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Biomedical subjects

A Stier

Publications and source records attributed to A Stier.

At least 19 recordsLinked to original sources
PubMed1995

Temperature dependence of Ca2+ wave properties in cardiomyocytes: implications for the mechanism of autocatalytic Ca2+ release in wave propagation.

Digital imaging microscopy of fluo-3 fluorescence was used to study the velocity and shape of intracellular Ca2+ waves in isolated rat cardiomyocytes as a function of temperature. Decreasing the temperature from 37 to 17 degrees C reduced the longitudinal wave velocity by a factor of 1.8 and remarkably slowed the decay of [Ca2+]i in the trailing flank of a wave. Using image analysis, rise times, and half-maximum decay times of local Ca2+ transients, which characterize the processes of local Ca2+ release and removal, were determined as a function of temperature. Apparent activation energies for wave front propagation, local Ca2+ release, and local Ca2+ removal were derived from Arrhenius plots and amounted to -23, -28, and -46 kJ/mol, respectively. The high activation energy of Ca2+ removal, which arises from the activity of the sarcoplasmic reticulum (SR) Ca2+ ATPase, relative to those of longitudinal wave propagation and local Ca2+ release excludes the hypothetical mechanism of regenerative "spontaneous Ca2+ release," in which Ca2+ that has been taken up from the approaching wavefront triggers Ca2+ release at a luminal site of the SR. It is consistent, however, with the hypothesis that Ca2+ wave propagation is based on Ca(2+)-induced Ca2+ release where Ca2+ triggers release on the cytosolic face of the SR.

Aniline Compounds↗
PubMed1994

Heterogeneity in rabbit liver cytochrome P-450 LM2 observed by cation exchange HPLC: partial biochemical characterization of the two major LM2 subfractions.

Cytochrome P450 LM2 (CYPIIB4) from phenobarbital-induced rabbit liver microsomes, purified to only one band in SDS-PAGE, was further resolved in five peaks by cation exchange HPLC. The two major peaks were partially characterized. Both of them have the amino terminal sequence Met-Glu and the same Cys content. They exhibited the same spectral absorption maximum and similar binding constants for 1-benzylimidazole and imidazole. However, binding of benzphetamine was different. One subfraction presented a Michaelis-Menten type binding curve, but the other presents a non-typical one with an additional high affinity binding site. These subfractions of cytochrome P450 LM2 slightly differed in their catalytic activities with benzyloxy- and pentoxyresorufin substrates. On the contrary, no heterogeneity was observed for P450 LM4.

Animals↗
PubMed1994

Conformational analysis of mitochondrial and microsomal cytochrome P-450 by resonance Raman spectroscopy.

Mitochondrial and microsomal cytochromes P-450SCC and P-450LM2 in the ferric substrate-free and substrate-bound states were studied by resonance Raman spectroscopy. In the spectra of cytochrome P-450SCC two conformational states (A and B) were detected, each of them constituting an equilibrium between a six-coordinated low-spin and a high-spin form. Both the conformational and the spin equilibria are pH- and temperature-dependent, which is in line with previously published results [Lange, R., Larroque, C., & Anzenbacher, P. (1992) Eur. J. Biochem. 207, 69-73)]. On the basis of well-resolved resonance Raman spectra, measured at different pH and temperatures, these equilibria were analyzed quantitatively. Both low-spin configurations of A and B exhibit different band patterns in the spin state marker band region, indicating differences in the active-site structures. While in the high-spin configuration of state A the heme iron remains weakly bound by a sixth ligand, the high-spin form of state B is five-coordinated. Binding of cholesterol to cytochrome P-450SCC causes a significant population of the high-spin forms, particularly of state A (62%). On the other hand, binding of 22R-hydroxycholesterol to the substrate-free enzyme leaves the overall spin equilibrium largely unchanged, i.e., six-coordinated low spin (76% A and 24% B). In both substrate-bound complexes, interactions between the substrate and the heme lead to small but distinct differences in the resonance Raman spectra of the low-spin form of state A. In contrast to cytochrome P-450SCC, the resonance Raman spectra of microsomal cytochrome P-450LM2 provide no indications for multiple conformers at 22 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex↗
PubMed1994

X-ray diffraction studies of whole rat heart during anoxic perfusion.

