[Hyperammonia, hypoglycemia and thrombocytopenia in a newborn after materanl treatment with valproate].
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Adult↗
Biomedical subjects
L Levesque
Publications and source records attributed to L Levesque.
Effects of radiation therapy on vascular responsiveness.
The use of radiation therapy to inhibit vascular proliferative diseases has produced encouraging results in several clinical trials. However, little is known about the possible side effects of radiation on vascular responsiveness. Our goal was to study the in vitro vascular responses of the rabbit aorta to various agonists immediately after several regimens of radiation therapy administered at doses prescribed in clinical protocols and at two different dose rates. High-dose-rate radiation was administered either by brachytherapy, using a gamma source, iridium 192, or an external electron beam producing beta radiation. Low-dose-rate radiation was administered by brachytherapy using a liquid-filled balloon with the beta emitter 32P. Vascular reactivity after the various regimens of irradiation was determined using the organ bath pharmacology assay. Various agonists were applied to the rabbit aorta to produce full cumulative concentration-response curves. Radiation, administered using an external electron beam, did not alter endothelium-dependent relaxation of the aorta induced by acetylcholine. However, the use of a catheter-based system to deliver radiation disrupted the endothelial cell lining of the vessel, causing a lack of relaxation by acetylcholine. Therefore, to compare all modalities of radiation therapy on vascular responsiveness, the agonists used in this study are known to act directly on the smooth muscle. Radiation therapy had no effect on the contractile responses induced by the following agonists: phenylephrine and potassium chloride. Vascular dilatation induced by nitroglycerin, a nitric oxide donor, was unaffected by radiation therapy. The contractile response induced by des-Arg9-bradykinin, a kinin B1 receptor agonist, was significantly increased twofold to threefold by all types of irradiation under study. This enhanced response is attributable to an increase of mRNA levels coding for this receptor. In all cases, radiation therapy did not alter the effective concentration producing 50% of maximal responsiveness (EC50) and did not reduce the vascular responsiveness induced by agonists. Taken together, we conclude that radiation therapy does not hinder endothelium-independent vascular responsiveness and increases the kinin B1 receptor-mediated vasoconstriction.
Acetylcholine↗
NXT1 (p15) is a crucial cellular cofactor in TAP-dependent export of intron-containing RNA in mammalian cells.
TAP, the human homologue of the yeast protein Mex67p, has been proposed to serve a role in mRNA export in mammalian cells. We have examined the ability of TAP to mediate export of Rev response element (RRE)-containing human immunodeficiency virus (HIV) RNA, a well-characterized export substrate in mammalian cells. To do this, the TAP gene was fused in frame to either RevM10 or RevDelta78-79. These proteins are nonfunctional Rev mutant proteins that can bind to HIV RNA containing the RRE in vivo but are unable to mediate the export of this RNA to the cytoplasm. However, the fusion of TAP to either of these mutant proteins gave rise to chimeric proteins that were able to complement Rev function. Significantly, cotransfection with a vector expressing NXT1 (p15), an NTF2-related cellular factor that binds to TAP, led to dramatic enhancement of the ability of the chimeric proteins to mediate RNA export. Mutant-protein analysis demonstrated that the domain necessary for nuclear export mapped to the C-terminal region of TAP and required the domain that interacts with NXT1, as well as the region that has been shown to interact with nucleoporins. RevM10-TAP function was leptomycin B insensitive. In contrast, the function of this protein was inhibited by DeltaCAN, a protein consisting of part of the FG repeat domain of CAN/Nup214. These results show that TAP can complement Rev nuclear export signal function and redirect the export of intron-containing RNA to a CRM1-independent pathway. These experiments support the role of TAP as an RNA export factor in mammalian cells. In addition, they indicate that NXT1 serves as a crucial cellular cofactor in this process.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Carboxyl-terminal fragments of Alzheimer beta-amyloid precursor protein accumulate in restricted and unpredicted intracellular compartments in presenilin 1-deficient cells.
