Biomedical subjects

D Schubert

Publications and source records attributed to D Schubert.

At least 37 records · Page 2Linked to original sources

PubMed2002

Enzymatic and histological alterations in the isolated perfused rat pancreas under conditions of oxidative stress.

BACKGROUND: Oxidative stress is a relevant event in the pathogenesis of acute pancreatitis. Investigations in vivo are limited because of the complexity of the organism and the short half-life of free radicals. The isolated perfused rat pancreas could be useful for investigations in the early phase of acute pancreatitis especially under conditions of oxidative stress. METHODS: External perfusions of the pancreatic glands of Wistar rats were carried out using a modified Krebs-Ringer buffer including an additive of the detergents Triton X-100 and a perfusion including hydrogen peroxide (0.0012%) or tert-butylhydroperoxide (0.0042%) or xanthine oxidase (0.1 U/ml). Changes in amylase, lipase, LDH in the portal outflow fluid and for histological alterations were analyzed. RESULTS: Damage to pancreatic parenchyma using Triton X-100 was indicated by increased levels of pancreatic enzymes in the perfusion medium. During perfusion with hydrogen peroxide or tert-butylhydroperoxide we found no changes in pancreatic enzymes in the portal outflow. In contrast, perfusion with xanthine oxidase induced a significant elevation in lipase and amylase in the effusion fluid after 30 min. We found a significant increase in edema in the hydrogen peroxide and in the xanthine oxidase group. Focal necroses of the pancreatic parenchyma were detected in all groups of oxidative stress. CONCLUSIONS: The isolated perfused rat pancreas is a valuable experimental model for investigating the early phase of pathophysiology in acute pancreatitis, for instance, the effect of oxidative stress as an early event in acute pancreatitis. Using hydrogen peroxide tert-butylhydroperoxide or xanthine oxidase, only xanthine oxidase was able to induce a typical elevation in the pancreas enzymes in the effusion fluid.

Acute Disease↗

PubMed2001

Oxidative glutamate toxicity can be a component of the excitotoxicity cascade.

Along with ionotropic and metabotropic glutamate receptors, the cystine/glutamate antiporter x(c)(-) may play a critical role in CNS pathology. High levels of extracellular glutamate inhibit the import of cystine, resulting in the depletion of glutathione and a form of cell injury called oxidative glutamate toxicity. Here we show that a portion of the cell death associated with NMDA receptor-initiated excitotoxicity can be caused by oxidative glutamate toxicity. In primary mouse cortical neurons the cell death resulting from the short-term application of 10 microm glutamate can be divided into NMDA and NMDA receptor-independent phases. The NMDA receptor-independent component is associated with high extracellular glutamate and is inhibited by a variety of reagents that block oxidative glutamate toxicity. These results suggest that oxidative glutamate toxicity toward neurons lacking functional NMDA receptors can be a component of the excitotoxicity-initiated cell death pathway.

2-Amino-5-phosphonovalerate↗

PubMed2001

The activation of dopamine D4 receptors inhibits oxidative stress-induced nerve cell death.

Oxidative stress is thought to be the cause of nerve cell death in many CNS pathologies, including ischemia, trauma, and neurodegenerative disease. Glutamate kills nerve cells that lack ionotropic glutamate receptors via the inhibition of the cystine-glutamate antiporter x(c)(-), resulting in the inhibition of cystine uptake, the loss of glutathione, and the initiation of an oxidative stress cell death pathway. A number of catecholamines were found to block this pathway. Specifically, dopamine and related ligands inhibit glutamate-induced cell death in both clonal nerve cell lines and rat cortical neurons. The protective effects of dopamine, apomorphine, and apocodeine, but not epinephrine and norepinephrine, are antagonized by dopamine D4 antagonists. A dopamine D4 agonist also protects, and this protective effect is inhibited by U101958, a dopamine D4 antagonist. Although the protective effects of some of the catecholamines are correlated with their antioxidant activities, there is no correlation between the protective and antioxidant activities of several other ligands. Normally, glutamate causes an increase in reactive oxygen species (ROS) and intracellular Ca(2+). Apomorphine partially inhibits glutamate-induced ROS production and blocks the opening of cGMP-operated Ca(2+) channels that lead to Ca(2+) elevation in the late part of the cell death pathway. These data suggest that the protective effects of apomorphine on oxidative stress-induced cell death are, at least in part, mediated by dopamine D4 receptors via the regulation of cGMP-operated Ca(2+) channels.

