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M Eder

Publications and source records attributed to M Eder.

At least 37 records · Page 2Linked to original sources
PubMed2000

Divergent enzyme kinetics and structural properties of the two human mitochondrial creatine kinase isoenzymes.

The mitochondrial isoenzymes of creatine kinase (MtCK), ubiquitous uMtCK and sarcomeric sMtCK, are key enzymes of oxidative cellular energy metabolism and play an important role in human health and disease. Very little is known about uMtCK in general, or about sMtCK of human origin. Here we have heterologously expressed and purified both human MtCK isoenzymes to perform a biochemical, kinetic and structural characterization. Both isoenzymes occurred as octamers, which can dissociate into dimers. Distinct Stokes' radii of uMtCK and sMtCK in solution were indicative for conformational differences between these equally sized proteins. Both human MtCKs formed 2D-crystals on cardiolipin layers, which revealed further subtle differences in octamer structure and stability. Octameric human sMtCK displayed p4 symmetry with lattice parameters of 145 A, indicating a 'flattening' of the octamer on the phospholipid layer. pH optima and enzyme kinetic constants of the two human isoenzymes were significantly different. A pronounced substrate binding synergism (Kd > Km) was observed for all substrates, but was most pronounced in the forward reaction (PCr production) of uMtCK and led to a significantly lower Km for creatine (1.01 mM) and ATP (0.11 mM) as compared to sMtCK (creatine, 7.31 mM; ATP, 0.68 mM).

Creatine Kinase↗
PubMed2000

[Value of esophageal scintigraphy in exploration of a gastro-esophageal reflux in a respiratory patient].

Patients with respiratory pathologies in the forms of tracheitis, rhino-sinusitis, and asthma sometimes have symptoms of gastro-oesophageal reflux, that should be taken into consideration in order to treat the co-factors associated with the etiopathology. However, these patients, because of their respiratory handicaps, are frequently unwilling to submit themselves to traumatic investigations. Oesophageal scintigraphy, because of its perfect tolerance and reliability, seems to be an examination that is adapted to the situation to resolve this dilemma.

Adolescent↗
PubMed2000

[Solubility and dissolution rate of digoxin from Digitalis lanata drug extracts] .

The influence on solubility and dissolution rate was investigated for digoxin as a model drug with a very low solubility in water. The investigations were carried out with different fractions of extracts from leaves of Digitalis lanata. These fractions differ in the composition of concomitant compounds. The solubility of digoxin from the extract fractions is increased up to 42 times, with considerable differences between the fractions. The solubility depends on the weight of the extract fraction; a limit of solubility exists. Even after separation of the solved extract components the solubility of digoxin in the residues is larger than that of the pure digoxin. The dissolution rate of digoxin of "Vorgereinigter Gesamtglykosidextrakt (VE)" and the glycosid fraction G 1 is influenced significantly, whereas digoxin in the glycosid fraction G 4 has such a degree of purity that the solubility properties are not influenced by the small amount of concomitant compounds. After 10 min already 50.4% of the digoxin in the extract fraction G 1 are dissolved, while only 21.7% of the pure digoxin are dissolved in that interval. The extract fractions exhibit different wettability properties, so that the increased dissolution rate could be attributed to improved wettability of the extract fractions. Physical mixtures of crystal-line digoxin and compounds of the extracts of the almost digoxin free fraction G 2 did not exert an influence on the dissolution behavior. Different batches of the extract fractions showed different solubility in spite of comparable digoxin content.

Cardiotonic Agents↗
PubMed1999

Functional analysis of a single chain chimeric alpha/beta-granulocyte-macrophage colony-stimulating factor receptor. Importance of a glutamate residue in the transmembrane region.

To analyze the function of each subunit of the receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF), GMR, we previously generated a single-chain chimeric receptor by fusion of the extracellular and transmembrane domain from the alpha-subunit (alpha-GMR) to the intracellular part of the beta-subunit (beta-GMR) introducing an additional glutamate residue at the fusion site (alpha/beta-GMR). We demonstrated the capacity of alpha/beta-GMR to bind GM-CSF with low affinity and to induce GM-CSF-dependent activation of tyrosine kinase activity and proliferation in transfected Ba/F3 cells. To further compare the functions of wild type and chimeric receptors, we now report that this alpha/beta-GMR is sufficient to mediate morphological changes, expression of alpha(4)- and beta(1)-integrin receptor subunits, and serine-phosphorylation of Akt kinase. To analyze the function of the glutamate residue at the fusion region of alpha/beta-GMR various point mutants changing this amino acid and its position were expressed in Ba/F3 cells. None of these mutants was capable of supporting GM-CSF-dependent proliferation; however, when beta-GMR was coexpressed, GM-CSF mediated short and long term proliferation. Interestingly, some mutants but not alpha/beta-GMR can induce proliferation in the presence of an anti-alpha-GMR antibody. These data demonstrate the significance of a glutamate residue in the transmembrane region of alpha/beta-GMR for ligand-induced receptor activation.