Equatorial x-ray reflections were recorded from the ventricular region of whole rat heart, during the transition from normoxia to anoxia. The intensity ratio of the 1.0 and 1.1 equatorial reflections (I 1.0/I 1.1) was 2.96 during normoxic perfusion, decreasing to 0.37 after 115 minutes of anoxic perfusion and closely parallel those reported between partially dissected relaxed and rigor vertebrate heart muscle (1.2). The peak positions of these reflections both increased by ca. 5% during anoxic perfusion indicative of a lateral expansion in the sarcomere filament lattice. These results indicate that the process of anoxia leads to the condition of rigor in which the majority of the myosin cross-bridges bind to the thin filament. Using this technique global changes in whole heart structure can be studied, and due to the ease of perfusion of the heart, biochemical and physiological problems may be investigated in relation to the structure of the heart as a whole. This may be of clinical interest, particularly in terms of investigations into organ preservation and transplantation. This is believed to be the first occasion where intact muscular organs have been studied in this way.

Animals↗
PubMed1994

Determination of membrane-bound fragments of cytochrome P-450 2B4.

Membrane-bound sites of cytochrome P-450 2B4 (LM2) were determined by means of two different methods, photoactivated binding of membrane phospholipids to the protein and epitope mapping by antibodies. Phospholipids bearing photoreactive labels at different distances from the their polar 'head' were used in the former case. Phosphatidylcholine labelled at the apolar end of the fatty acid chain bound only to the N-terminal region of the hemoprotein. Other phospholipids labelled nearer to the head group bound not only to the N-terminus but also to the segments 273-314 and 427-491. Epitope mapping of the domain next to the N-terminus (residues 21-119) of the isolated hemoprotein was performed with the help of a peptide-scanning method, a programmable peptide synthesis on pins followed by ELISA testing with the polyclonal antiserum against cytochrome P-450 2B4. This domain was shown to possess a considerable density of sites with high antigenic activity. No membrane-penetrating part of this domain was found except for the fragment 1-21. A model of structure of P-450 2B4 was computed by comparison with the structure of cytochrome P-450cam on the basis of an alignment of 47 cytochromes P-450 with the former hemoprotein. Major parts of the protein sequences photoreacting with the phospholipid probes, but not the antibody-reactive epitopes of the region 21-119, are located at the membrane-facing side in this model.

Amino Acid Sequence↗
PubMed1994

Binding of anti-trifluoroacetyl antibodies to isolated hepatocytes observed by digital fluorescence microscopy.

These experiments were designed to observe specific binding of fluorescein-conjugated FAB'2 secondary antibodies to epitopes on the surface of isolated hepatocytes. The hepatocytes were attached as monolayers on microscope cover slips and an antigenic adduct known to be formed during metabolism of halothane, -trifluoroacetyl-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, was exchanged into their surface. Then the monolayers of hepatocytes were incubated with primary rabbit antibodies specific for the trifluoroacetyl group. Each coverslip was mounted in a perfusion chamber on a fluorescence microscope and a set of digital fluorescence images was made. Then fluorescein-conjugated goat-anti-rabbit FAB'2 secondary antibodies were flowed over the monolayer, the perfusion chamber was washed with buffer, and a second set of digital fluorescence images was made. The difference of these two sets of images demonstrated intense fluorescence superimposed on the outline of the cells. This intense fluorescence was not observed in control experiments in which the primary antibodies were omitted.

Animals↗
PubMed1994

Anisotropic propagation of Ca2+ waves in isolated cardiomyocytes.

