Biomedical subjects

E Bürger

Publications and source records attributed to E Bürger.

13 recordsLinked to original sources

PubMed2001

The neuroprotective actions of FK506 binding protein ligands: neuronal survival is triggered by de novo RNA synthesis, but is independent of inhibition of JNK and calcineurin.

The immunosuppressant FK506 displays substantial neuroprotective and neuroregenerative effects. It is not fully understood to which extent these effects depend on the inhibition of the calcineurin phosphatase (PP2B). The present study has re-addressed this issue using Lie120, a novel highly specific inhibitor of calcineurin, which does not block the enzymatic activity of FKBPs or cyclophilins, respectively. We have determined the effect of FK506 (10-500 nM), V-10,367 (a FK506 derivative which does not block calcineurin; 1-5 microM) and Lie120 (a novel specific inhibitor of calcineurin, 0.1-5 microM) on the cellular survival and the pro-degenerative JNK activity of PC12 and Neuro2A cells following application of 200 microM H(2)O(2). FK506 and V-10,367, but not Lie120, protected both cell lines against H(2)O(2)-mediated death, whereas an increase in JNK1 activity was blocked by FK506 and Lie120, but not by V-10,367. Co-incubation of FK506 and V-10,367 with the mRNA synthesis inhibitor actinomycin D abolished the protective effect of FK506 and V-10,367. This antagonization was effective when actinomycin D was applied 30 min or 1 h, but not 2 or 4 h, after H(2)O(2) suggesting that FKBP-ligands confer their neuroprotection by rapid de novo synthesis of (functionally) anti-apoptotic proteins. The search for the corresponding effector genes revealed that the expression of FKBP25, FKBP38 and FKBP52 (analysis by reverse transcription-polymerase chain reaction (RT-PCR) did not change following H(2)O(2) or FK506, and this was also true for the expression of apoptosis-related genes caspase 3, bax, bcl-2 and bcl-xL (analysis by Multiplex-PCR). Summarizing, neuronal protection by FKBP-ligands is not mediated either by calcineurin or by JNK1 in this experimental set-up, whereas the FK506 mediated inhibition of JNK1 is realized by the inhibition of calcineurin, an effective activator of JNK1 in neurons.

Animals↗

PubMed2000

The immunophilin ligand FK506, but not GPI-1046, protects against neuronal death and inhibits c-Jun expression in the substantia nigra pars compacta following transection of the rat medial forebrain bundle.

The immunophilin ligand FK506 (Tacrolimus) is used for prevention of graft rejection following organ transplantation. FK506 is a high-affinity ligand for FK506-binding proteins, an immunophilin subgroup of peptidyl-prolyl-cis/trans-rotamases abundant in the mammalian brain. Here, we demonstrate that FK506 is a potent survival factor that prevents neuronal cell death following axotomy of central intrinsic neurons. Administration of FK506 (2 mg/kg, s.c., per day for two days pre-axotomy and for up to eight days post-axotomy) effectively delayed and reduced the death of axotomized neurons in the substantia nigra pars compacta following transection of the medial forebrain bundle. In saline-treated controls, 75%, 89% and 92% of nigral neurons died after 25, 50 and 60 days post-axotomy, respectively. In contrast, application of FK506 resulted in survival of 46%, 44% and 28% of the axotomized nigral neurons, and the majority of these surviving neurons showed continuous expression of tyrosine hydroxylase, the pacemaker enzyme for dopamine synthesis. Moreover, FK506 significantly reduced the expression of the inducible transcription factor c-Jun and its N-terminal phosphorylation and prevented the axotomy-induced suppression of the constitutive transcription factor ATF-2 in neurons of the substantia nigra and mammillary body. The latter is also axotomized by the coincident transection of the mammillothalamic tract, but the mammillary neurons survive the axotomy. In contradistinction to FK506, the non-immunosuppressive FK506-binding protein ligand GPI-1046 (25 or 12.5 mg/kg, applied once or twice per day for two days pre-axotomy and for eight days post-axotomy) was completely ineffective for all these parameters investigated. Finally, FK506, but not GPI-1046, impressively accelerated the recovery from surgery. Our data provide the first evidence that FK506 acts as a neuroprotective molecule that rescues axotomized otherwise degenerating central intrinsic neurons in the adult mammalian brain by mechanisms that interfere with the transcriptional program of the axotomy-induced cell body response, such as activating transcription factor-2 suppression and c-Jun expression and phosphorylation.