Animals↗
PubMed1999

Precisely localized LTD in the neocortex revealed by infrared-guided laser stimulation.

In a direct approach to elucidate the origin of long-term depression (LTD), glutamate was applied onto dendrites of neurons in rat neocortical slices. An infrared-guided laser stimulation was used to release glutamate from caged glutamate in the focal spot of an ultraviolet laser. A burst of light flashes caused an LTD-like depression of glutamate receptor responses, which was highly confined to the region of "tetanic" stimulation (<10 micrometers). A similar depression of glutamate receptor responses was observed during LTD of synaptic transmission. A spatially highly specific postsynaptic mechanism can account for the LTD induced by glutamate release.

Animals↗
PubMed1999

Monitoring of BCR-ABL expression using real-time RT-PCR in CML after bone marrow or peripheral blood stem cell transplantation.

To analyze the value of real time RT-PCR for monitoring of bcr-abl expression in CML patients after allogeneic or autologous stem cell transplantation (SCT), we generated pairs of PCR-primers and TaqMan probes specific for either the b2a2- or the b3a2-variant of bcr-abl. Either variant could be detected specifically from cDNA from a single K562 (b3a2) and BV173 (b2a2) cell with the respective TaqMan probe. Bcr-abl expression was normalized by comparison with GAPDH expression, and samples were quantitated using standard cDNA dilutions from K562 or BV173 cells. In a retrospective analysis 13 patients with CML after allogeneic (n = 10) or autologous (n = 3) SCT including patients with relapsed or persistent CML were analyzed by both real-time and conventional nested RT-PCR. In addition chimerism was monitored by FISH analysis of sex chromosomes in three patients with relapsed disease. The bcr-abl/GAPDH ratio dropped at least 1000-fold in all seven patients evaluable prior to and after allogeneic SCT as estimated by real-time RT-PCR, and conventional RT-PCR became negative in 6/7 patients. In five patients with relapsed or persistent disease after allogeneic SCT the bcr-abl/GAPDH ratio eventually increased again, and real-time RT-PCR was as sensitive as conventional RT-PCR for detection of bcr-abl. Donor lymphocyte infusions (DLI) were given to all five patients, and the bcr-abl/GAPDH ratio dropped to undetectable levels in two patients both remaining in continuing molecular remission. In contrast, in three other patients the bcr-abl/GAPDH ratio decreased only or did not change significantly after DLI. In three patients undergoing autologous SCT the bcr-abl/GAPDH ratio dropped only 1.1 to 30-fold, and the patients were tested positive with real-time RT-PCR at all time points. These data demonstrate that real-time RT-PCR is valuable to quantitate bcr-abl expression in CML patients after transplantation.

Bone Marrow Transplantation↗
PubMed1999

Crystal structure of brain-type creatine kinase at 1.41 A resolution.

Excitable cells and tissues like muscle or brain show a highly fluctuating consumption of ATP, which is efficiently regenerated from a large pool of phosphocreatine by the enzyme creatine kinase (CK). The enzyme exists in tissue--as well as compartment-specific isoforms. Numerous pathologies are related to the CK system: CK is found to be overexpressed in a wide range of solid tumors, whereas functional impairment of CK leads to a deterioration in energy metabolism, which is phenotypic for many neurodegenerative and age-related diseases. The crystal structure of chicken cytosolic brain-type creatine kinase (BB-CK) has been solved to 1.41 A resolution by molecular replacement. It represents the most accurately determined structure in the family of guanidino kinases. Except for the N-terminal region (2-12), the structures of both monomers in the biological dimer are very similar and closely resemble those of the other known structures in the family. Specific Ca2+-mediated interactions, found between two dimers in the asymmetric unit, result in structurally independent heterodimers differing in their N-terminal conformation and secondary structure. The high-resolution structure of BB-CK presented in this work will assist in designing new experiments to reveal the molecular basis of the multiple isoform-specific properties of CK, especially regarding different subcellular locations and functional interactions with other proteins. The rather similar fold shared by all known guanidino kinase structures suggests a model for the transition state complex of BB-CK analogous to the one of arginine kinase (AK). Accordingly, we have modeled a putative conformation of CK in the transition state that requires a rigid body movement of the entire N-terminal domain by rms 4 A from the structure without substrates.

