[Show abstract][Hide abstract] ABSTRACT: Neuropsychiatric adverse events have been reported in influenza patients with and without exposure to oseltamivir (Tamiflu), triggering speculation as to whether oseltamivir may be interacting with any human receptors and contributing to such neuropsychiatric events. In this study, the in vitro selectivity profile of oseltamivir prodrug and active metabolite was investigated. Both compounds lacked clinically relevant pharmacological activities on human, rodent and primate neuraminidases and on a panel of 155 other molecular targets, including those relevant for mood, cognition and behavior. Neuropsychiatric adverse events observed in influenza patients are likely a phenomenon caused by the infection rather than by oseltamivir.
European journal of pharmacology 11/2009; 628(1-3):6-10. DOI:10.1016/j.ejphar.2009.11.020 · 2.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Degeneration of cholinergic basal forebrain neurons (CBFN) is a hallmark in the pathology of Alzheimer's disease (AD). Critically depending upon the neurotrophic support through nerve growth factor (NGF), CBFN in the AD brain face elevated concentrations of the pro-form of NGF (proNGF) and suffer from an imbalance between TrkA and p75(NTR) expression. Research for the underlying mechanisms of CBFN death suggested a pro-apoptotic activity of proNGF. However, this finding could not be confirmed by all investigators and other studies even observed a neurotrophic function of proNGF. In the presence of these controversial findings we investigated the activity of proNGF in PC12 cells with specific emphasis on its neurotoxic versus neurotrophic action. In this study, we show that proNGF can mediate TrkA receptor signaling directly, yet in the manner of a partial agonist with a lower maximum activity than NGF. A pro-apoptotic activity of proNGF could not be confirmed in our cellular system. Interestingly and surprisingly, pre-incubation with proNGF at low, sub-active concentrations inhibited TrkA-mediated neurotrophic NGF signaling in PC12 cells. Our data support a novel hypothesis for the role of elevated proNGF levels in CBFN pathology in AD. Thus, proNGF can indirectly contribute to the slow neurodegeneration in AD by reducing NGF-mediated trophic support.
Journal of Neurochemistry 10/2008; 107(5):1294-303. DOI:10.1111/j.1471-4159.2008.05690.x · 4.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: (R)-Configured isophthalic hydroxyethylamines play an important role in the inhibition of beta-secretase (BACE1). We present the synthesis of a number of (S)-configured hydroxyethylamine derivatives via 2-iodoethanol intermediates and the comparison with the (R)-analogues. An N-substituted indole was investigated as a substitute for the isophthalamide moiety. (c) 2007 Elsevier Ltd. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Antibodies against beta-amyloid peptides (Abetas) are considered an important therapeutic opportunity in Alzheimer's disease. Despite the vast interest in Abeta no thermodynamic data on the interaction of antibodies with Abeta are available as yet. In the present study we use isothermal titration calorimetry (ITC) and surface plasmon resonance to provide a quantitative thermodynamic analysis of the interaction between soluble monomeric Abeta(1-40) and mouse monoclonal antibodies (mAb). Using four different antibodies directed against the N-terminal, middle, and C-terminal Abeta epitopes, we measured the thermodynamic parameters for the binding to Abeta. Each antibody species was found to have two independent and equal binding sites for Abeta with binding constants in the range of 10(7) to 10(8) M(-1). The binding reaction was essentially enthalpy driven with a reaction enthalpy of DeltaH(0)(Abeta) approximately -19 to -8 kcal/mol, indicating the formation of tight complexes. The loss in conformational freedom was supported by negative values for the reaction entropy DeltaS(0)(Abeta). We also measured the heat capacity change of the 1mAb:2Abeta reaction. DeltaC(0)(p, abeta) was large and negative but could not be explained exclusively by the hydrophobic effect. The free energy of binding was found to be linearly correlated with the size of the epitope.
