Biomedical subjects

D Schubert

Publications and source records attributed to D Schubert.

At least 55 records · Page 3Linked to original sources

PubMed2000

Isolation and characterization of novel presenilin binding protein.

Approximately 50% of familial Alzheimer's disease (AD) cases are linked to the presenilin (PS) gene. This suggests that an altered function of mutated PSs accounts for a fundamental process leading to AD. Here we identify a new PS binding protein, PBP, which is highly expressed in cerebral cortex and hippocampus. immunohistochemical studies and cell fractionation analysis show that PBP redistributes from cytoplasm to membranes in the presence of PS. In addition, PBP is deficient in the soluble fraction of sporadic AD brains.

Alzheimer Disease↗

PubMed2000

Interpretative reporting and selective antimicrobial susceptibility release in non-critical microbiology results.

The action taken in response to 169 positive sputum, urine and wound culture reports was examined. All reports included interpretative comments. Antimicrobial susceptibilities were released in 29 (17%). Therapy was significantly more likely to be started or altered in response to reports where susceptibilities were released (13 of 29, 45%) versus those without susceptibility release (31 of 140, 22%). Susceptibility release did not influence the appropriateness of antibiotic therapy. The clinical microbiology team was contacted for therapeutic advice in response to 32 (19%) reports. Of the remaining 137 reports, therapy was started or altered in response to 30 (22%) reports, but was considered appropriate in only seven (22%) of these.

Anti-Bacterial Agents↗

PubMed1999

Controlled Arrangement of Supramolecular Metal Coordination Arrays on Surfaces.

Metallo-supramolecular systems have been adsorbed in a controlled way onto graphite surfaces and visualized with molecular resolution for the first time. A parallel or orthogonal arrangement of the metal coordination arrays is evident depending on the specific ligands (see picture). Furthermore, simple nanomanipulations were performed by extracting single grids from the layer.

Journal Article↗

PubMed1999

The up-regulation of endosomal-lysosomal components in amyloid beta-resistant cells.

The abundance of amyloid beta peptide (A beta) and the selective loss of neurons are characteristics of Alzheimer's disease. However, subpopulations of brain cells survive, including neurons near A beta-rich plaques. The surviving neurons may have gene expression profiles that allow them to be resistant to A beta toxicity. Here we use the differential display technique to compare the profiles of gene expression in an A beta-resistant cell line with its parental cells. Prominent among the changes are two components of the endosomal-lysosomal system, insulin growth factor II receptor/mannose-6-phosphate receptor and arylsulfatase B. Both are more highly expressed in the A beta-resistant clone, and arylsulfatase is inducible by A beta and hydrogen peroxide. Another lysosomal enzyme, beta-glucuronidase, is also up-regulated in A beta-resistant cells. These results are consistent with the observation that the endosomal-lysosomal system is highly activated in Alzheimer's disease brains, and they raise the possibility that the high expression of endosomal-lysosomal components is important for neuronal survival in the presence of A beta.

Amyloid beta-Peptides↗

PubMed1999

Propofol hemisuccinate protects neuronal cells from oxidative injury.

Oxidative stress contributes to the neuronal death observed in neurodegenerative disorders and neurotrauma. Some antioxidants for CNS injuries, however, have yet to show mitigating effects in clinical trials, possibly due to the impermeability of antioxidants across the blood-brain barrier (BBB). Propofol (2,6-diisopropylphenol), the active ingredient of a commonly used anesthetic, acts as an antioxidant, but it is insoluble in water. Therefore, we synthesized its water-soluble prodrug, propofol hemisuccinate sodium salt (PHS), and tested for its protective efficacy in neuronal death caused by non-receptor-mediated, oxidative glutamate toxicity. Glutamate induces apoptotic death in rat cortical neurons and the mouse hippocampal cell line HT-22 by blocking cystine uptake and causing the depletion of intracellular glutathione, resulting in the accumulation of reactive oxygen species (ROS). PHS has minimal toxicity and protects both cortical neurons and HT-22 cells from glutamate. The mechanism of protection is attributable to the antioxidative property of PHS because PHS decreases the ROS accumulation caused by glutamate. Furthermore, PHS protects HT-22 cells from oxidative injury induced by homocysteic acid, buthionine sulfoximine, and hydrogen peroxide. For comparison, we also tested alpha-tocopherol succinate (TS) and methylprednisolone succinate (MPS) in the glutamate assay. Although TS is protective against glutamate at lower concentrations than PHS, TS is toxic to HT-22 cells. In contrast, MPS is nontoxic but also nonprotective against glutamate. Taken together, PHS, a water-soluble prodrug of propofol, is a candidate drug to treat CNS injuries owing to its antioxidative properties, low toxicity, and permeability across the BBB.