Animals↗

PubMed1997

Role of prohormone convertases in pro-neuropeptide Y processing: coexpression and in vitro kinetic investigations.

Proneuropeptide Y (ProNPY) undergoes cleavage at a single dibasic site Lys38-Arg39 resulting in the formation of 1-39 amino acid NPY which is further processed successively by carboxypeptidase-like and peptidylglycine alpha-amidating monooxygenase enzymes. To investigate whether prohormone convertases are involved in ProNPY processing, a vaccinia virus derived expression system was used to coexpress recombinant ProNPY with each of the prohormone convertases PC1/3, PC2, furin, and PACE4 in Neuro2A and NIH 3T3 cell lines as regulated neuroendocrine and constitutive prototype cell lines, respectively. The analysis of processed products shows that only PC1/3 generates NPY in NIH 3T3 cells while both PC1/3 and PC2 are able to generate NPY in Neuro2A cells. The convertases furin and PACE4 are unable to process ProNPY in either cell line. Moreover, comparative in vitro cleavage of recombinant NPY precursor by the enzymes PC1/3, PC2 and furin shows that only PC1/3 and PC2 are involved in specific cleavage of the dibasic site. Kinetic studies demonstrate that PC1/3 cleaves ProNPY more efficiently than PC2. The main difference between the cleavage efficiency is observed in the Vmax values whereas no major difference is observed in Km values. In addition the cleavage by PC1/3 and PC2 of two peptides reproducing the dibasic cleavage site with different amino acid sequence lengths namely (20-49)-ProNPY and (28-43)-ProNPY was studied. These shortened ProNPY substrates, when recognized by the enzymes, are more efficiently cleaved than ProNPY itself. The shortest peptide is not cleaved by PC2 while it is by PC1/3. On the basis of these observations it is proposed, first, that the constitutive secreted NPY does not result from the cleavage carried out by ubiquitously expressed enzymes furin and PACE4; second, that PC1/3 and PC2 are not equipotent in the cleavage of ProNPY; and third, substrate peptide length might discriminate PC1/3 and PC2 processing activity.

3T3 Cells↗

PubMed1993

A tetrameric complex of membrane proteins in the endoplasmic reticulum.

The translocation site (translocon), at which nascent polypeptides pass through the endoplasmic reticulum membrane, contains a component previously called 'signal sequence receptor' that is now renamed as 'translocon-associated protein' (TRAP). Two glycosylated subunits of the TRAP complex have been identified before (alpha and beta subunits). We now show that TRAP complex is actually comprised of four membrane proteins (alpha, beta, gamma, delta), present in a stoichiometric relation, which are genuine neighbours in intact microsomes. The amino acid sequences of the additional, non-glycosylated subunits were deduced from cloning of the corresponding cDNAs. The delta subunit spans the membrane only once and has its major portion, containing a disulfide bridge, at the lumenal side. The gamma subunit is predicted to span the membrane four times.

Amino Acid Sequence↗

PubMed1993

Establishment of a cellular assay system for G protein-linked receptors: coupling of human NK2 and 5-HT2 receptors to phospholipase C activates a luciferase reporter gene.