Absence of functional presenilin 1 (PS1) protein leads to loss of gamma-secretase cleavage of the amyloid precursor protein (betaAPP), resulting in a dramatic reduction in amyloid beta peptide (Abeta) production and accumulation of alpha- or beta-secretase-cleaved COOH-terminal fragments of betaAPP (alpha- or beta-CTFs). The major COOH-terminal fragment (CTF) in brain was identified as betaAPP-CTF-(11-98), which is consistent with the observation that cultured neurons generate primarily Abeta-(11-40). In PS1(-/-) murine neurons and fibroblasts expressing the loss-of-function PS1(D385A) mutant, CTFs accumulated in the endoplasmic reticulum, Golgi, and lysosomes, but not late endosomes. There were some subtle differences in the subcellular distribution of CTFs in PS1(-/-) neurons as compared with PS1(D385A) mutant fibroblasts. However, there was no obvious redistribution of full-length betaAPP or of markers of other organelles in either mutant. Blockade of endoplasmic reticulum-to-Golgi trafficking indicated that in PS1(-/-) neurons (as in normal cells) trafficking of betaAPP to the Golgi compartment is necessary before alpha- and beta-secretase cleavages occur. Thus, although we cannot exclude a specific role for PS1 in trafficking of CTFs, these data argue against a major role in general protein trafficking. These results are more compatible with a role for PS1 either as the actual gamma-secretase catalytic activity or in other functions indirectly related to gamma-secretase catalysis (e.g. an activator of gamma-secretase, a substrate adaptor for gamma-secretase, or delivery of gamma-secretase to betaAPP-containing compartments).
Alzheimer Disease↗
Nicastrin modulates presenilin-mediated notch/glp-1 signal transduction and betaAPP processing.
Nicastrin, a transmembrane glycoprotein, forms high molecular weight complexes with presenilin 1 and presenilin 2. Suppression of nicastrin expression in Caenorhabditis elegans embryos induces a subset of notch/glp-1 phenotypes similar to those induced by simultaneous null mutations in both presenilin homologues of C. elegans (sel-12 and hop-1). Nicastrin also binds carboxy-terminal derivatives of beta-amyloid precursor protein (betaAPP), and modulates the production of the amyloid beta-peptide (A beta) from these derivatives. Missense mutations in a conserved hydrophilic domain of nicastrin increase A beta42 and A beta40 peptide secretion. Deletions in this domain inhibit A beta production. Nicastrin and presenilins are therefore likely to be functional components of a multimeric complex necessary for the intramembranous proteolysis of proteins such as Notch/GLP-1 and betaAPP.
Amino Acid Sequence↗
[Filiform ankyloblepharon adnatum].
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Blepharitis↗
Proliferative effect of lipoprotein lipase on human vascular smooth muscle cells.
Vascular smooth muscle cell (VSMC) proliferation is a key event in the development and progression of atherosclerotic lesions. Accumulating evidence suggests that lipoprotein lipase (LPL) produced in the vascular wall may exert proatherogenic effects. The aim of the present study was to examine the effect of LPL on VSMC proliferation. Incubation of growth-arrested human VSMCs with purified endotoxin-free bovine LPL for 48 and 72 hours, in the absence of any added exogenous lipoproteins, resulted in a dose-dependent increase in VSMC growth. Addition of VLDLs to the culture media did not further enhance the LPL effect. Treatment of growth-arrested VSMCs with purified human or murine LPL (1 microg/mL) led to a similar increase in cell proliferation. Neutralization of bovine LPL by the monoclonal 5D2 antibody, irreversible inhibition, or heat inactivation of the lipase suppressed the LPL stimulatory effect on VSMC growth. Moreover, preincubation of VSMCs with the specific protein kinase C inhibitors calphostin C and chelerythrine totally abolished LPL-induced VSMC proliferation. In LPL-treated VSMCs, a significant increase in protein kinase C activity was observed. Treatment of VSMCs with heparinase III (1 U/mL) totally inhibited LPL-induced human VSMC proliferation. Taken together, these data indicate that LPL stimulates VSMC proliferation. LPL enzymatic activity, protein kinase C activation, and LPL binding to heparan sulfate proteoglycans expressed on VSMC surfaces are required for this effect. The stimulatory effect of LPL on VSMC proliferation may represent an additional mechanism through which the enzyme contributes to the progression of atherosclerosis.