Animals↗

PubMed1999

Species-specific, postentry barriers to primate immunodeficiency virus infection.

By using replication-defective vectors derived from human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV(mac)), and murine leukemia virus (MuLV), all of which were pseudotyped with the vesicular stomatitis virus (VSV) G glycoprotein, the efficiency of postentry, early infection events was examined in target cells of several mammalian species. Titers of HIV-1 vectors were significantly lower than those of SIV(mac) and MuLV vectors in most cell lines and primary cells from Old World monkeys. By contrast, most New World monkey cells exhibited much lower titers for the SIV(mac) vector compared with those of the HIV-1 vector. Prosimian cells were resistant to both HIV-1 and SIV(mac) vectors, although the MuLV vector was able to infect these cells. Cells from other mammalian species were roughly equivalent in susceptibility to the three vectors, with the exception of rabbit cells, which were specifically resistant to the HIV-1 vector. The level of HIV-1 vector expression was very low in transduced cells of rodent, rabbit, cow, and pig origin. Early postentry restriction of primate immunodeficiency virus infection exhibits patterns largely coincident with species borders and applies to diverse cell types within an individual host, suggesting the involvement of species-specific, widely expressed cellular factors.

Animals↗

PubMed1999

Cellular morphology and physiology of the perinatal rat cerebral cortex.

The cellular morphology and electrophysiology of the rat neocortex between embryonic day (E) 18 and postnatal day (P) 3 was studied in vitro by extracellular biocytin injections and whole-cell recordings, respectively. Most neurons were characterized by a small number of short-range dendrites and a main axon that was directed towards the white matter. Biocytin injections into the marginal zone and the cortical plate labeled far-reaching connections extending up to 2 mm in horizontal direction, indicating the existence of a dense network of long-range intrinsic projections in the neonatal cortex. Action potentials could be elicited as early as E18 and repetitive firing could first be observed at P0. Electrical stimulation of the immature cortex at various positions elicited polyphasic and long-lasting (up to 1 s) excitatory postsynaptic potentials and currents, which were significantly reduced in amplitude by a selective N-methyl-D-aspartate receptor antagonist. Our data indicate that the perinatal cortex manifests the structural and functional conditions for powerful excitatory interactions, which increase the likelihood for the generation of epileptiform activity during this developmental period.

Animals↗

PubMed1998

Lyotropic-salt-induced changes in monomer/dimer/tetramer association equilibrium of formyltransferase from the hyperthermophilic Methanopyrus kandleri in relation to the activity and thermostability of the enzyme.

