Biomedical subjects

O Glatter

Publications and source records attributed to O Glatter.

At least 19 recordsLinked to original sources

PubMed2001

Electrospray ionization mass spectrometry, circular dichroism and SAXS studies of the (S)-hydroxynitrile lyase from Hevea brasiliensis.

We report on experiments pertaining to solution properties of the (S)-hydroxynitrile lyase from Hevea brasiliensis (HbHNL). Small angle X-ray scattering unequivocally established the enzyme to occur in solution as a dimer, presumably of the same structure as in the crystal. The acid induced, irreversible deactivation of HbHNL was examined by electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD) and by measuring the enzyme activity. The deactivation is paralleled by an unfolding of the enzyme. ESI-MS of this 30000 Da per monomer heavy protein demonstrated that unfolding took place in several stages which are paralleled by a decrease in enzyme activity. Unfolding can also be observed by CD spectroscopy, and there is a clear correlation between enzyme activity and unfolding as detected by ESI-MS and CD.

Aldehyde-Lyases↗

PubMed2001

Room temperature activates human blood platelets.

Temperatures ranging from room temperature (20 degrees C) to 42 degrees C are generally not considered to have an activating effect on platelets. However, this assumption is not supported by clinical phenomena that result in hemostatic failure related to hypothermia. In this study, we investigated the effect of temperatures between room temperature (20 degrees C) and 42 degrees C on human blood platelets and found that room temperature causes marked activation of platelets. Major changes in platelet morphology were seen at 20 degrees C compared to resting platelets at 37 degrees C. Platelet morphology was investigated with noninvasive live cell techniques (light microscopy and dynamic and static light scattering), as well as with transmission and scanning electron microscopy. The changes in platelet morphology correlated with the expression of the activation marker, activated glycoprotein (GP) IIb-IIIa, measured by flow cytometry. Twenty-five percent to 30% of platelets expressed activated GPIIb-IIIa after exposure to 20 degrees C for 10 minutes. In the presence of serotonin re-uptake inhibitors, the serotonin content of platelets at 20 degrees C was twice that of resting platelets. In comparison, moderate heat shock conditions (42 degrees C for 10 minutes) caused no signs of platelet activation as indicated by the absence of morphological alterations, no expression of activated GPIIb-IIIa, and no changes in serotonin content. These results show that room temperature by itself significantly activates platelets and has an effect on the platelet serotonin content. This may contribute to both the functional lesion associated with 22 degrees C storage of platelets for transfusion and the in vivo hemostatic failure after hypothermia.

Blood Platelets↗

PubMed2001

Lipoprotein lipase mediates the uptake of glycated LDL in fibroblasts, endothelial cells, and macrophages.

The nonenzymatic glycation of LDL is a naturally occurring chemical modification of apolipoprotein (apo)-B lysine residues by glucose. Once glycated, LDL is only poorly recognized by lipoprotein receptors including the LDL receptor (LDL-R), the LDL-R-related protein (LRP), and scavenger receptors. Glycated LDL (gLDL) is a preferred target for oxidative modifications. Additionally, its presence initiates different processes that can be considered "proatherogenic." Thus, LDL glycation might contribute to the increased atherosclerotic risk of patients with diabetes and familial hypercholesterolemia. Here we investigate whether lipoprotein lipase (LPL) can mediate the cellular uptake of gLDL. The addition of exogenous LPL to the culture medium of human skin fibroblasts, porcine aortic endothelial cells, and mouse peritoneal macrophages enhanced the binding, uptake, and degradation of gLDL markedly, and the relative effect of LPL on lipoprotein uptake increased with the degree of apoB glycation. The efficient uptake of gLDL by LDL-R-deficient fibroblasts and LRP-deficient Chinese hamster ovary cells in the presence of LPL suggested a mechanism that was independent of the LDL-R and LRP. In macrophages, the uptake of gLDL was also correlated with their ability to produce LPL endogenously. Mouse peritoneal macrophages from genetically modified mice, which lacked LPL, exhibited a 75% reduction of gLDL uptake compared with normal macrophages. The LPL-mediated effect required the association of the enzyme with cell surface glycosaminoglycans but was independent of its enzymatic activity. The uptake of gLDL in different cell types by an LPL-mediated process might have important implications for the cellular response after gLDL exposure as well as the removal of gLDL from the circulation.