Animals↗

PubMed2001

Projection structure and oligomeric properties of a bacterial core protein translocase.

The major route for protein export or membrane integration in bacteria occurs via the Sec-dependent transport apparatus. The core complex in the inner membrane, consisting of SecYEG, forms a protein-conducting channel, while the ATPase SecA drives translocation of substrate across the membrane. The SecYEG complex from Escherichia coli was overexpressed, purified and crystallized in two dimensions. A 9 A projection structure was calculated using electron cryo-microscopy. The structure exhibits P12(1) symmetry, having two asymmetric units inverted with respect to one another in the unit cell. The map shows elements of secondary structure that appear to be transmembrane helices. The crystallized form of SecYEG is too small to comprise the translocation channel and does not contain a large pore seen in other studies. In detergent solution, the SecYEG complex displays an equilibrium between monomeric and tetrameric forms. Our results therefore indicate that, unlike other known channels, the SecYEG complex can exist as both an assembled channel and an unassembled smaller unit, suggesting that transitions between the two states occur during a functional cycle.

Bacterial Proteins↗

PubMed2001

Layer-specific intracolumnar and transcolumnar functional connectivity of layer V pyramidal cells in rat barrel cortex.

Layer V pyramidal cells in rat barrel cortex are considered to play an important role in intracolumnar and transcolumnar signal processing. However, the precise circuitry mediating this processing is still incompletely understood. Here we obtained detailed maps of excitatory and inhibitory synaptic inputs onto the two major layer V pyramidal cell subtypes, intrinsically burst spiking (IB) and regular spiking (RS) cells, using a combination of caged glutamate photolysis, whole-cell patch-clamp recording, and three-dimensional reconstruction of biocytin-labeled cells. To excite presynaptic neurons with laminar specificity, the release of caged glutamate was calibrated and restricted to small areas of 50 x 50 microm in all cortical layers and in at least two neighboring barrel-related columns. IB cells received intracolumnar excitatory input from all layers, with the largest EPSP amplitudes originating from neurons in layers IV and VI. Prominent transcolumnar excitatory inputs were provided by presynaptic neurons also located in layers IV, V, and VI of neighboring columns. Inhibitory inputs were rare. In contrast, RS cells received distinct intracolumnar inhibitory inputs, especially from layers II/III and V. Intracolumnar excitatory inputs to RS cells were prominent from layers II-V, but relatively weak from layer VI. Conspicuous transcolumnar excitatory inputs could be evoked solely in layers IV and V. Our results show that layer V pyramidal cells are synaptically driven by presynaptic neurons located in every layer of the barrel cortex. RS cells seem to be preferentially involved in intracolumnar signal processing, whereas IB cells effectively integrate excitatory inputs across several columns.

Action Potentials↗

PubMed2001

Concentrations of hepatocyte growth factor in cerebrospinal fluid under normal and different pathological conditions.

Hepatocyte growth factor (HGF) and its specific receptor, MET, are expressed in the developing and adult mammalian brain. Recent studies have shown a neurotrophic activity of HGF in the nervous system. The present study focused on HGF concentrations in the cerebrospinal fluid (CSF) and serum in normal persons and in different central nervous system (CNS) diseases considering blood-CSF barrier (BCB) function. Concentrations of HGF were analyzed using an enzyme-linked immunosorbent assay (ELISA). HGF was present in normal human CSF (346+/-126 pg/ml) representing approximately half of the HGF serum concentrations. The CSF HGF levels were not significantly changed in chronic CNS disease and in aseptic meningitis (419+/-71 pg/ml), but significantly increased in patients with bacterial meningitis (6101+/- 5200 pg/ml). The HGF levels in CSF were not influenced by increased serum concentrations in patients with normal or mildly affected BCB function. The results show that HGF is present in normal CSF and does not appear to cross the CSF barrier significantly unless it is severely disrupted. So far, strong increases of HGF concentration in CSF are only present in acute bacterial meningitis.

Acute Disease↗

PubMed2001

Regulation of antioxidant metabolism by translation initiation factor 2alpha.