Formyltransferase from Methanopyrus kandleri is composed of only one type of subunits of molecular mass 32 kDa. The enzyme requires the presence of lyotropic salts for activity and thermostability. We report here that the enzyme is in a monomer/dimer/tetramer association equilibrium, the association constant being affected by lyotropic salts. At 0.01 M K2HPO4/KH2PO4, pH 7.2, the enzyme (0.4 mg/ml) was mainly present in a monomeric form. Upon increase of the phosphate concentration, the concentration of the dimer increased up to a phosphate concentration of 0.6 M, then decrease at the expense of tetramer formation up to a phosphate concentration of 1.0 M. The specific activity at 4 C increased from <0.1 U/mg at 0.01 M, over 1.5 U/mg at 0.6 M to 3.6 U/mg at 1.0 M. Similar results were obtained with ammonium sulfate as lyotropic salt. The findings indicate that both oligomerization and activity increase with increasing salt concentrations, suggesting that there is a causal connection. To determine this, we exploited the observation that oligomer formation was not induced by the weak lyotropic salt NaCl up to a concentration of 1.5 M and that the dissociation of the dimer into the monomer at 4 degrees C proceeded very slowly (50% in approximately 6 h). This allowed us to study the effect of NaCl on the activity of the oligomers at NaCl concentrations not sufficient to induce oligomerization. At 4 degrees C, the activity of the oligomers increased from 0.3 U/mg at 0.25 M NaCl to 3.4 U/mg at 1.0 M NaCl. At these NaCl concentrations, the monomers were inactive. The findings indicate that oligomerization is a prerequisite for enzyme activity in the presence of NaCl. The salt-dependent induction of oligomerization was parallelled by an increase in thermostability; strong lyotropic salts conferred thermostability at much lower concentrations than the weak lyotropic NaCl.

Biopolymers↗

PubMed1998

Amyloid beta peptide alters intracellular vesicle trafficking and cholesterol homeostasis.

Amyloid beta peptide (Abeta) is thought to play a central role in the pathogenesis of Alzheimer disease (AD). How Abeta induces neurodegeneration in AD is not known. A connection between AD and cholesterol metabolism is suggested by the finding that people with the apolipoprotein E4 allele, a locus coding for a cholesterol-transporting lipoprotein, have a modified risk for both late-onset AD and cardiovascular disease. In the present study we show that both Abeta and submicromolar concentrations of free cholesterol alter the trafficking of a population of intracellular vesicles that are involved in the transport of the reduced form of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT formazan), the formation of which is a widely used cell viability assay. Treatments that change cellular free cholesterol levels also modulate the trafficking of the MTT formazan-containing vesicles, suggesting that the trafficking of these vesicles may be regulated by free cholesterol under physiological conditions. In addition, Abeta decreases cholesterol esterification and changes the distribution of free cholesterol in neurons. These results suggest that the MTT formazan-transporting vesicles may be involved in cellular cholesterol homeostasis and that the alteration of vesicle transport by Abeta may be relevant to the chronic neurodegeneration observed in AD.

Amyloid beta-Peptides↗

PubMed1998

The activation of metabotropic glutamate receptors protects nerve cells from oxidative stress.

Metabotropic glutamate receptors (mGluRs) have been implicated in a variety of cellular responses to glutamic acid. The work described in this manuscript extends the role of mGluRs to include protection from oxidative stress-induced programmed cell death. Glutamate analogs regulate inositol-1,4,5 triphosphate mass accumulation in accordance with their ability to protect cells from oxidative glutamate toxicity, and protection appears to take place at the level of glutathione metabolism. Short-term exposure of cells to low concentrations of glutamate desensitizes cells to a subsequent challenge from glutamate. Glutamate exposure upregulates the expression of mGluR5 in hippocampal HT-22 cells and mGluR1 in cortical primary cultures. Finally, group I mGluR agonists also protect cells from death programs initiated by glucose starvation and cystine deprivation.

Animals↗

PubMed1998

Phosphatidylcholine-specific phospholipase C regulates glutamate-induced nerve cell death.