Acetylation↗

PubMed2000

Comparison of HOCl traps with myeloperoxidase inhibitors in prevention of low density lipoprotein oxidation.

In this study, the production of the highly toxic oxidant hypochlorous acid (HOCl) by the phagocytic enzyme myeloperoxidase (MPO) was quantitated and the concomitant alterations of low density lipoprotein (LDL) were analyzed in view of the potential role of LDL in atherosclerosis. Using the monochlorodimedone assay, it was found that HOCl is produced in micromolar concentrations. The kinetics of the decrease of tryptophan fluorescence appeared to be a sensitive method to monitor LDL alterations under near in vivo conditions. Therefore, this method was used to subsequently compare the effectiveness of MPO inhibitors that block production of HOCl with compounds that act as HOCl traps. The efficiency of MPO inhibitors to prevent LDL damage increased in the series benzohydroxamic acid < salicylhydroxamic acid < 3-amino-1,2,4-triazole < sodium azide < potassium cyanide < p-hydroxy-benzoic acid hydrazide, while for the HOCl traps the protective efficiency increased in the series glycine < taurine < methionine. We conclude that HOCl traps may have high potential therapeutic impact in vivo due to their low toxicity, although high concentrations of them would have to reach sites of inflammation. In contrast, only low concentrations of a specific MPO inhibitor would be required to irreversibly inhibit the enzyme.

Arteriosclerosis↗

PubMed2000

Lipoprotein-associated alpha-tocopheryl-succinate inhibits cell growth and induces apoptosis in human MCF-7 and HBL-100 breast cancer cells.

alpha-Tocopheryl succinate (alpha-TS) is a potent inhibitor of tumor cell proliferation. The goal of the present study was to investigate whether and to what extent alpha-TS associates with plasma lipoproteins and if alpha-TS-enriched lipoproteins inhibit breast cancer cell growth in a manner comparable to the free drug. In vitro enrichment of human plasma revealed that alpha-TS readily associated with the main lipoprotein classes, findings confirmed in vivo in mice. At the highest alpha-TS concentrations, lipoproteins carrying 50000 (VLDL), 5000 (LDL) and 700 (HDL) alpha-TS molecules per lipoprotein particle were generated. alpha-TS enrichment generated lipoprotein particles with slightly decreased density and increased particle radius. To study whether the level of LDL-receptor (LDL-R) expression affects alpha-TS uptake from apoB/E containing lipoprotein particles human breast cancer cells with low (MCF-7) and normal (HBL-100) LDL-R expression were used. The uptake of free, VLDL- and (apoE-free) HDL(3)-associated alpha-TS was nearly identical for both cell lines. In contrast, uptake of LDL-associated alpha-TS by HBL-100 cells (normal LDL-R expression) was about twice as high as compared to MCF-7 cells (low LDL-R expression). VLDL and LDL-associated alpha-TS inhibited proliferation most effectively at the highest concentration of alpha-TS used (100% inhibition of MCF-7 growth with 20 microg/ml of lipoprotein-associated alpha-TS). However, also alpha-TS-free VLDL and LDL inhibited HBL-100 cell proliferation up to 55%. In both cell lines, alpha-TS-enriched HDL(3) inhibited cell growth by 40-60%. Incubation of both cell lines in the presence of free or lipoprotein-associated alpha-TS resulted in DNA fragmentation indicative of apoptosis. Collectively, the present findings demonstrate that: (1) alpha-TS readily associates with lipoproteins in vitro and in vivo; (2) the lipoprotein-enrichment efficacy was dependent on the particle size and/or the triglyceride content of the lipoprotein; (3) uptake of LDL-associated alpha-TS was apparently dependent on the level of LDL-R expression; and (4) lipoproteins were efficient alpha-TS carriers inducing reduced cell proliferation rates and apoptosis in human breast cancer cells as observed for the free drug.