Digital imaging microscopy of fluor-3 fluorescence was used to study the propagation of intracellular Ca2+ waves in isolated adult rat cardiomyocytes from 17 to 37 degrees C. Ca2+ waves spread in both transverse and longitudinal direction of a myocyte. Transverse propagation was pronounced in waves starting from a focus at the edge of a myocyte and in waves following an irregular, curved path (spiral waves). For the former type of waves, propagation velocities were determined. Both transverse and longitudinal wave components propagated at constant velocity ranging from 30 to 125 micron/s. Myocytes were anisotropic with respect to wave propagation: waves propagated faster in the longitudinal than in the transverse direction. The ratio between longitudinal and transverse velocity increased from 1.30 at 17 degrees C to 1.55 at 37 degrees C. Apparent activation energies for transverse and longitudinal wave propagation were estimated to be -20 kJ/mol, suggesting that these processes are limited by diffusion of Ca2+. Direction-dependent propagation velocities are interpreted to result from the highly ordered structure of the myocytes, especially from the anisotropic arrangement of diffusion obstacles such as myofilaments and mitochondria.

Aniline Compounds↗
PubMed1994

[Jejunum pouch after total gastrectomy--clinical and scintigraphic studies of function and quality of life].

The formation of a pouch is a surgical procedure to restore the lost of reservoir function after resection of the stomach. After total gastrectomy the intestinal passage can be reconstructed by a jejunal pouch performing a Siewert/Peiper esophagojejunoplication. Regarding the postoperative quality of life we supposed that there is an advantage for this reconstruction method compared to simple esophagojejunostomy. The following study investigated whether the pouch reconstruction by jejunoplication can develop a reservoir function and therefore a better clinical course compared to jejunostomy. Without evidence of a local recurrence after total gastrectomy 18 patients with and 18 without pouch reconstruction were evaluated by alimentary scintigraphy for at least 6 months after operation. Only patients were included with a tumor stage not exceeding T2 (UICC). The results of the transit times with a solid radioactively labelled test meal were correlated with the complaints and nutritional status of the patients, evaluated with a standardized questionary for creating an individual score note. The transit in a jejunal pouch follows a linear decreasing function and is significantly slower compared to the exponential passage of the jejunostomy. Both patterns remain still significantly accelerated compared to the physiological ranges of gastric emptying. Patients with a pouch judge their postoperative individual state better than patients with simple jejunostomy indicated with an on average symptom related score note better than 3. As long as the tumor stage is associated with a beneficial prognosis and the tumor localisation allows the reconstruction by an esophagojejunoplication the formation of a pouch after total gastrectomy is recommended because of an improved transit pattern and clinical course.

Adult↗
PubMed1993

Rotation of cytochrome P450SCC (CYP11A1) in proteoliposomes studied by delayed fluorescence depolarization.

The rotational diffusion of cytochrome P450SCC (CYP45011A1) was investigated in proteoliposomes measuring the time-resolved delayed fluorescence anisotropy of diiodofluorescein iodoacetamide covalently and specifically attached to Cys264 of P450SCC. Rotation strongly depends on the lipid composition of the membrane, especially on the cardiolipin content. In proteoliposomes having a lipid composition similar to the inner membrane of bovine adrenal mitochondria P450SCC is rotating uniaxially with a relaxation time phi rot about 53 microseconds and almost no immobile P450 is present. Addition of high KCl concentration has no effect. The results and the absence of any intramembrane particles observed by freeze-fracturing indicate that P450SCC probably exists in the liposomal membrane as oligomer not penetrating the bilayer. It is suggested to bind tightly within the outer monolayer with large parts exposed to the aqueous solution surrounding the membrane.

Adrenal Glands↗
PubMed1990

NMR studies of the lipid metabolism of T47D human breast cancer spheroids.

The in vivo 31P NMR spectrum of T47D human breast cancer cells grown as spheroids shows changes in the phosphomonoester lipid precursors as a function of spheroid size. The ratio of phosphorylethanolamine (PE) to phosphorylcholine (PC) was 1.0 +/- 0.3 for 3-day-old, 150 microns spheroids. This ratio increased to 2.4 +/- 0.4 for spheroids 7 days and older and which were at least 300 microns in diameter. To investigate the phosphatidylethanolamine to phosphatidylcholine (PdylE/PdylC) ratio in the membranes, chloroform/methanol extracts of spheroids were performed. The 31P spectrum of these extracts showed no change with spheroid size, namely the PdylE/PdylC ratio was 0.5 +/- 0.06 for spheroids of all ages suggesting that membrane composition is strongly regulated at the precursor level.