Adenosine Triphosphate↗
PubMed1998

Substrate activation by acyl-CoA dehydrogenases: transition-state stabilization and pKs of involved functional groups.

The mechanism by which acyl-CoA dehydrogenases initiate catalysis was studied by using p-substituted phenylacetyl-CoAs (substituents-NO2, -CN, and CH3CO-), 3S-C8-, and 3'-dephospho-3S-C8CoA. These analogues lack a beta C-H and cannot undergo alpha,beta-dehydrogenation. Instead they deprotonate at alpha C-H at pH > or = 14 to form delocalized carbanions having strong absorbancies in the near UV-visible spectrum. The pKas of the corresponding phenylacetone analogues were determined as approximately 13.6 (-NO2), approximately 14.5 (-CN), and approximately 14.6 (CH3CO-). Upon binding to human wild-type medium-chain acyl-CoA dehydrogenase (MCADH), all analogues undergo alpha C-H deprotonation. While the extent of deprotonation varies, the anionic products from charge-transfer complexes with the oxidized flavin. From the pH dependence of the dissociation constants (Kd) of p-NO2-phenylacetyl-CoA (4NPA-CoA), 3S-C8-CoA, and 3'-dephospho-3S-C8CoA, four pKas at approximately 5, approximately 6, approximately 7.3, and approximately 8 were identified. They were assigned to the following ionizations: (a) pKa approximately 5, ligand (L-H) in the MCADH approximately ligand complex; (b) pKa approximately 6, Glu376-COOH in uncomplexed MCADH; (c) pKa approximately 7.3, Glu99-COOH in uncomplexed MCADH (Glu99 is a residue that flanks the bottom of the active-center cavity; this pK is absent in the mutant Glu99Gly-MCADH); and (d) pK approximately 8, Glu99-COOH in the MCADH approximately 4NPA-CoA complex. The pKa approximately 6 (b) is not significantly affected in the MCADH approximately 4NPA-CoA complex, but it is increased by > or = 1 pK unit in that with 3S-C8CoA and further in the presence of C8-CoA, the best substrate. The alpha C-H pKas of 4NPA-CoA, of 3S-C8-CoA, and of 3'-dephospho-3S-C8CoA in the complex with MCADH are approximately 5, approximately 5, and approximately 6. Compared to those of the free species these pKa values are therefore lowered by 8 to > or = 11 pH units (50 to > or = 65 kJ mol-1) and are close to the pKa of Glu376-COOH in the complex with substrate/ligand. This effect is ascribed mainly to the hydrogen-bond interactions of the thioester carbonyl group with the ribityl-2'-OH of FAD and Glu376-NH. It is concluded that the pKa shifts induced with normal substrates such as n-octanoyl-CoA are still higher and of the order of 9-13 pK units. With 4NPA-CoA and MCADH, alpha C-H abstraction is fast (kapp approximately 55 s-1 at pH 7.5 and 25 degrees C, deuterium isotope effect approximately 1.34). However, it does not proceed to completion since it constitutes an approach to equilibrium with a finite rate for reprotonation in the pH range 6-9.5. The extent of deprotonation and the respective rates are pH-dependent and reflect apparent pKas of approximately 5 and approximately 7.3, which correspond to those determined in static experiments.

Acetyl Coenzyme A↗
PubMed1998

Some new aspects of creatine kinase (CK): compartmentation, structure, function and regulation for cellular and mitochondrial bioenergetics and physiology.

Creatine kinase (CK) isoenzymes, specifically located at places of energy demand and energy production, are linked by a phosphocreatine/creatine (PCr/Cr) circuit, found in cells with intermittently high energy demands. Cytosolic CKs, in close conjunction with Ca(2+)-pumps, play a crucial role for the energetics of Ca(2+)-homeostasis. Mitochondrial Mi-CK, a cuboidal-shaped octamer with a central channel, binds and crosslinks mitochondrial membranes and forms a functionally coupled microcompartment with porin and adenine nucleotide translocase for vectorial export of PCr into the cytosol. The CK system is regulated by AMP-activated protein kinase via PCr/Cr and ATP/AMP ratios. Mi-CK stabilizes and cross-links cristae- or inner/outer membranes to form parallel membrane stacks and, if overexpressed due to creatine depletion or cellular energy stress, forms those crystalline intramitochondrial inclusions seen in some mitochondrial cytopathy patients. Mi-CK is a prime target for free radical damage by peroxynitrite. Mi-CK octamers, together with CK substrates have a marked stabilizing and protective effect against mitochondrial permeability transition pore opening, thus providing a rationale for creatine supplementation of patients with neuromuscular and neurodegenerative diseases.