The Journal of Physical Chemistry B 03/2007; 111(5):1238-43. DOI:10.1021/jp0664059 · 3.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The combination of the enantioselective, organocatalytic alpha-animation of aldehydes by diazodicarboxylates and the Passerini reaction provides rapid access to norstatine-based peptidomimetics. These intermediates were elaborated further by deprotection and cleavage of the N-N bond to provide useful building blocks for aspartic protease inhibitors. Coupling of the compounds 76-86 with the mono-isophthalamide 91 provided moderate inhibitors of human P-secretase (BACE) 92-102, ((c) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006).
[Show abstract][Hide abstract] ABSTRACT: Until recently, there was a lack of selective radioligands for the subtypes of metabotropic glutamate (mGlu) receptors. [(3)H]LY354740 ((+)-2-aminobicyclo[3,1,0]hexane-2,6-dicarboxylic acid), a selective agonist for group II receptors (mGlu2 and -3, which are negatively coupled to cAMP production), has now been used to map their brain distribution and abundance by in vitro binding and quantitative radioautography. The selective cation dependence of its binding allowed the discrimination between mGlu2 and mGlu3 receptor labeling. Thus, in the presence of Ca(2+) and Mg(2+) ions, the agonist bound selectively to mGlu2 receptors as evidenced by: 1) the correlative distribution and abundance of binding sites (highest in the lacunosum moleculare of the hippocampus and lowest in white matter) with mGlu2 receptor mRNA and protein revealed by in situ hybridization histochemistry and immunohistochemistry, respectively; 2) its selective pharmacology; and 3) the distribution of LY354740-stimulated [(35)S]GTPgammaS binding (25-97% above basal, according to the brain region), revealing G protein-coupled receptor coupling to G(i) proteins. Nonspecific binding (in the presence of 10 muM DCG-IV, a group II-selective, mGlu2-preferring, receptor agonist) was <10% of total. In adjacent sections, the distribution of binding sites for [(3)H]DCG-IV was very similar. This extensive study paves the way for investigations of the regional expression and regulation of mGlu2 receptors in human CNS diseases, such as Alzheimer's disease, which may reveal their functional roles and identify potential therapeutic drug targets. Indeed, it has recently been demonstrated (Higgins et al.  Neuropharmacology 46:907-917) that pharmacological manipulation of mGlu2 receptors influences cognitive performance in the rodent.
The Journal of Comparative Neurology 06/2005; 487(1):15-27. DOI:10.1002/cne.20538 · 3.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Most of the transgenic mice generated to model Alzheimer's disease express human amyloid precursor protein (APP) mutants alone or in conjunction with presenilin mutants. We have generated a mouse model by overexpressing human BACE and human APP with the V717F mutation. The combination of a mutation at the gamma-secretase cleavage site of APP and of increased beta-secretase activity should favour the production of amyloid peptides. We analysed double BACE/APPIn and single APPIn transgenic mice at 16-18 months for amyloid load, brain histopathology and behavioural deficits. We show that overexpression of BACE induces an increase in APP CTFbeta and total brain Abeta peptides. Brain histopathology shows clearly enhanced amyloid deposits in the cortex, hippocampus and in brain vasculature when compared to single APPIn transgenic mice. Amyloid deposits are mostly diffuse and predominantly composed of Abeta(42). A strong inflammatory reaction is evidenced by the presence of microglial cells around the most mature amyloid deposits and astrocytosis over the entire cerebral cortex. At the same age, the APPIn single-transgenic mice show only very limited pathology. When assessed for their cognitive performance at 12 months, BACE/APPIn mice show impaired spatial acquisition in the Morris water maze test. However, these deficits are not greater than those observed in the APPIn single-transgenic animals.