Animals↗

PubMed2000

Lateral microheterogeneity of diphenylhexatriene-labeled choline phospholipids in the erythrocyte ghost membrane as determined by time-resolved fluorescence spectroscopy.

Choline phospholipids are the major constituents of the outer layer of the erythrocyte membrane. To investigate their lateral membrane organization we determined the fluorescence lifetime properties of diphenylhexatriene analogues of phosphatidylcholine, choline plasmalogen, (the respective enolether derivative), and sphingomyelin inserted into the outer layer of hemoglobin-free ghosts. Fluorescence lifetimes were recorded by time-resolved phase and modulation fluorometry and analyzed in terms of Continuous Lorentzian distributions. To assess the influence of membrane proteins on the fluorescence lifetime of the labeled lipids in the biomembrane, lipid vesicles were used as controls. In general, the lifetime distributions in the ghost membranes are broad compared to vesicles. Phosphatidylcholine and sphingomyelin exhibit very similar lifetime distributions in contrast to an increased plasmalogen lifetime heterogeneity in both systems. Orientational effects of side chain mobilities on the observed lifetimes can be excluded. Fluorescence anisotropies revealed identical values for all three labeled phospholipids in the biomembrane.

Choline↗

PubMed2000

Synthetic phospholipid analogs: a structural investigation with scattering methods.

The phase behavior of the newly synthesized phospholipid analogs is necessary to be known because of the importance of the structure of the phospholipid analogs for the activity against tumor cell lines. The type of aggregates formed under defined conditions, the size distribution and the internal structure of the particles as well as stability upon storage are examined by scattering methods. Octadecyl-methyl-glycero-phosphocholine (OMGPC) and Octadecyl-imidazoyl-deoxy-glycero-phosphocholine (OIDGPC) are cytostatic systems designed synthetically for the medical application and, therefore, prepared and measured usually at 37 degrees C. Both phospholipid analogs behave under the same conditions almost similarly. They show also the similar behavior in long term studies like the biological ganglioside GM1. Under physiological conditions, OMGPC and OIDGPC form spherical micelles with a maximum dimension of about 8 nm, which do not change with concentration.

Molecular Structure↗

PubMed1999

Characterization of Enzymatically Induced Aggregation of Casein Micelles in Natural Concentration by in Situ Static Light Scattering and Ultra Low Shear Viscosimetry.

The aggregation of casein micelles in undiluted skim milk after the addition of chymosin was studied by static light scattering and ultra low shear viscometry. The static light scattering measurements were made with two different sample thicknesses, 72 and 16 µm. The scattering data were analyzed by indirect Fourier transformation and by the polydispersity inversion technique which led to pair distance distribution functions and size distribution function, respectively. The minimum scattering angle was 1 degrees, which allows for the determination of particle sizes up to a maximum diameter of 12 µm. The fractal dimension determined from double logarithmic plots of intensity versus scattering vector resulted in values between 1.9 and 2.0. The influence of multiple scattering was determined by comparison of the measurements with the different sample thicknesses. The measurements show no significant influence of multiple scattering when the transmission is above 0.85. Due to the very complex and porous structure of the casein aggregates the Rayleigh-Debye-Gans scattering theory has been used in the data analysis. Measurements with a new instrument using ultra low shear showed good agreement with theory. Copyright 1999 Academic Press.

Journal Article↗

PubMed1999

Molecular dynamics of microbial lipases as determined from their intrinsic tryptophan fluorescence.

We have studied the intrinsic tryptophan fluorescence of the lipases from Chromobacterium viscosum (CVL), Pseudomonas species (PSL), and Rhizopus oryzae (ROL) in aqueous buffer, zwitterionic detergent micelles, and isopropanol-water mixtures. It was the purpose of this study to obtain information about biophysical properties of the respective enzymes under conditions that modulate enzyme activities and stereoselectivities to a significant extent. According to their decay-associated emission spectra, CVL tryptophans are located in the hydrophobic interior of the protein. In contrast, the PSL and ROL tryptophans are probably confined to the core and the surface of the lipase. From the tryptophan lifetime distributions it can be concluded that the conformation of CVL is not much affected by detergent or organic solvent (isopropanol). Accordingly, CVL is enzymatically active in these systems and most active in the presence of isopropanol. In contrast, ROL and PSL show high conformational mobility, depending on the solvent, because their lifetime distributions are very different in the presence and absence of detergent or isopropanol. Time-resolved anisotropy studies provided evidence that the lipases exhibit very high internal molecular flexibility. This peculiar feature of lipases is perhaps the key to the great differences in activity and stereoselectivity observed in different reaction media. Furthermore, information about self-association of the lipases in different solvents could be obtained. PSL, but not CVL and ROL, forms aggregates in water. Lipase aggregation can be reversed by the addition of detergent or isopropanol, which competes for the hydrophobic surface domains of this protein. This dissociation could efficiently contribute to the increase in lipase activity in the presence of a detergent or isopropanol.