A functional cellular assay system was developed for the detection of substances modulating the activity of G protein-coupled receptors, linked to the phospholipase C second messenger system. The human adenocarcinoma cell line A549 was transformed with the Photinus pyralis luciferase gene under the control of the ICAM-1 gene 5'regulatory region and, subsequently, stably transfected with the human neurokinin 2 (NK2) receptor gene. The ICAM-1 promoter is known to be inducible via the phospholipase C signal transduction pathway. In this NK2 receptor test cell line, expression of luciferase was inducible by neurokinin A and other NK2-specific agonists. The order of potency of the three neurokinins substance P, neurokinin A and neuromedin K was consistent with published data and results from ligand binding studies performed with the same NK2 test cell line. The agonistic effect of neurokinin A could be inhibited in a dose-dependent manner by simultaneous addition of NK2-specific antagonists or protein kinase C-inhibitors. Similarly, a stable test cell line expressing the human serotonin 2 receptor was established. Agonist-induced luciferase expression in this cell line was abolished in the presence of 5-HT2-specific antagonists. These cellular assay systems can be employed for the identification of competitive, non-competitive and allosteric modulators of the NK2 and the 5-HT2 receptor, and they represent prototypes for analogous test cell lines for other phospholipase C-coupled receptors.

Biological Assay↗

PubMed1993

Interrelations between electrical and biochemical processes in ischemic porcine livers at low temperature.

Recently it has been shown that during liver ischemia at 25 degrees C the presence of glycogen, by supporting glycolytic supply, not only retards ATP decay but also leads to a corresponding delay of the rise of the electrical impedance of the ischemic organ. A sudden rise of impedance during ischemia is supposed to indicate the closure of gap junctions. Although similar effects on energy state do exist at low temperature, the impact of glycogen on the electrical impedance under storage conditions has still to be evaluated. Therefore, in a model with protected porcine livers, we examined the intraischemic effects of a preischemic glucose and potassium feeding on impedance changes, lactate production and ATP-content at a storing temperature of 5 degrees C. Impedance was measured both in the low frequency alpha- and the higher frequency beta-dispersion range. In addition, the same parameters were determined in a group of unprotected livers. In this group all animals had received glucose and potassium orally prior to ischemia. Whereas in case of preischemic glucose feeding the rise of impedance in the range of the beta-dispersion (e.g. 5kHz) roughly coincided with the exhaustion of ATP, the corresponding impedance changes in the protected group without a glucose premedication only occurred when glycolysis had already stopped and ATP had reached basal values for some hours. In contrast, in the alpha-dispersion range the impedance changes in the latter group just began at the time when ATP became exhausted and lactate production ceased.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗

PubMed1991

Influence of tissue acidification and halothane anesthesia on hepatic electrical and biochemical properties during ischemia.

In order to further corroborate the recent findings on liver ischemia after perfusion with solutions containing an additive of heptanol, that the intraischemic loss of cell-to-cell communication in protected livers can be detected by electrical impedance measurement (1), we tried to induce uncoupling in porcine liver by tissue acidification applying acidified protective solutions. Moreover, the effects of preischemic inhaling of high concentrations of the decoupling anesthetic halothane were examined in unprotected ischemic livers (2-5). Electrical impedance, biochemical analyses, and pH measurements were applied in parallel. In addition, typical time courses of impedance parameters of unprotected liver and skeletal muscle were compared, because the latter is devoid of gap junctions. In spite of overlapping side-effects of the respective uncoupling measure, the results suggest that the loss of cell-to-cell communication in the liver is associated with measureable effects on the electrical impedance.

Acid-Base Equilibrium↗

PubMed1990

Heptanol effects on protected livers.

Heptanol, an agent known for inducing closure of gap junctions in a variety of organs, was used to evaluate the influence of uncoupling on the electrical impedance of livers during ischemia. Heptanol was added to a modified HTK solution or to Belzer's UW-CSS solution. Livers of swine were then perfused for 8 min with either one of the solutions containing heptanol or a solution devoid of this additive. During the following ischemia the phase angle of impedance at 5 kHz, pH and different biochemical parameters were determined. Heptanol fundamentally changed the time course of impedance and made the otherwise characteristic fast increase of the phase angle of impedance disappear. Already early during ischemia the phase angle was raised in a dose-dependent manner up to even highest values at the beginning of the whole observation period in case of a fully developed effect. Heptanol also stimulated anaerobic energy turnover. The results suggest that, besides unspecific effects, heptanol induces uncoupling which is detectable by electrical impedance measurement.

Alcohols↗

PubMed1988

Peptide hormones and neuropeptides. Proteolytic processing of the precursor regulatory peptides.