Alkaloids↗
Presenilin 1 controls gamma-secretase processing of amyloid precursor protein in pre-golgi compartments of hippocampal neurons.
Mutations of presenilin 1 (PS1) causing Alzheimer's disease selectively increase the secretion of the amyloidogenic betaA4(1-42), whereas knocking out the gene results in decreased production of both betaA4(1-40) and (1-42) amyloid peptides (De Strooper et al. 1998). Therefore, PS1 function is closely linked to the gamma-secretase processing of the amyloid precursor protein (APP). Given the ongoing controversy on the subcellular localization of PS1, it remains unclear at what level of the secretory and endocytic pathways PS1 exerts its activity on APP and on the APP carboxy-terminal fragments that are the direct substrates for gamma-secretase. Therefore, we have reinvestigated the subcellular localization of endogenously expressed PS1 in neurons in vitro and in vivo using confocal microscopy and fine-tuned subcellular fractionation. We show that uncleaved PS1 holoprotein is recovered in the nuclear envelope fraction, whereas the cleaved PS fragments are found mainly in post-ER membranes including the intermediate compartment (IC). PS1 is concentrated in discrete sec23p- and p58/ERGIC-53-positive patches, suggesting its localization in subdomains involved in ER export. PS1 is not found to significant amounts beyond the cis-Golgi. Surprisingly, we found that APP carboxy-terminal fragments also coenrich in the pre-Golgi membrane fractions, consistent with the idea that these fragments are the real substrates for gamma-secretase. Functional evidence that PS1 exerts its effects on gamma-secretase processing of APP in the ER/IC was obtained using a series of APP trafficking mutants. These mutants were investigated in hippocampal neurons derived from transgenic mice expressing PS1wt or PS1 containing clinical mutations (PS1(M146L) and PS1(L286V)) at physiologically relevant levels. We demonstrate that the APP-London and PS1 mutations have additive effects on the increased secretion of betaA4(1-42) relative to betaA4(1-40), indicating that both mutations operate independently. Overall, our data clearly establish that PS1 controls gamma(42)-secretase activity in pre-Golgi compartments. We discuss models that reconcile this conclusion with the effects of PS1 deficiency on the generation of betaA4(1-40) peptide in the late biosynthetic and endocytic pathways.
Amyloid Precursor Protein Secretases↗
Glial expression of presenilin epitopes in human brain with cerebral infarction and in astrocytoma.
Presenilins, some gene mutations of which are associated with familial Alzheimer's disease (AD), are expressed mainly in neurons in normal brains and brains from patients clinicopathologically diagnosed as AD. They are thought to be related to cell death and survival. We studied the immunolocalization of presenilin to investigate its possible relation to cell death and glial proliferation, using two antibodies against different portions of the presenilin 1 protein, in human brains with cerebral infarction and in astrocytoma, where abundant cell death and glial proliferation are present. Expression of presenilin epitopes was more marked in glial cells than in neurons in and around the ischemic focus, and it was also robust in astrocytoma cells. These findings suggest that presenilins are functioning not only in neurons but also in glial cells in reactive and neoplastic proliferation.
Aged↗
Presenilin mutations associated with Alzheimer disease cause defective intracellular trafficking of beta-catenin, a component of the presenilin protein complex.
The presenilin proteins are components of high-molecular-weight protein complexes in the endoplasmic reticulum and Golgi apparatus that also contain beta-catenin. We report here that presenilin mutations associated with familial Alzheimer disease (but not the non-pathogenic Glu318Gly polymorphism) alter the intracellular trafficking of beta-catenin after activation of the Wnt/beta-catenin signal transduction pathway. As with their effect on betaAPP processing, the effect of PS1 mutations on trafficking of beta-catenin arises from a dominant 'gain of aberrant function' activity. These results indicate that mistrafficking of selected presenilin ligands is a candidate mechanism for the genesis of Alzheimer disease associated with presenilin mutations, and that dysfunction in the presenilin-beta-catenin protein complexes is central to this process.
Alzheimer Disease↗
Presenilins interact with armadillo proteins including neural-specific plakophilin-related protein and beta-catenin.