Phosphatidylcholine-specific phospholipase C (PC-PLC) is a necessary intermediate in transducing apoptotic signals for tumor necrosis factor and Fas/Apo-1 ligands in nonneuronal cells. The data presented here show that PC-PLC also is required in oxidative glutamate-induced programmed cell death of both immature cortical neurons and a hippocampal nerve cell line, HT22. In oxidative glutamate toxicity, which is distinct from excitotoxicity, glutamate interferes with cystine uptake by blocking the cystine/glutamate antiporter, indirectly causing a depletion of intracellular glutathione. A PC-PLC inhibitor blocks oxidative glutamate toxicity, and exogenous PC-PLC potentiates glutamate toxicity. The inhibition of PC-PLC uncouples the cystine uptake from glutamate inhibition, allowing the maintenance of glutathione synthesis and cell viability. These data suggest that PC-PLC modulates neuronal cell death through a mechanism that is distinct from that involved in nonneuronal apoptosis.

Animals↗

PubMed1998

The regulation of reactive oxygen species production during programmed cell death.

Reactive oxygen species (ROS) are thought to be involved in many forms of programmed cell death. The role of ROS in cell death caused by oxidative glutamate toxicity was studied in an immortalized mouse hippocampal cell line (HT22). The causal relationship between ROS production and glutathione (GSH) levels, gene expression, caspase activity, and cytosolic Ca2+ concentration was examined. An initial 5-10-fold increase in ROS after glutamate addition is temporally correlated with GSH depletion. This early increase is followed by an explosive burst of ROS production to 200-400-fold above control values. The source of this burst is the mitochondrial electron transport chain, while only 5-10% of the maximum ROS production is caused by GSH depletion. Macromolecular synthesis inhibitors as well as Ac-YVAD-cmk, an interleukin 1beta-converting enzyme protease inhibitor, block the late burst of ROS production and protect HT22 cells from glutamate toxicity when added early in the death program. Inhibition of intracellular Ca2+ cycling and the influx of extracellular Ca2+ also blocks maximum ROS production and protects the cells. The conclusion is that GSH depletion is not sufficient to cause the maximal mitochondrial ROS production, and that there is an early requirement for protease activation, changes in gene expression, and a late requirement for Ca2+ mobilization.

Animals↗

PubMed1998

Mechanisms of resistance to oxidative stress in Alzheimer's disease.

It is likely that amyloid beta-protein (A beta) mediates nerve cell death in Alzheimer's disease (AD). Some nerve cell populations, however, remain undamaged in AD brain. To understand the biochemical basis for resistance to A beta toxicity, a series of cell lines were isolated which are resistant to A beta toxicity. It is shown that a major component of the resistance mechanism is the transcriptional elevation of two H2O2 degrading enzymes, glutathione peroxidase and catalase. These data support other evidence for the role of oxidative damage in A beta toxicity, and suggest strategies for clinical approaches to the disease.

Alzheimer Disease↗

PubMed1998

Cellular mechanisms of resistance to chronic oxidative stress.

Oxidative stress is implicated in several pathologies such as AIDS, Alzheimer's disease, and Parkinson's disease, as well as in normal aging. As a model system to study the response of cells to oxidative insults, glutamate toxicity on a mouse nerve cell line, HT-22, was examined. Glutamate exposure kills HT-22 via a nonreceptor-mediated oxidative pathway by blocking cystine uptake and causing depletion of intracellular glutathione (GSH), leading to the accumulation of reactive oxygen species and, ultimately, apoptotic cell death. Several HT-22 subclones that are 10-fold resistant to exogenous glutamate were isolated and the mechanisms involved in resistance characterized. The expression levels of neither heat shock proteins nor apoptosis-related proteins are changed in the resistant cells. In contrast, the antioxidant enzyme catalase, but not glutathione peroxidase nor superoxide dismutase, is more highly expressed in the resistant than in the parental cells. In addition, the resistant cells have enhanced rates of GSH regeneration due to higher activities of the GSH metabolic enzymes gamma-glutamylcysteine synthetase and GSH reductase, and GSH S-transferases activities are also elevated. As a consequence of these alterations, the glutamate resistant cells are also more resistant to organic hydroperoxides and anticancer drugs that affect these GSH enzymes. These results indicate that resistance to apoptotic oxidative stress may be acquired by coordinated changes in multiple antioxidant pathways.