2-Propanol↗

PubMed1999

Thermodynamic properties and DNA binding of the ParD protein from the broad host-range plasmid RK2/RP4 killing system.

ParD is a small, acidic protein from the partitioning system of the plasmid RK2/RP4. The ParD protein exhibits specific DNA binding activity and, as the antidote component of a toxin-antidote plasmid addiction system, ParD forms a tight complex in solution with its toxin antagonist, the ParE protein. Unopposed ParE acts as a toxin that causes growth retardation and killing of plasmid cured cells. ParD negatively autoregulates its expression by binding to an operator sequence in the parDE promoter region. This DNA binding activity is crucial for the regulation of the relative abundance of toxin and antidote which ultimately determines life or death for the bacterial host and its daughter cells. In light scattering studies and gel filtration chromatography we observed the existence of a stable dimer of ParD in solution. The stoichiometry of ParD-DNA complex formation appeared to be 4:1, the molecular mass of the complex was 72.1 kDa. The alpha-helical content of ParD as determined by CD-spectrometry was 35%. The protein exhibited high thermostability with a T(M) of 64 degrees C and deltaH of 25 kcal/mol as shown by differential scanning calorimetry. Upon complex formation the T(M) increased by 10 degrees C. The thermal unfolding of the ParD protein was highly reversible as observed in repeated DSC scans of the same sample. The recovery of the native fold was proven by CD-spectroscopy.

Bacterial Proteins↗

PubMed1998

The solution structure of functionally active human proliferating cell nuclear antigen determined by small-angle neutron scattering.

The function of proliferating cell nuclear antigen (PCNA) in DNA replication and repair is to form a sliding clamp with replication factor C (RF-C) tethering DNA polymerase delta or epsilon to DNA. In addition, PCNA has been found to interact directly with various proteins involved in cell cycle regulation. The crystal structure of yeast PCNA shows that the protein forms a homotrimeric ring lining a hole through which double-stranded DNA can thread, thus forming a moving platform for DNA synthesis. Human and yeast PCNA are highly conserved at a structural and functional level. We determined the solution structure of functionally active human PCNA by small-angle neutron scattering. Our measurements strongly support a trimeric ring-like structure of functionally active PCNA in solution, and the data are in good agreement with model calculations based on the crystal structure from yeast PCNA. The human PCNA used in the small-angle neutron scattering experiments was active before and after the measurements in a RF-C independent and a RF-C dependent assay suggesting that the trimeric structure is the in vivo functional form.

Buffers↗

PubMed1998

Time and temperature dependent aggregation behaviour of the ganglioside GM1 in aqueous solution.

Gangliosides are marked amphiphiles with several ramified sugar rings and a ceramide moiety. Therefore they form micelles in aqueous solution. This aggregation behavior is studied with static and dynamic light scattering and small angle X-ray scattering. The shape and the size of the GM1 micelles in dependence of time and temperature are investigated. Some data have been published recently where two stable states for the micelles are found. This bistability is said to be achieved by temperature rise from room temperature up to 55 degrees C. The authors did not find such an internal structural change of the micelle in the experiments. Instead the authors found some large aggregates of the ganglioside in parallel to the micelle caused by the low solubility of this system at room temperature. These aggregates are dissolved quickly with increasing temperature or slowly with time at room temperature. So the variation of the scattering intensity is explainable by dissolving the large particles, meanwhile the structure of the GM1 micelles is the same all the time.