Adenine Nucleotides↗
PubMed1998

Locust dorsal unpaired median (DUM) neurones directly innervate and modulate hindleg proprioceptors

A subgroup of the large efferent octopaminergic dorsal unpaired median (DUM) neurones of the third thoracic ganglion, the DUM3,4,5 neurones, directly innervates the tendons of certain proprioceptors of the locust hindleg, the so-called strand receptors. The terminals of the DUM neurones occur in regions of the strands that also contain the dendrites of the mechanoreceptive sensory cells. Both stimulation of the DUM3,4,5 neurones and bath application of octopamine change the responses of strand receptor units to mechanical stimulation. In both situations, most single strand receptor units show an increased response to mechanical stimulation. Some units, however, decrease their sensitivity to mechanical stimulation in response to octopamine application or DUM neurone stimulation.

Journal Article↗
PubMed1998

[The importance of concomitant compounds in plant extracts].

Plant extracts are multi-composed mixtures that can be subdivided into main active substances and concomitant compounds. Concomitant compounds are called co-effectors because they can change the physicochemical properties of the main active substances and therefore influence the biopharmaceutical parameters, e.g. solubility and bioavailability. The composition and the properties of plant extracts depend on a multitude of different factors such as quality and degree of reduction of the plant, and the process of extraction and drying. Concomitant compounds affect the physical and chemical stability of plant extracts. The hygroscopicity also depends on the composition of the concomitant compounds. It is an important property for the further processing of the plant extract to solid dosage form, e.g. tablets. Solubility and dissolution rate can be influenced by the concomitant compounds. Both properties are important for the absorption of the active substances though in many cases the responsible substances and mechanisms are still unknown. Solubilisation by surface-active agents, formation of soluble drug-concomitant compound-associates and solid dispersion, in which the drug is finely dispersed or X-ray amorphous, are often responsible for the increased solubility. Improved wettability in the presence of the concomitant compounds results in better dissolution. Supersaturated solutions of the active substances are stabilized for months by inhibition of crystallization. Numerous examples are known for influencing the absorption by natural concomitant compounds. The absorption is enhanced by the increased amount of dissolved drug or by the alteration of membrane permeability. Adsorption or complex formation with the concomitant compounds decrease the absorption rate, in some cases also the extent of absorption.

Animals↗
PubMed1998

Functional aspects of the X-ray structure of mitochondrial creatine kinase: a molecular physiology approach.

Mitochondrial creatine kinase (Mi-CK) is a central enzyme in energy metabolism of tissues with high and fluctuating energy requirements. In this review, recent progress in the functional and structural characterization of Mi-CK is summarized with special emphasis on the solved X-ray structure of chicken Mib-CK octamer (Fritz-Wolf et al., Nature 381, 341-345, 1996). The new results are discussed in a historical context and related to the characteristics of CK isoforms as known from a large number of biophysical and biochemical studies. Finally, two hypothetical functional aspects of the Mi-CK structure are proposed: (i) putative membrane binding motifs at the top and bottom faces of the octamer and (ii) a possible functional role of the central 20 A channel.

Amino Acid Sequence↗
PubMed1997

Characterization of human and pig kidney long-chain-acyl-CoA dehydrogenases and their role in beta-oxidation.

Long-chain-acyl-CoA dehydrogenase (LCADH) has been produced by recombinant techniques from the human cDNA and purified after expression in Escherichia coli. Pig kidney LCADH was purified using an optimized method which also produces apparently pure short-chain-acyl-CoA dehydrogenase (SCADH) and medium-chain-acyl-CoA dehydrogenase (MCADH) in good yields. LCADH from both sources has a maximal turnover rate (Vmax of 650-700 min(-1) at pH 7.6) with the best substrates, which is approximately fivefold higher than reported previously. The human enzyme has an approximately fivefold higher Km compared with the pig kidney enzyme with substrates of chain length from C10 to C18 and a significantly different dependence of Vmax on the chain length. Pig kidney LCADH has a similar Vmax/Km with C10 to C14 substrates as MCADH does with C6 to C10 substrates. Recombinant human LCADH, however, is significantly less efficient (approximately fourfold with C12) than purified pig kidney enzyme. We conclude that human LCADH is either quantitatively less important in beta-oxidation than in the pig, or that post-translational modifications, not present in the recombinant human enzyme, are required to optimize human LCADH activity. Our results demonstrate that LCADH is as important as the other acyl-CoA dehydrogenases in fatty acid oxidation at physiological, mitochondrial pH with optimal substrates of chain length C10-C14. The extent of the LCADH-flavin cofactor reduction observed with most substrates and the rate of the subsequent reoxidation with oxygen are markedly different from those found with human medium chain acyl-CoA dehydrogenase. Both LCADH are inactivated by the substrate analogue 2-octynoyl-CoA, possibly via covalent modification of Glu261, the active-site residue involved in deprotonation of the substrate (alpha)C-H.