[Show abstract][Hide abstract] ABSTRACT: Transgenic mice, expressing mutant beta-amyloid precursor proteins (betaAPPs), have lead to a better understanding of the pathophysiological processes in Alzheimer's disease (AD). In many of these models, however, the temporal development of cognitive decline and the relationship to Abeta deposition and inflammation are unclear. We now report a novel transgenic mouse line, PS2APP (PS2N141I x APPswe), which develops a severe cerebral amyloidosis in discrete brain regions, and present a cross-sectional analysis of these mice at 4, 8, 12, and 16 months of age. Each age cohort was investigated for changes in behavior, electrophysiology of synapse efficacy, ELISA-determined Abeta load, histopathology, and in immunoelectron microscopy. Cognitive deficits were first observed at 8 months when Abeta deposits and inflammation were restricted to discrete brain regions, namely the subiculum and frontolateral (motor and orbital) cortex. As early as 5 months, electron microscopy revealed the presence, in these regions, of pre-plaque, immunogold-labeled extracellular fibrillar Abeta. At the same age, increased levels of insoluble Abeta were detected by ELISA, with Abeta1-40 levels exceeding those of Abeta1-42. Further cognitive decline occurred in an age-related manner, and this was accompanied by the spread of amyloidosis to ultimately affect not only neo- and limbic cortices, but also thalamic and pontine nuclei. Dentate gyrus post-tetanic potentiation was significantly attenuated at 17 months, and there were also significant differences in paired-pulse parameters. This systematic cross-sectional study of the behavioral and pathological changes in the PS2APP mouse indicates that it develops age-related cognitive decline associated with severe amyloidosis and inflammation in discrete brain regions and therefore is suitable for testing a range of potential symptomatic and disease-modifying therapies for AD.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 11/2003; 23(26):8989-9003. · 6.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Junctional adhesion molecules (JAMs) are a family of immunoglobulin-like single-span transmembrane molecules that are expressed in endothelial cells, epithelial cells, leukocytes and myocardia. JAM has been suggested to contribute to the adhesive function of tight junctions and to regulate leukocyte trans migration. We describe the crystal structure of the recombinant extracellular part of mouse JAM (rsJAM) at 2.5 A resolution. rsJAM consists of two immunoglobulin-like domains that are connected by a conformationally restrained short linker. Two rsJAM molecules form a U-shaped dimer with highly complementary interactions between the N-terminal domains. Two salt bridges are formed in a complementary manner by a novel dimerization motif, R(V,I,L)E, which is essential for the formation of rsJAM dimers in solution and common to the known members of the JAM family. Based on the crystal packing and studies with mutant rsJAM, we propose a model for homophilic adhesion of JAM. In this model, U-shaped JAM dimers are oriented in cis on the cell surface and form a two-dimensional network by trans-interactions of their N-terminal domains with JAM dimers from an opposite cell surface.
The EMBO Journal 09/2001; 20(16):4391-8. DOI:10.1093/emboj/20.16.4391 · 10.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Junctional adhesion molecule (JAM) is an integral membrane protein that belongs to the immunoglobulin superfamily, localizes at tight junctions, and regulates both paracellular permeability and leukocyte transmigration. To investigate molecular determinants of JAM function, the extracellular domain of murine JAM was produced as a recombinant soluble protein (rsJAM) in insect cells. rsJAM consisted in large part of noncovalent homodimers, as assessed by analytical ultracentrifugation. JAM dimers were also detected at the surface of Chinese hamster ovary cells transfected with murine JAM, as evaluated by cross-linking and immunoprecipitation. Furthermore, fluid-phase rsJAM bound dose-dependently solid-phase rsJAM, and such homophilic binding was inhibited by anti-JAM Fab BV11, but not by Fab BV12. Interestingly, Fab BV11 exclusively bound rsJAM dimers (but not monomers) in solution, whereas Fab BV12 bound both dimers and monomers. Finally, we mapped the BV11 and BV12 epitopes to a largely overlapping sequence in proximity of the extracellular amino terminus of JAM. We hypothesize that rsJAM dimerization induces a BV11-positive conformation which in turn is critical for rsJAM homophilic interactions. Dimerization and homophilic binding may contribute to both adhesive function and junctional organization of JAM.