Calorimetry, Differential Scanning↗

PubMed1997

Effects of reagent and enzymatically generated hypochlorite on physicochemical and metabolic properties of high density lipoproteins.

Myeloperoxidase (MPO), a protein secreted by activated phagocytes, may be a potential candidate for the generation of modified/oxidized lipoproteins in vivo via intermediate formation of HOCl, a powerful oxidant. During the present study, the effects of reagent NaOCl and OCl- generated by the MPO/H2O2/Cl- system on physicochemical and metabolic properties of high density lipoprotein (HDL) subclass 3 (HDL3) were investigated. Up to a molar oxidant:lipoprotein ratio of approximately 30:1, apolipoprotein A-I (apoA-I), the major HDL3 apolipoprotein component, represented the preferential target for OCl- attack (consuming 35-76% of the oxidant), thereby protecting HDL3 fatty acids (consuming between 17 and 30% of the oxidant) against OCl--mediated modification. At molar oxidant:HDL3 ratios >/= 60:1, we have observed pronounced consumption of HDL3 unsaturated fatty acids with concomitant formation of fatty acid chlorohydrins. Modification of HDL3 in the presence of the MPO/H2O2/Cl- system resulted in amino acid oxidation in a manner comparable with that found with reagent NaOCl only. Treatment of HDL3 with reagent NaOCl as well as modification by the MPO/H2O2/Cl- system resulted in significantly enhanced turnover rates of HDL3 by mouse peritoneal macrophages, an effect that was not a result of HDL3 aggregation as judged by dynamic and static light-scattering experiments. In comparison with native HDL3, the degradation by macrophages was enhanced by 4- and 15-fold when HDL3 was modified with reagent NaOCl or the MPO/H2O2/Cl- system. Finally, the ability of HDL3 to promote cellular cholesterol efflux from macrophages was significantly diminished after modification with reagent NaOCl. Collectively, these results demonstrate that the modification of HDL3 by hypochlorite (added as reagent or generated by the MPO/H2O2/Cl- system) transformed an antiatherogenic lipoprotein particle into a modified lipoprotein with characteristics similar to lipoproteins commonly thought to initiate foam cell formation in vivo.

Animals↗

PubMed1996

Characterization of heme-deficient neuronal nitric-oxide synthase reveals a role for heme in subunit dimerization and binding of the amino acid substrate and tetrahydrobiopterin.

Neuronal nitric-oxide (NO) synthase contains FAD, FMN, heme, and tetrahydrobiopterin as prosthetic groups and represents a multifunctional oxidoreductase catalyzing oxidation of L-arginine to L-citrulline and NO, reduction of molecular oxygen to superoxide, and electron transfer to cytochromes. To investigate how binding of the prosthetic heme moiety is related to enzyme activities, cofactor, and L-arginine binding, as well as to secondary and quaternary protein structure, we have purified and characterized heme-deficient neuronal NO synthase. The heme-deficient enzyme, which had preserved its cytochrome c reductase activity, contained FAD and FMN, but virtually no tetrahydrobiopterin, and exhibited only marginal NO synthase activity. By means of gel filtration and static light scattering, we demonstrate that the heme-deficient enzyme is a monomer and provide evidence that heme is the sole prosthetic group controlling the quaternary structure of neuronal NO synthase. CD spectroscopy showed that most of the structural elements found in the dimeric holoenzyme were conserved in heme-deficient monomeric NO synthase. However, in spite of being properly folded, the heme-deficient enzyme did bind neither tetrahydrobiopterin nor the substrate analog N(G)-nitro-L-arginine. Our results demonstrate that the prosthetic heme group of neuronal NO synthase is requisite for dimerization of enzyme subunits and for the binding of amino acid substrate and tetrahydrobiopterin.

Animals↗

PubMed1995

Physicochemical behavior of the ganglioside GM3 in dilute aqueous solution.