Regulatory peptides are initially synthesized as large protein precursors which must undergo proteolytic processing to yield the biologically active peptide. The proteolytic enzymes involved in this post-translational processing remove the signal peptide, a hydrophobic peptide located at the amino-terminus of the precursor. Further processing steps are the cleavage at single or two adjacent basic residues by endoproteases, followed by a removal of basic residues located at the C-terminus of the peptides by a carboxypeptidase-like enzyme. The selective processing may play an important role in determining which peptides are finally released by the cell. This article reviews the selectivities of various enzymes known to play a role in the proteolytic processing of various regulatory peptides.

Animals↗

PubMed1987

[Long-term stability of enzymes in solution].

Two proteases (thermitase, a thermostable serine protease from Thermoactinomyces vulgaris and subtilisin Carlsberg) and one non-proteolytic enzyme (urate oxidase from Penicillium spp.) were used for revealing the main influences leading to the inactivation of enzyme preparations during their long-time storage at low temperatures. The temperature dependences (0 degree C-60 degrees C) of inactivation resulted in a linear Arrhenius-plot for each of the three native enzymes as well as in the presence of all stabilizing substances tested. Therefore, a method is available which shortens the time considerably needed for the experiments aimed at the discovery of substances stabilizing enzymes under storage conditions (i.e. long time at low temperatures). Because of the linear Arrhenius-plots potential stabilizers can be tested experimentally at suitable higher temperatures and one can extrapolate on their influence on the enzyme at storage temperatures (0 degree C-10 degrees C). By using this method effective combinations of stabilizers for urate oxidase were found, and possible reasons of their stabilizing influence on the enzyme are discussed.

Cold Temperature↗

PubMed1981

Patterns of product inhibition of a bifunctional dehydrogenase; L-histidinol:NAD+ oxidoreductase.

The steady-state kinetic patterns of the bifunctional enzyme histidinol dehydrogenase from Salmonella typhimurium (EC 1.1.1.23) are compatible with a bi-uni uni-bi ping-pong mechanism. Studies of product inhibition make it possible to determine the sequence of substrate binding and product dissociation. Histidinol binds first to the enzyme, followed by the binding of NAD+; histidine is the last product to dissociate from histidinol dehydrogenase. Five of ten kinetic constants defined are determined from linear intercept and slope replots; Km for histidinol was found to be 16 +/- 3 microM and for NAD+ 1 +/- 0.3 mM; K2 for NAD+ was determined to be 0.8 +/- 0.4 mM and K3 for NADH to be 0.3 +/- 0.07 mM. K1 for histidine was found to be 2.1 +/- 0.5 mM.

Alcohol Oxidoreductases↗

PubMed1981

Evidence for an essential lysine at the active site of L-histidinol:NAD+ oxidoreductase; a bifunctional dehydrogenase.

Histidinol dehydrogenase (EC 1.1.1.23) from Salmonella typhimurium is inhibited by formaldehyde and pyridoxal 5-phosphate (pyridoxal-P). epsilon-Pyridoxyl-lysine is isolated upon acid hydrolysis of pyridoxal-P-treated enzyme reduced by sodium borohydride. In the presence of formylhistidinol and formylhistidine (specific ligands of the enzyme) inactivation of histidinol dehydrogenase by pyridoxal-P is prevented. Extrapolation of the initial part of the inactivation curve caused by pyridoxal-P indicates that modification of two essential lysine residues results in inactivation of the dimeric enzyme. The essential lysine residues appear to participate in the reversible oxidation/reduction reaction converting histidinol to histidinal.

Alcohol Oxidoreductases↗

PubMed1979

The catalytically active form of histidinol dehydrogenase from Salmonella typhimurium.

The active-enzyme-sedimentation procedure was used to identify the catalytically competent form of histidinol dehydrogenase (EC 1.1.1.23) isolated from Salmonella typhimurium. At pH 9.4 the active species has a sedimentation coefficient S20,W of 5.4S, indicating that the dimer with a mol.wt. of approx. 83 000 is the enzymically active form.

Alcohol Oxidoreductases↗