Animals↗

PubMed1998

Steroid hormones block amyloid fibril-induced 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) formazan exocytosis: relationship to neurotoxicity.

Perhaps the most reproducible early event induced by the interaction of amyloid beta peptide (A beta) with the cell is the inhibition of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. We recently demonstrated that cytotoxic amyloid peptides such as A beta and human amylin inhibit cellular MTT reduction by dramatically enhancing MTT formazan exocytosis. We now show the following: (a) Insulin and glucagon, when converted to fibrils with beta-pleated sheet structure, induce MTT formazan exocytosis that is indistinguishable from that induced by A beta. NAC35, an amyloidogenic fragment of alpha-synuclein (or NACP), also induces MTT formazan exocytosis. (b) All protein fibrils with the beta-pleated sheet structure examined are toxic to rat hippocampal neurons. (c) Many sterol sex hormones (e.g., estradiol and progesterone) block amyloid fibril-enhanced MTT formazan exocytosis as well as MTT formazan exocytosis in control cells by acting at a common late step in the exocytic pathway. Steroids fail, however, to protect hippocampal neurons from acute amyloid fibril toxicity. These findings suggest that the ability to enhance MTT formazan exocytosis and to induce neurotoxicity are common biological activities of protein fibrils with beta-pleated sheet structure but that enhanced MTT formazan exocytosis is not sufficient for acute A beta neurotoxicity.

Amyloid↗

PubMed1997

Requirement for cGMP in nerve cell death caused by glutathione depletion.

Glutathione depletion occurs in several forms of apoptosis and is associated with Parkinson's disease and HIV toxicity. The neurotransmitter glutamate kills immature cortical neurons and a hippocampal nerve cell line via an oxidative pathway associated with glutathione depletion. It is shown here that soluble guanylyl cyclase (sGC) activity is required for nerve cell death caused by glutathione depletion. Inhibitors of sGC block glutamate toxicity and a cGMP analogue potentiates cell death. Glutamate also induces an elevation of cGMP that occurs late in the cell death pathway. The resultant cGMP modulates the increase in intracellular calcium that precedes cell death because sGC inhibitors prevent calcium elevation and the cGMP analogue potentiates the increase in intracellular calcium. These results suggest that the final pathway of glutamate induced nerve cell death is through a cGMP-modulated calcium channel.

Animals↗

PubMed1997

Beta amyloid toxicity does not require RAGE protein.

It has been suggested that a receptor for advanced glycation end products (RAGE) is the nerve cell receptor for amyloid beta protein (A beta). To determine if this is indeed the case, two neural cell lines as well as rat cortical neurons were examined for the presence of the mRNA for RAGE by PCR and northern blot analysis. Although lung was strongly positive, in no case was RAGE mRNA detected in the cultured neural cells. Glycated-albumin is a major ligand for RAGE and the cell surface RAGE protein is trypsin sensitive. In agreement with the mRNA data, trypsin treatment did not alter A beta toxicity, nor did glycated albumin modify the A beta response. It follows that RAGE is not the neural receptor for A beta.

Amyloid beta-Peptides↗

PubMed1997

The role of protein phosphorylation in beta amyloid toxicity.

Recent evidence suggests that amyloid beta protein (A beta) mediates the neurotoxicity observed in Alzheimer's disease (AD). Little is known, however, about the cytotoxic pathway leading to nerve cell death. Using a rat brain cell line which is sensitive to A beta, it is shown that a 50-60 kDa protein becomes more phosphorylated when cells are exposed to A beta. Several kinase and phosphatase inhibitors block both the increase in phosphorylation of the 50-60 kDa protein and A beta toxicity. In contrast, a tyrosine kinase inhibitor blocks toxicity at a step which is distinct from the phosphorylation of this protein. A beta also causes a general increase in overall phosphatase activity. It is therefore likely that a protein phosphorylation cascade is involved in A beta toxicity.

Amyloid beta-Peptides↗