Oxidative stress and highly specific decreases in glutathione (GSH) are associated with nerve cell death in Parkinson's disease. Using an experimental nerve cell model for oxidative stress and an expression cloning strategy, a gene involved in oxidative stress-induced programmed cell death was identified which both mediates the cell death program and regulates GSH levels. Two stress-resistant clones were isolated which contain antisense gene fragments of the translation initiation factor (eIF)2alpha and express a low amount of eIF2alpha. Sensitivity is restored when the clones are transfected with full-length eIF2alpha; transfection of wild-type cells with the truncated eIF2alpha gene confers resistance. The phosphorylation of eIF2alpha also results in resistance to oxidative stress. In wild-type cells, oxidative stress results in rapid GSH depletion, a large increase in peroxide levels, and an influx of Ca(2+). In contrast, the resistant clones maintain high GSH levels and show no elevation in peroxides or Ca(2+) when stressed, and the GSH synthetic enzyme gamma-glutamyl cysteine synthetase (gammaGCS) is elevated. The change in gammaGCS is regulated by a translational mechanism. Therefore, eIF2alpha is a critical regulatory factor in the response of nerve cells to oxidative stress and in the control of the major intracellular antioxidant, GSH, and may play a central role in the many neurodegenerative diseases associated with oxidative stress.

Antioxidants↗

PubMed2001

Flavonoids protect neuronal cells from oxidative stress by three distinct mechanisms.

Flavonoids are a family of antioxidants found in fruits and vegetables as well as in popular beverages such as red wine and tea. Although the physiological benefits of flavonoids have been largely attributed to their antioxidant properties in plasma, flavonoids may also protect cells from various insults. Nerve cell death from oxidative stress has been implicated in a variety of pathologies, including stroke, trauma, and diseases such as Alzheimer's and Parkinson's. To determine the potential protective mechanisms of flavonoids in cell death, the mouse hippocampal cell line HT-22, a model system for oxidative stress, was used. In this system, exogenous glutamate inhibits cystine uptake and depletes intracellular glutathione (GSH), leading to the accumulation of reactive oxygen species (ROS) and an increase in Ca(2+) influx, which ultimately causes neuronal death. Many, but not all, flavonoids protect HT-22 cells and rat primary neurons from glutamate toxicity as well as from five other oxidative injuries. Three structural requirements of flavonoids for protection from glutamate are the hydroxylated C3, an unsaturated C ring, and hydrophobicity. We also found three distinct mechanisms of protection. These include increasing intracellular GSH, directly lowering levels of ROS, and preventing the influx of Ca(2+) despite high levels of ROS. These data show that the mechanism of protection from oxidative insults by flavonoids is highly specific for each compound.

Animals↗

PubMed2001

Presenilin binding protein is associated with neurofibrillary alterations in Alzheimer's disease and stimulates tau phosphorylation.

A novel presenilin binding protein, PBP, has recently been identified. PBP is localized to the particulate fraction of extracts of Alzheimer's disease brain but is found in the soluble fractions of brain from age matched normal controls. It is shown here that PBP is associated with neurofibrillary tangles in Alzheimer's disease brain. In addition, the expression of PBP increases the phosphorylation of tau in cultured cells. Therefore PBP may have a regulatory role in tau phosphorylation and in the genesis of neurofibrillary tangles.

Aged↗

PubMed2001

The role of Bax in glutamate-induced nerve cell death.

The role of the Bax gene product was examined in three forms of cortical nerve cell death in primary cultures. These include spontaneous cell death, oxidative glutamate toxicity, in which exogenous glutamate inhibits cystine uptake resulting in toxic oxidative stress, and ionotropic glutamate receptor-mediated excitotoxicity following a brief exposure to 10 microM glutamate. Primary cortical and hippocampal neuron cultures were established from embryos of Bax -/+ x Bax -/+ matings and the embryos genotyped and assayed for cell death in the three experimental paradigms. Cell death induced by oxidative glutamate toxicity and glutamate-mediated excitotoxicity was not altered in the Bax -/- homozygous knockout animals. In contrast, there was an approximately 50% inhibition of spontaneous cell death. These results suggest that a classical Bax-dependent apoptotic pathway contributes to the spontaneous cell death that takes place when nerve cells are initially exposed to cell culture conditions. A Bax-dependent programmed cell death pathway is not, however, utilized in oxidative glutamate toxicity and NMDA receptor-mediated excitotoxicity following a brief exposure to low concentrations of glutamate.

Animals↗

PubMed2001

[Enzymatic and histological alterations in the isolated perfused rat pancreas in the taurocholate and cerulein model of acute pancreatitis].