The aggregative properties of the ganglioside GM3 have been studied with small-angle X-ray scattering and dynamic light scattering in dilute aqueous solutions. Dynamic light scattering experiments show that GM3 solutions are very polydisperse containing a large amount of small aggregates (hydrodynamic radius of 7-9 nm) in addition to a quite broad distribution of aggregates with an average hydrodynamic radius of 50-60 nm and a small fraction of very large aggregates (> 200 nm). This together with small-angle X-ray scattering scattering experiments and model calculations suggests the coexistence of a lamellar phase (vesicles or extended lamellae) with small aggregates (modelled as lamellar platelets). The latter can also be viewed as lamellar fragments coming from GM3 vesicles which constantly break and reform.

G(M3) Ganglioside↗

PubMed1995

Structural analysis of porcine brain nitric oxide synthase reveals a role for tetrahydrobiopterin and L-arginine in the formation of an SDS-resistant dimer.

Nitric oxide synthases (NOSs), which catalyze the formation of the ubiquitous biological messenger molecule nitric oxide, represent unique cytochrome P-450s, containing reductase and mono-oxygenase domains within one polypeptide and requiring tetrahydrobiopterin as cofactor. To investigate whether tetrahydrobiopterin functions as an allosteric effector of NOS, we have analyzed the effect of the pteridine on the conformation of neuronal NOS purified from porcine brain by means of circular dichroism, velocity sedimentation, dynamic light scattering and SDS-polyacrylamide gel electrophoresis. We report for the first time the secondary structure of NOS, showing that the neuronal isozyme contains 30% alpha-helix, 14% antiparallel beta-sheet, 7% parallel beta-sheet, 19% turns and 31% other structures. The secondary structure of neuronal NOS was neither modulated nor stabilized by tetrahydrobiopterin, and the pteridine did not affect the quaternary structure of the protein, which appears to be an elongated homodimer with an axial ratio of approximately 20/1 under native conditions. Low temperature SDS-polyacrylamide gel electrophoresis revealed that tetrahydrobiopterin and L-arginine synergistically convert neuronal NOS into an exceptionally stable, non-covalently linked homodimer surviving in 2% SDS and 5% 2-mercaptoethanol. Ligand-induced formation of an SDS-resistant dimer is unprecedented and suggests a novel role for tetrahydrobiopterin and L-arginine in the allosteric regulation of protein subunit interactions.

Allosteric Regulation↗

PubMed1994

Phase behavior of the antineoplastic ether lipid 1-O-octadecyl-2-O-methyl-glycero-3-phosphochloline.

The physicochemical properties of the antineoplastic etherphospholipid 1-O-octadecyl-2-O-methyl-glycero-3-phosphochline were examined in the concentration range 1-35% (w/w) lipid, as a function of temperature (range -10 degrees C to 40 degrees C) and of different aqueous solvents by dynamic light scattering, small- and wide-angle X-ray scattering, differential scanning calorimetry and ultrasonic speed measurements. On cooling the lipid dispersion undergoes a phase transition near 6 degrees C, transforming slowly from a micellar into a lamellar gel phase with interdigitating hydrocarbon chains. The lamellar repeat distance is nearly constant over the hydration range 65-90% buffer (d = 5.09-5.14 nm). The size of the micelles in terms of the hydrodynamic radius is 3.8 +/- 0.1 nm, the polydispersity is low. Their average shape is spherical. The electron density distribution across the micelle gives 2.5 nm for the extension of the hydrocarbon chains and 1.5 nm for the polar moiety. The existence of micelles was verified up to a concentration of 35% lipid. Throughout this concentration range size and shape do not change significantly. The kinetics of formation of the low-temperature phase is slow on cooling, increasing with increasing concentration. Upon heating the phase behavior shows a hysteresis. The extended lamellar organizations start to break down into smaller aggregates near 3 degrees C. The micellar phase is reformed near 20 degrees C.

Antineoplastic Agents↗

PubMed1992

Shape change of human blood platelets: reliable and fast detection by quasi-elastic light scattering.

Quasi-elastic light scattering has been used for the first time to obtain reliable information about the morphology of platelets under physiological conditions within a short time. By measuring two independent parameters (electrophoretic mobility and diffusion coefficient) it is possible to distinguish between different stages of shape change on the one hand, and between shape change and binding of particles to the platelet surface without shape change on the other hand.

Adult↗