Acyl-CoA Dehydrogenase↗
PubMed1997

IL-3 in the clinic.

Since the cloning of human interleukin 3 (IL-3) in 1986 [1] and the demonstration of its proliferative effects on multiple hematopoietic progenitor cells, IL-3 has been widely studied to treat different states of bone marrow failure or hematologic malignancies, to mobilize or expand hematopoietic progenitor cells for transplantation, and to support engraftment after bone marrow transplantation. However, no condition for the clinical use of IL-3 has been established so far despite its theoretical advantages as an early-acting cytokine and in contrast to erythropoietin (EPO), G-CSF, or GM-CSF all of which have already been approved for several clinical modalities. Here we shortly review our current knowledge about the effects of IL-3 on the molecular and cellular level, summarize recent clinical studies with IL-3, and discuss further perspectives for the use of this cytokine.

Cells, Cultured↗
PubMed1997

Comparison of the effects of different low molecular weight heparins on the hemostatic system activation in vivo in man.

In a double-blind, randomized, cross-over study the effects of single subcutaneous doses of 120 anti-Xa units/kg body wt. of three different low molecular weight heparin (LMWH) preparations were investigated in 15 healthy subjects by determination of thrombin-antithrombin III complex (TAT), prothrombin fragment 1.2 (f1.2), and beta-thromboglobin (beta-TG) in shed blood and in venous blood. Certoparin, dalteparin, and enoxaparin significantly inhibited coagulation activation marker formation in shed blood. The substantial inhibition of TAT and f1.2 formation was slightly more pronounced in response to certoparin. beta-TG was decreased following certoparin and enoxaparin, but not following dalteparin. However, no difference between groups was detectable. A small but consistent decrease of f1.2 formation in venous blood was noted for all LMWHs and dalteparin and enoxaparin, but not certoparin, inhibited TAT formation. Only a minor impact of the three LMWH preparations was noted on beta-TG plasma concentrations. Our data indicate that the studied LMWH preparations have a major impact on blood clotting in the activated state and inhibit in vivo the hemostatic system to a comparable extent.

Adult↗
PubMed1996

Defective activation of mitogen-activated protein kinase after allogeneic bone marrow transplantation.

Allogeneic bone marrow transplant (BMT) recipients have increased susceptibility to infections for prolonged periods after phenotypic reconstitution of donor cells. This immunodeficiency status is characterized by multiple T-cell functional abnormalities. This study was designed to investigate several signaling pathways involved in T-cell activation during this period of immune deficiency. In initial experiments using equal numbers of CD3+ cells or highly purified T-cell subpopulations obtained from normal controls and BMT recipients, we confirmed that abnormal T-cell proliferation after CD3 cross-linking, phytohemagglutinin stimulation, or phorbol myristate acetate (PMA) stimulation of peripheral blood mononuclear cells from BMT recipients was due to a qualitative T-cell deficiency rather than to low numbers of circulating T cells. We next investigated the ability of the T-cell receptor/CD3 complex to transduce signals via receptor-associated protein tyrosine kinases. In all BMT recipients, CD3 cross-linking induced protein tyrosine phosphorylation of several proteins in a similar fashion to that seen in controls, including phosphorylation of a 21-kD protein that represents the zeta subunit of the receptor itself. Further investigation showed that CD3 cross-linking and PMA stimulation did not increase 42-44-kD mitogen-activated protein kinase (MAPK) activity. The failure of MAPK activation in BMT recipients occurred despite tyrosine phosphorylation of the 42-44-kD proteins, which, in normal controls, parallels enzyme activation. Our results indicate that T-cell immunodeficiency in BMT recipients is associated with a selective failure of MAPK activation, possibly related to abnormal posttranslational positive regulation of this enzyme.

Adult↗