Breast Neoplasms↗
PubMed1990

Transient increases of intracellular Ca2+ induced by volatile anesthetics in rat hepatocytes.

The affects of volatile anesthetics on mobilization of intracellular Ca2+ was monitored in primary cultures of rat hepatocytes using the fluorescent Ca2+ probe Fura-2. The use of Fura-2 was limited by several factors which complicated the quantitative analysis of the results, such as: (i) a high rate of dye leakage; (ii) changes in the redox state of the hepatocytes which interfered with the fluorescence produced by the dye at various excitation wavelengths; (iii) compartmentalization of the dye producing high local intracellular concentrations; and, of particular importance for this study, (iv) enhanced photobleaching of the dye in the presence of halothane. To aid in the interpretation of the Fura-2 data, the Ca2(+)-sensitive photoprotein aequorin was also used to monitor changes in [Ca2+]i. The aequorin and Fura-2 techniques qualitatively yielded the same result, that the volatile anesthetic agents halothane, enflurane, and isoflurane induce an immediate and transient increase of [Ca2+]i. The durations of these transients were approximately between 5 and 10 min and were not related to any evident acute cell toxicity. The [Ca2+]i increases induced by the volatile anesthetic agents were dose-dependent, with halothane the most potent. The exact mechanism governing these increases in [Ca2+]i induced by these anesthetics in rat hepatocytes is unknown, but is likely to involve effects on both the cell surface membrane and endoplasmic reticulum components of the signal transducing system.

Aequorin↗
PubMed1989

Resonance Raman study on the structure of the active sites of microsomal cytochrome P-450 isozymes LM2 and LM4.

The isozymes 2 and 4 of rabbit microsomal cytochrome P-450 (LM2, LM4) have been studied by resonance Raman spectroscopy. Based on high quality spectra, a vibrational assignment of the porphyrin modes in the frequency range between 100-1700 cm-1 is presented for different ferric states of cytochrome P-450 LM2 and LM4. The resonance Raman spectra are interpreted in terms of the spin and ligation state of the heme iron and of heme-protein interactions. While in cytochrome P-450 LM2 the six-coordinated low-spin configuration is predominantly occupied, in the isozyme LM4 the five-coordinated high-spin form is the most stable state. The different stability of these two spin configurations in LM2 and LM4 can be attributed to the structures of the active sites. In the low-spin form of the isozymes LM4 the protein matrix forces the heme into a more rigid conformation than in LM2. These steric constraints are removed upon dissociation of the sixth ligand leading to a more flexible structure of the active site in the high-spin form of the isozyme LM4. The vibrational modes of the vinyl groups were found to be characteristic markers for the specific structures of the heme pockets in both isozymes. They also respond sensitively to type-I substrate binding. While in cytochrome P-450 LM4 the occupation of the substrate-binding pocket induces conformational changes of the vinyl groups, as reflected by frequency shifts of the vinyl modes, in the LM2 isozyme the ground-state conformation of these substituents remain unaffected, suggesting that the more flexible heme pocket can accommodate substrates without imposing steric constraints on the porphyrin. The resonance Raman technique makes structural changes visible which are induced by substrate binding in addition and independent of the changes associated with the shift of the spin state equilibrium: the high-spin states in the substrate-bound and substrate-free enzyme are structurally different. The formation of the inactive form, P-420, involves a severe structural rearrangement in the heme binding pocket leading to drastic changes of the vinyl group conformations. The conformational differences of the active sites in cytochromes P-450 LM2 and LM4 observed in this work contribute to the understanding of the structural basis accounting for substrate and product specificity of cytochrome P-450 isozymes.

Animals↗
PubMed1989

Protein-protein interactions in microsomal cytochrome P-450 isozyme LM2 and their effect on substrate binding.