METHODS: The pancreas of 24 male Wistar rats was perfused extracoporally by modified Krebs-Ringer-buffer for 80 minutes (including a 20 minutes equilibration period). To verify any organ damage we measured the activity of pancreatic enzymes like amylase, lipase and lactatdehydrogenase in the portal effluent. Furthermore histological changes were analysed after perfusion. Organ damage was induced by adding cerulein in a physiological dose (10(-10) M, n = 6) and in a supramaximal dose (10(-8) M, n = 6) and by intraductal injection of taurocholate (3.5 %, n = 6). RESULTS: Already 10 minutes after stimulation with cerulein (10(-8) M) and after intraductal injection of taurocholate increased activities (p < 0.01) of amylase and lipase were measured in the portal effluent compared to the group without any treatment. Lactatdehydrogenase levels did not changed. Apart from marked oedema in both groups considerable zones of necrosis could be noticed especially in the taurocholate group. CONCLUSION: Our data suggest that the isolated perfused rat pancreas (IPRP) is a valuable experimental tool to verify pathophysiological changes in the early phase of acute pancreatitis (AP). Various established models of AP such as by cerulein hyperstimulation or intraductal injection of taurocholate, could be applied to the IPRP. We conclude that this method enlarges the spectrum of established experimental models of acute pancreatitis.

Acute Disease↗

PubMed2001

[The isolated perfused rat pancreas - an experimental model for investigation the early events in the pathogenesis of acute pancreatitis].

METHODS: External perfusions of the pancreatic glands of Wistar-rats were done, using a modified Krebs-Ringer-Buffer (KRB). We looked for an elevation of amylase, lipase and lactate-dehydrogenase in the effusion fluid (portal outflow fluid). We investigated a normal perfusion (KRB, 60 minutes), a long term perfusion (KRB, 240 minutes) and a perfusion (60 minutes) including an additive of the detergents triton x-100 or the cholecystokinin analogue ceruletid (10(-8) M). RESULTS: An isolated external perfusion of a rat pancreas is possible without inducing any increase of parameters of damage such as amylase, lipase or lactate-dehydrogenase in the outflow medium. The perfusion time should be limited to 80 minutes including a 20 minutes equilibration period. A damage of pancreatic parenchyma is indicated by increased levels of pancreatic enzymes in the perfusion medium. Such damage can be induced by various noxious substances like detergents or cerulein, which has a significance in the pathophysiology of experimental acute pancreatitis. A significant increase (p < 0.01) of lactate-dehydrogenase, lipase and amylase was found 10, 20 and 30 minutes after an application of triton x-100. During a perfusion with the cholecystokinin analogue ceruletid (10(-8) M) we found an increase of lipase (p < 0.05) after 30 minutes and an increase of amylase (p < 0.05) after 50 minutes perfusion. CONCLUSIONS: The isolated perfused rat pancreas is a valuable experimental model to investigate the early phase of pathophysiology in acute pancreatitis.

Acute Disease↗

PubMed2001

Oxytosis: A novel form of programmed cell death.

Extensive nerve cell death occurs during the development of the central nervous system as well as in episodes of trauma and in neurodegenerative disease. The mechanistic details of how these cells die are poorly understood. Here we describe a unique oxidative stress-induced programmed cell death pathway called oxytosis, and outline pharmacological approaches which interfere with its execution. Oxidative glutamate toxicity, in which exogenous glutamate inhibits cystine uptake through the cystine/glutamate antiporter leading to a depletion of glutathione, is used as an example of oxytosis. It is shown that there is a sequential requirement for de novo macromolecular synthesis, lipoxygenase activation, reactive oxygen species production, and the opening of cGMP-gated channels which allow the influx of extracellular calcium. The translation initiation factor elF2alpha plays a central role in this pathway by regulating the levels of glutathione. Finally, examples are given in which the reduction in glutathione, the production of reactive oxygen species, and calcium influx can be experimentally manipulated to prevent cell death. Data are reviewed which suggest that oxytosis may be involved in nerve cell death associated with nervous system trauma and disease.

Apoptosis↗

PubMed2000

Identification of mating-type dependent genes by non-radioactive, arbitrarily primed PCR in Schizophyllum commune.