Missense substitutions in the presenilin 1 (PS1) and presenilin 2 (PS2) proteins are associated with early-onset familial Alzheimer's disease. We have used yeast-two-hybrid and coimmunoprecipitation methods to show that the large cytoplasmic loop domains of PS1 and PS2 interact specifically with three members of the armadillo protein family, including beta-catenin, p0071, and a novel neuronal-specific armadillo protein--neural plakophilin-related armadillo protein (NPRAP). The PS1:NPRAP interaction occurs between the arm repeats of NPRAP and residues 372-399 at the C-terminal end of the large cytoplasmic loop of PS1. The latter residues contain a single arm-like domain and are highly conserved in the presenilins, suggesting that they form a functional armadillo protein binding site for the presenilins.
Alzheimer Disease↗
Localisation of presenilin 2 in human and rodent pancreatic islet beta-cells; Met239Val presenilin 2 variant is not associated with diabetes in man.
Mutations in presenilin 1 and 2 are causative factors for early onset familial Alzheimer's disease and possible roles for presenilins include protein trafficking, regulation of apoptosis and/or calcium homeostasis. Presenilin 2 mRNA is expressed in brain, muscle and pancreas but the role of pancreatic presenilin 2 and its relationship to diabetes are unknown. Presenilin 2 immunoreactivity was localised in human and rodent pancreas to islet cells and found in granules of beta-cells. Presenilin 2 was identified in primitive islet and duct cells of human foetal pancreas and in proliferating exocrine duct cells in human pancreatitis but not found in islet amyloid deposits in Type 2 diabetic subjects. Full length, approximately 50 kDa, and the approximately 30 kDa N-terminal fragment of presenilin 2 were identified by western blotting in extracted rodent pancreas but only the 30 kDa fragment was detected in mouse islets and human insulinoma. Post-mortem pancreatic morphology was normal in a subject with the presenilin 2 Met239Val variant and early onset familial Alzheimer's disease. Oral glucose tolerance tests on subjects with the presenilin 2 Met239Val mutation unaffected by early onset familial Alzheimer's disease (mean age 35 years) and on their first-degree relatives without the mutation demonstrated no evidence of glucose intolerance or increased proinsulin secretion. PS2 is a novel &bgr;-cell protein with potential roles in development or protein processing but pancreatic islet structure and function appear to be unaffected by the Met239Val mutation.
Adult↗
The presenilin 1 protein is a component of a high molecular weight intracellular complex that contains beta-catenin.
The presenilin (PS) genes associated with Alzheimer disease encode polytopic transmembrane proteins which undergo physiologic endoproteolytic cleavage to generate stable NH2- and COOH-terminal fragments (NTF or CTF) which co-localize in intracellular membranes, but are tightly regulated in their stoichiometry and abundance. We have used linear glycerol velocity and discontinuous sucrose gradient analysis to investigate the distribution and native conformation of PS1 and PS2 during this regulated processing in cultured cells and in brain. The PS1 NTF and CTF co-localize in the endoplasmic reticulum (ER) and in the Golgi apparatus, where they are components of a approximately 250-kDa complex. This complex also contains beta-catenin but not beta-amyloid precursor protein (APP). In contrast, the PS1 holoprotein precursor is predominantly localized to the rough ER and smooth ER, where it is a component of a approximately 180-kDa native complex. PS2 forms similar but independent complexes. Restricted incorporation of the presenilin NTF and CTF along with a potentially functional ligand (beta-catenin) into a multimeric complex in the ER and Golgi apparatus may provide an explanation for the regulated accumulation of the NTF and CTF.
Alzheimer Disease↗
Depletion of protein kinase C-alpha by antisense oligonucleotides alters beta-adrenergic function and reverses the phorbol ester-induced reduction of isoproterenol-induced adenosine 3'-5'-cyclic monophosphate accumulation in murine Swiss 3T3 fibroblasts.