The effects of protein-protein interactions and substrate binding on the structure of the active site of rabbit liver microsomal cytochrome P-450 LM2 have been analyzed by resonance Raman spectroscopy of the monomeric and oligomeric protein in solution. Also H2O2-dependent catalytic activities of the two states have been compared. The two vinyl substituents of the heme exhibit different orientations, as indicated by the frequencies and intensities of their stretching vibrations. One group lies in the plane of the heme and remains unchanged in the two states of cytochrome P-450 LM2, the other is tilted out of the plane. The tilting angle in oligomers was smaller than in monomers. These vinyl stretching modes together with some porphyrin modes, were found to be sensitive indicators of the quaternary structure and of substrate binding. In both the oligomer and the monomer, substrate binding causes changes of the relative intensities of some porphyrin modes and the vinyl stretching vibrations which may reflect modifications of the electronic transitions due to hydrophobic interactions between the bound substrate and the heme. In contrast to the monomeric cytochrome P-450 LM2, benzphetamine binding to the oligomers of this isozyme additionally produces a shift of the spin-state equilibrium. This indicates that in the oligomer the substrate-binding pocket is converted by protein-protein interaction to a structure that forces substrates to interfere with the sixth ligands, inducing an increase of the five-coordinated high-spin configuration. In the monomer the substrate-binding pocket can accommodate benzphetamine without affecting the spin state. Binding of imidazole to the monomeric and oligomeric cytochrome P-450 LM2 produces essentially the same resonance Raman spectra. Apparently the replacement of the native sixth ligand by imidazole disturbs the structure of the active site in such a way that it becomes insensitive to protein-protein interactions. H2O2-dependent N-demethylation of benzphetamine and aniline p-hydroxylation by cytochrome P-450 LM2 did not depend on its state of aggregation.

Animals↗
PubMed1989

31P-NMR spectroscopy of phosphate compartmentation during ischaemia in hearts protected by cardioplegic treatment.

Four tissue compartments, differing in proton and inorganic phosphate concentration, were resolved by 31P-NMR spectroscopy in samples from dog hearts after cardioplegic treatment with HTK solution. Inversion of the physiological cytoplasmic-mitochondrial pH gradient was observed. The considerable ensuing acidosis of the matrix is discussed with regard to a possible delocalization of ferrous ions.

Animals↗
PubMed1988

Resonance Raman study of the cytochrome P-450 LM2-halothane intermediate complex.

Resonance Raman (RR) and absorption spectroscopic studies of purified rabbit liver cytochromes P-450 show that the form 2 isomer (LM2) but not the form 4 isomer (LM4) forms a long-lived complex with halothane after dithionite reduction, absorbing light at 470 nm, in which ferric 6-coordinated heme iron in the low-spin configuration is liganded to 2-chloro-1,1-difluoroethylene. The RR data exclude the possibility that the CF3CHCl- carbanion is a ligand and are consistent with the involvement of an active-site pocket in the cytochrome P-450 polypeptide.

Animals↗
PubMed1988

Surface enhanced resonance Raman study of phenobarbital-induced rabbit liver cytochrome P-450 LM2.

Surface enhanced resonance Raman (SERR) spectroscopy has been used to study the vibrational spectra of the heme of purified rabbit liver cytochrome P-450 LM2 which was adsorbed on colloidal silver suspensions or on a silver electrode. Bases on a comparison with the resonance Raman (RR) spectra of the 'solute' species the high sensitivity of the SERR technique is demonstrated. Two different features were chosen in order to determine the structural and functional integrity of the adsorbed P-450. Both, substrate-induced spin state changes on the oxidized P-450 and the effect of the thiolate ligand on the oxidation state marker band v4 in the reduced P-450 could be observed in the SERR spectra of the adsorbed as well as in the RR spectra of the dissolved enzyme. These findings indicate that the protein structure near the substrate binding site and the coordination by thiolate are not affected by the interaction with the metal surface. Both structural elements are crucial for the function of P-450. Thus the elementary processes of the enzymatic action of P-450 can be investigated by this highly sensitive version of RR spectroscopy.

Adsorption↗