[Show abstract][Hide abstract] ABSTRACT: A hallmark of infectious meningitis is the invasion of leukocytes into the subarachnoid space. In experimental meningitis triggered by tumor necrosis factor-alpha and interleukin-1beta, the interaction of leukocytes with endothelial cells and the subsequent migration of the cells through the vessel wall can be inhibited by an antibody to the junctional adhesion molecule (JAM). In contrast to the cytokine-induced meningitis model, anti-JAM antibodies failed to prevent leukocyte influx into the central nervous system after infection of mice with Listeria monocytogenes or lymphocytic choriomeningitis virus. Furthermore, in bacterial meningitis, anti-JAM IgG antibodies, but not Fab fragments, caused disruption of the endothelium. Likewise complement-dependent antibody-mediated cytotoxicity was observed in cultured brain endothelial cells treated with anti-JAM IgG but not with its Fab fragment.
The Journal of Infectious Diseases 10/2000; 182(3):978-82. DOI:10.1086/315765 · 5.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mutant presenilins (PS) contribute to the pathogenesis of familial Alzheimer's disease (FAD) by enhancing the production of Abeta42 from beta-amyloid precursor protein. Presenilins are endoproteolytically processed to N-terminal and C-terminal fragments, which together form a stable 1:1 complex. We have mapped the cleavage site in the PS2 protein by direct sequencing of its C-terminal fragment isolated from mouse liver. Three different N-terminal residues were identified starting at Val-299, Thr-301, and Leu-307 that correspond closely to the previously described N termini of the C-terminal fragment of human PS1. Mutational analysis of the PS2 cleavage site indicates that the principal endoproteolytic cleavage occurs at residues Met-298/Val-299 and that the N terminus is subsequently modified by secondary proteolytic cleavages. We have generated cleavage defective PS2 constructs, which accumulate exclusively as full-length polypeptides in transfected Neuro2a cells. Functional analysis of such cleavage defective PS2 carrying the FAD mutation Asn-141 --> Ile showed that its Abeta42 producing activity was strongly reduced compared with cleavage-competent FAD PS2. In contrast, cleavage defective PS2 was active in rescuing the egg-laying defect of a sel-12 mutant in Caenorhabditis elegans. We conclude that PS2 endoproteolytic cleavage is not an absolute requirement for its activities but may rather selectively enhance or stabilize its functions.
[Show abstract][Hide abstract] ABSTRACT: The mechanisms that govern leukocyte transmigration through the endothelium are not yet fully defined. Junctional adhesion molecule (JAM) is a newly cloned member of the immunoglobulin superfamily which is selectively concentrated at tight junctions of endothelial and epithelial cells. A blocking monoclonal antibody (BV11 mAb) directed to JAM was able to inhibit monocyte transmigration through endothelial cells in in vitro and in vivo chemotaxis assays. In this study, we report that BV11 administration was able to attenuate cytokine-induced meningitis in mice. The intravenous injection of BV11 mAb significantly inhibited leukocyte accumulation in the cerebrospinal fluid and infiltration in the brain parenchyma. Blood-brain barrier permeability was also reduced by the mAb. We conclude that JAM may be a new target in limiting the inflammatory response that accompanies meningitis.