Beta-adrenergic agonists are well known to increase the activity of adenylate cyclase, yielding increases of the intracellular concentration of cAMP. It has been reported that activation of protein kinase C (PKC) by phorbol esters reduces the amplitude of isoproterenol-induced cAMP production in a 3T3-L1 cell line. In this study, we investigated whether PKC-alpha is involved in this process in murine Swiss 3T3 fibroblasts. A 20-mer phosphorothioate oligonucleotide designed to hybridize to the AUG initiation codon of the murine PKC-alpha mRNA, which contains 2'-O-methoxyethyl modifications incorporated into the 5' and 3' segments of the oligonucleotide, was used to assess the putative role of PKC-alpha in the beta-adrenergic receptor regulation. ISIS 14012 reduced PKC-alpha mRNA for over 72 hr after the initial treatment and the reduction was concentration dependent, whereas the mismatch control, ISIS 13818, had no effect. This depletion was found to be selective; ISIS 14012 had no effect on the mRNA expression of PKC-delta and PKC-zeta. ISIS 14012 reduced in a time and concentration-dependent fashion the levels of immunoreactive PKC-alpha protein by over 85% at 72 hr after treatment. Depletion of PKC-alpha inhibited the effect of isoproterenol-induced cAMP production by phorbol dibutyrate (PdBu). This finding is corroborated by the use of a nonspecific inhibitor of PKC, GF-109203x, which also prevented the effect of PdBu. Depletion of PKC-alpha by ISIS 14012 potentiated isoproterenol-induced cAMP production in cells untreated with PdBu. However, neither depletion of PKC-alpha nor PKC activation by a phorbol ester altered beta-adrenergic receptor affinity and density. PKC activation by PdBu did not alter forskolin-induced cAMP levels, but enhanced cAMP production by cholera toxin. PKC-alpha inhibition by ISIS 14012 had no effect on either cholera toxin-induced increases in cAMP or the acute effects of phorbol esters on cholera toxin in induction of cAMP. Thus, PKC-alpha appears to be involved in the regulation of beta-adrenergic receptor coupling to adenylate cyclase, possibly by phosphorylating the Gs protein, but other PKC isotypes must be involved in the effects observed when cells are treated with cholera toxin.
3T3 Cells↗
Evidence for phosphorylation and oligomeric assembly of presenilin 1.
Pathogenic mutations in presenilin 1 (PS1) are associated with approximately 50% of early-onset familial Alzheimer disease. PS1 is endoproteolytically cleaved to yield a 30-kDa N-terminal fragment (NTF) and an 18-kDa C-terminal fragment (CTF). Using COS7 cells transfected with human PS1, we have found that phorbol 12, 13-dibutyrate and forskolin increase the state of phosphorylation of serine residues of the human CTF. Phosphorylation of the human CTF resulted in a shift in electrophoretic mobility from a single major species of 18 kDa to a doublet of 20-23 kDa. This mobility shift was also observed with human PS1 that had been transfected into mouse neuroblastoma (N2a) cells. Treatment of the phosphorylated CTF doublet with phage lambda protein phosphatase eliminated the 20- to 23-kDa doublet while enhancing the 18-kDa species, consistent with the interpretation that the electrophoretic mobility shift was due to the addition of phosphate to the 18-kDa species. The NTF and CTF eluted from a gel filtration column at an estimated mass of over 100 kDa, suggesting that these fragments exist as an oligomerized species. Upon phosphorylation of the PS1 CTF, the apparent mass of the NTF- or CTF-containing oligomers was unchanged. Thus, the association of PS1 fragments may be maintained during cycles of phosphorylation/dephosphorylation of the PS1 CTF.
Animals↗
Phosphorylation, subcellular localization, and membrane orientation of the Alzheimer's disease-associated presenilins.
Presenilins 1 and 2 are unglycosylated proteins with apparent molecular mass of 45 and 50 kDa, respectively, in transfected COS-1 and Chinese hamster ovary cells. They colocalize with proteins from the endoplasmic reticulum and the Golgi apparatus in transfected and untransfected cells. In COS-1 cells low amounts of intact endogeneous presenilin 1 migrating at 45 kDa are detected together with relative larger amounts of presenilin 1 fragments migrating between 18 and 30 kDa. The presenilins have a strong tendency to form aggregates (mass of 100-250 kDa) in SDS-polyacrylamide gel electrophoresis, which can be partially resolved when denatured by SDS at 37 degrees C instead of 95 degrees C. Sulfation, glycosaminoglycan modification, or acylation of the presenilins was not observed, but both proteins are posttranslationally phosphorylated on serine residues. The mutations Ala-246 --> Glu or Cys-410 --> Tyr that cause Alzheimer's disease do not interfere with the biosynthesis or phosphorylation of presenilin 1. Finally, using low concentrations of digitonin to selectively permeabilize the cell membrane but not the endoplasmic reticulum membrane, it is demonstrated that the two major hydrophilic domains of presenilin 1 are oriented to the cytoplasm. The current investigation documents the posttranslational modifications and subcellular localization of the presenilins and indicates that postulated interactions with amyloid precursor protein metabolism should occur in the early compartments of the biosynthetic pathway.