Using arbitrary primers for a PCR reaction on cDNA from different mating interactions, genes differentially expressed in a mating-type dependent manner have been identified. The cDNAs were prepared from mRNA extracted from liquid grown cultures of the fungus in order to prevent further steps in development and increase the possibility of identifying genes directly under control of the mating type system. A reaction using a monokaryotic strain was compared to a semicompatible mating reaction induced for B-regulated development and a reaction using a fully compatible mating. In the comparison of silver-stained, non-denaturing polyacrylamide gel separation of the PCR products bands differentially detected between the samples were cloned and subjected to further analysis. Bands were characterized which were present in both mating reactions but absent in the monokaryon while the third band was specifically absent in the fully compatible mating interaction. Sequence analysis allowed design of specific primers for verification of differential expression in a semi-quantitative PCR. Thus, use of radioactivity was not necessary for the fast and reproducible method identifying genes specifically regulated by mating reactions.

Cloning, Molecular↗

PubMed2000

Evidence for high affinity nickel transporter genes in heavy metal resistant Streptomyces spec.

We have isolated 25 new strains of streptomycetes from soil samples of a polluted site at the former uranium mine, Wismut, in eastern Thuringia, Germany. The strains grew on medium containing 1 mM NiCl2 and thus were resistant to the heavy metal ion. Seven of the strains were further characterized. All of these strains were resistant to heavy metals in various degrees with up to 10 mM resistance against NiCl2 supplied with the liquid minimal growth medium. The high level of resistance prompted us to look for high affinity nickel transporter genes thought to provide a means to eliminate the excess nickel ions form the cells. Degenerate oligonucleotide primers derived from sequences of P-type ATPase transporter genes of Gram negative bacteria identified a fragment which shows deduced amino acid sequence similarities to known high affinity nickel transporters. Investigation of two genes obtained from the isolates Streptomyces spec. E8 and F4 showed high sequence divergence. This was unexpected since a transmissible plasmid had been thought to convey heavy metal resistance.

Amino Acid Sequence↗

PubMed2000

Signaling by reactive oxygen species in the nervous system.

Free radicals and reactive oxygen species (ROS) are involved in a variety of different cellular processes ranging from apoptosis and necrosis to cell proliferation and carcinogenesis. Cells contain multiple sites for ROS production and a few mechanisms for their degradation. Which of these sites is activated by a given stimulus may play a role in dictating the subsequent downstream effects of the ROS generated on cellular function. Even when the ultimate outcome is similar, such as when ROS production leads to cell death, the specific cellular changes can be quite different depending on the initial stimulus and the type of cell involved. These data, along with the evidence that ROS can modify a number of intracellular signaling pathways including protein phosphatases, protein kinases and transcription factors, suggest that the majority of the effects of ROS on cells are through their actions on signaling pathways rather than via nonspecific damage of intracellular macromolecules.

Animals↗

PubMed2000

Arbuscular mycorrhiza development regulates the mRNA abundance of Mtaqp1 encoding a mercury-insensitive aquaporin of Medicago truncatula.

The genome of the model legume Medicago truncatula Gaertn. was screened for the presence of genes encoding tonoplast intrinsic proteins, and a gene family was identified. The cDNA fragments of two members of the multigene family were cloned from roots inoculated with an arbuscular mycorrhizal fungus. Transcript accumulation in roots could be detected for both cDNA fragments, but only one gene was induced in the symbiosis when compared to non-mycorrhizal control roots. A full-length cDNA clone was obtained from the arbuscular-mycorrhiza-regulated gene, and injection of in-vitro-transcribed RNA into Xenopus oocytes revealed that the encoded protein MtAQP1 specifically facilitates water transport. The possible role of MtAQP1 in buffering osmotic fluctations in the highly compartmented vacuole of arbuscule cells is discussed.

Amino Acid Sequence↗

PubMed2000

A mutation affecting the association equilibrium of formyltransferase from the hyperthermophilic Methanopyrus kandleri and its influence on the enzyme's activity and thermostability.

Formyltransferase from Methanopyrus kandleri is composed of only one type of subunit of molecular mass 32 kDa. The enzyme is in a monomer/dimer/tetramer association equilibrium, the association constant being affected by lyotropic salts. Oligomerization is required for enzyme activity and thermostability. We report here on a subunit interface mutation (R261E) which affects the dimer/tetramer part of the association equilibrium of formyltransferase. With the mutant protein it was shown that tetramerization is not required for activity but is necessary for high thermostability.

Amino Acid Substitution↗