Journal of Experimental Medicine 12/1999; 190(9):1351-6. DOI:10.1084/jem.190.9.1351 · 13.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the present paper, we characterize an antibody, mAb BV13, directed to mouse vascular endothelial (VE)-cadherin, a major adhesive protein of interendothelial adherens junctions. When added to cultured endothelial cells, BV13 induces a redistribution of VE-cadherin from intercellular junctions. VE-cadherin redistribution did not change the localization of platelet endothelial cell adhesion molecule or tight junction markers such as zonula occludens 1, cingulin, and junctional adhesion molecule. Intravenous administration of mAb BV13 induced a concentration- and time-dependent increase in vascular permeability in heart and lungs. By electron microscopy, interstitial edema and accumulation of mixed types of inflammatory cells in heart and lungs were observed. Injection of (rhodamine-labeled) Ricinus communis I lectin showed focal spots of exposed basement membrane in the alveolar capillaries and in some larger pulmonary vessels. These data indicate that VE-cadherin is required for vascular integrity and normal organ functions.
Proceedings of the National Academy of Sciences 09/1999; 96(17):9815-20. DOI:10.1073/pnas.96.17.9815 · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Alzheimer's disease (AD) associated presenilin (PS) proteins are proteolytically processed. One of the processing pathways involves cleavage by caspases. Pharmacological inhibition of caspases is currently being discussed as a treatment for a variety of neurodegenerative diseases, including AD. We therefore inhibited caspase mediated processing of PS-1 and PS-2 in cells transfected with wt and mutant PS by mutagenizing the substrate recognition site or by using specific peptide aldehydes known to block caspases. We found that the inhibition of caspase mediated processing of PS proteins does not decrease its amyloidogenic activity. PS cDNA constructs with mutations in the caspase cleavage site are biologically active in Caenorhabditis elegans such as the wt human PS proteins, demonstrating that caspase-mediated cleavage is not required for the physiological PS function in NOTCH signaling.
[Show abstract][Hide abstract] ABSTRACT: Presenilin 1 (PS1) and presenilin 2 (PS2) are endoproteolytically processed in vivo and in cell transfectants to yield 27-35-kDa N-terminal and 15-24-kDa C-terminal fragments. We have studied the cleavage of PS1 and PS2 in transiently and stably transfected hamster kidney and mouse and human neuroblastoma cells by immunoblot and pulse-chase experiments. C-terminal fragments were isolated by affinity chromatography and SDS-polyacrylamide gel electrophoresis and sequenced. The processing sites identified in PS1 and PS2 (Asp345/Ser346 and Asp329/Ser330, respectively) are typical for caspase-type proteases. Specific caspase inhibitors and cleavage site mutations confirmed the involvement of caspase(s) in PS1 and PS2 processing in cell transfectants. Fluorescent peptide substrates carrying the PS-identified cleavage sites were hydrolyzed by proteolytic activity from mouse brain. The PS2-derived peptide substrate was also cleaved by recombinant human caspase-3. Additional processing of PS2 by non-caspase-type proteases was also observed.
[Show abstract][Hide abstract] ABSTRACT: Eleven metastatic cancer patients were studied during three different regimens of immunotherapy with interleukin-2 (IL-2) and/or interferon alpha (IFN alpha): group A received 4 days of IL-2 i.a. infusion (n = 3), group B IFN alpha s.c. during 5 days (n = 4), followed on day 3 by 5 days of a continuous IL-2 i.v. infusion, and group C had 4 days of IL-2 i.v. infusion together with s.c. IFN alpha on days 1 and 4 (n = 4). Soluble tumor necrosis factor receptors (sTNFR) p55 and p75 and TNF alpha concentrations in serum were analyzed before therapy and daily during 8 days of the first therapy cycle. sTNFR was measured by radioimmunoassay. sTNFR p55 increased in all patient groups from a baseline value of 5.2 +/- 0.9 ng/ml to a maximum of 13.6 +/- 1.2 ng/ml by days 3-4 (P = 0.003). sTNFR p75 increased from 7.6 +/- 1.1 ng/ml to peak values of 30.1 +/- 2.6 ng/ml in groups A and B (P = 0.02). In group C the sTNFR p75 response was weak (NS). In group B, the increase of both p55 and p75 occurred only after addition of IL-2 to IFN alpha. TNF alpha increased weakly during treatment with IFN alpha alone (group B); it rose strongly during IL-2 and the combined treatment (groups A-C) from 8 +/- 2 pg/ml to 115 +/- 13 pg/ml (P = 0.003). In group B, it reached the maximum 24 h after addition of IL-2 to IFN alpha and decreased thereafter. There was a significant relationship between TNF alpha and sTNFR p55 or sTNFR p75 in groups A and C, (P = 0.001), but not in group B. Group C was also investigated during the third therapy cycle. The increase of sTNFR p75 was stronger (P = 0.01) and that of TNF alpha weaker than in the first cycle; the sTNFR p55 response was similar in both cycles. In conclusion sTNFR p55 and p75 are rapidly induced during IL-2 and IL-2+ IFN alpha treatment, the increase of sTNF receptors parallels or exceeds that of TNF alpha and may influence the immunomodulatory effects of TNF alpha during cytokine therapy.