Alzheimer Disease↗
Antisense oligonucleotides targeting human protein kinase C-alpha inhibit phorbol ester-induced reduction of bradykinin-evoked calcium mobilization in A549 cells.
Regulation of the bradykinin-evoked increase in intracellular Ca2+ concentration by protein kinase C (PKC)-alpha was investigated in A549 human lung carcinoma cells. Bradykinin, a potent and selective kinin B2 receptor agonist, induces calcium mobilization in a concentration-dependent fashion in this cell line. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a potent activator of PKC, is known to reduce the amplitude of agonist-induced calcium mobilization in various cell lines. Because PKC-alpha is a major PKC isozyme in A549 cells, we investigated whether this isozyme plays a role in this process. A 20-mer phosphorothioate oligonucleotide targeting the 3'-untranslated region of the human PKC-alpha mRNA, which contains 2'-methoxyethyl modifications incorporated into the 5' and 3' segments of the oligonucleotide, was used to assess the putative role of PKC-alpha in the receptor regulation. ISIS 9606 reduced PKC-alpha mRNA for > or = 72 hr after the initial treatment and the reduction was concentration dependent, whereas the mismatch control, ISIS 13009, had no effect. Concentrations of ISIS 9606 of 150 nM specifically reduced the level of immunoreactive PKC-alpha protein by 66.3 +/- 2.5% at 72 hr after treatment, without an effect on immunoreactive PKC-delta protein. This reduction in PKC-alpha was sufficient to inhibit the reduction of bradykinin-induced calcium mobilization by TPA. This finding is corroborated by the use of staurosporine, a nonselective PKC inhibitor, that prevented the effect of TPA. These results suggest that PKC-alpha is involved in kinin B2 receptor regulation by phorbol esters in A549 cells.
Bradykinin↗
Pathologic leukocyte infiltration of the rabbit aorta confers a vasomotor effect to chemotactic peptides through cyclooxygenase-derived metabolites.
We determined the effect of chemotactic peptides FMLP and C5a, postulated to be relatively selective activators of phagocytes, on the thoracic aorta of rabbits subjected to experimental pathologies that allowed infiltration by leukocytes, i.e., dietary atherosclerosis and serum sickness. Aortic ring tissues isolated from hypercholesterolemic rabbits, precontracted or not by phenylephrine, exhibited a rapid and relatively sustained (10 to 20 min) contractile response when challenged by FMLP (10 nM and above); precontracted tissues also responded to C5a (2.5 nM and above). Aortic rings from rabbits with serum sickness (13 days post-BSA injection) exhibited brief contractions that were often followed by a relaxation in phenylephrine-precontracted tissues. In both models, tissues from normal weight-matched animals were not consistently responsive to these peptides. The cyclooxygenase inhibitor indomethacin extensively reduced the contractile effect of either peptide on precontracted aortic rings in both models. Chemotactic peptide-induced increased prostanoid secretion was evident only in the fluid bathing atherosclerotic aortic rings. Morphologic correlations included the demonstration of cells positive for the RAM-11 macrophage marker and the C5a receptors in tissues from rabbits with hypercholesterolemia (numerous clusters of cells) or serum sickness (modest infiltration). Control aortic rings responded to FMLP by a significant contraction if cultured for 2 h in the presence of resident peritoneal cells (84% macrophages), but not in the presence of a high density of PBL (less than 0.5% monocytes). Infiltrating or adherent macrophages in the blood vessel wall confer to some phagocyte activating peptides the role of eicosanoid-dependent vasoconstrictor agents.
Animals↗