease in some families. Secondly, alpha-synuclein is present in Lewy bodies of affected nerve cells in the predominant sporadic type of Parkinson's disease as well as in dementia with Lewy bodies. We demonstrate in the rat optic system that a portion of alpha-synuclein is carried by the vesicle-moving fast component of axonal transport and that it binds to rat brain vesicles through its amino-terminal repeat region. We find alpha-synuclein with the A30P mutation of familial Parkinson's disease devoid of vesicle-binding activity and propose that mutant alpha-synuclein may accumulate, leading to assembly into Lewy body filaments.

Amino Acid Sequence↗