Cancer Immunology and Immunotherapy 03/1994; 38(2):113-8. DOI:10.1007/BF01526206 · 3.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Two TNF binding proteins have been characterized as soluble fragments of TNF receptors. We measured the plasma concentrations of soluble type A (p75) and type B (p55) TNF receptors in patients with systemic lupus erythematodes (SLE), progressive systemic sclerosis (PSS), and mixed connective tissue disease (MCTD). In SLE and PSS patients plasma concentrations of both types of TNF receptors and in MCTD patients type A TNF receptors were significantly elevated compared to controls. Plasma concentrations of both soluble TNF receptors were highly correlated in SLE, PSS, and MCTD patients, indicating a possible coregulation of both TNF receptors. In contrast, soluble interleukin 2 receptor (sCD 25) plasma concentrations were not correlated and seem to be an independent parameter. The soluble forms of the TNF receptors neutralize TNF in cytotoxicity assays and are functionally active as TNF antagonists. In one patient with SLE, autoantibodies against type A TNF receptors were detected, TNF alpha, and TNF beta did not interfere with the autoantibody binding to the receptor.
[Show abstract][Hide abstract] ABSTRACT: In previous studies we showed that cultured human keratinocytes expressed the 55-kD TNF receptor (TNFR) and that its expression the important for TNF alpha-mediated upregulation of intercellular adhesion molecule-1 (ICAM-1) expression on keratinocytes. Because factors that either reduce or enhance TNFR expression are likely to have a major impact on the biological effects of TNF alpha on keratinocytes, these studies were conducted to determine the factors that regulate its expression on keratinocytes. Using reverse transcriptase polymerase chain reaction, human keratinocytes were shown to lack 75-kD TNFR expression, indicating that TNF responsiveness of human keratinocytes critically depended on regulation of 55-kD TNFR expression. Human keratinocyte 55-kD TNFR surface and mRNA expression was found to be regulated in vitro by recombinant human (rh) TNF alpha. Stimulation of keratinocytes with rhTNF alpha initially decreased, but later increased, 55-kD TNFR surface expression. This biphasic modulation of 55-kD TNFR surface expression was associated with concomitant changes in 55-kD TNFR mRNA expression. Ultraviolet B (UVB) radiation, a well-known inducer of synthesis and secretion of TNF alpha by human keratinocytes, was found to mimic TNF alpha-induced modulation of 55-kD TNFR surface and mRNA expression via a TNF alpha-mediated autocrine regulatory mechanism. Production of soluble 55-kD TNFR by human keratinocytes remained unaffected by TNF alpha stimulation or UVB irradiation. These studies provide clear evidence that membrane expression of the human 55-kD TNFR may be regulated in human keratinocytes by the ligand itself: TNF alpha. Since in previous studies UVB irradiation transiently inhibited TNF alpha-induced human keratinocyte ICAM-1 expression, it is proposed that UVB radiation-induced biphasic modulation of human keratinocyte 55-kD TNFR expression may affect the capacity of these cells to respond to TNF alpha.