Helmut Lindner

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.

Room Temperature Activates Human Blood Platelets

Temperatures ranging from room temperature (20° C) to 42° 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° C) and 42° C on human blood platelets and found that room temperature causes marked activation of platelets. Major changes in platelet morphology were seen at 20° C compared to resting platelets at 37° 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° C for 10 minutes. In the presence of serotonin re-uptake inhibitors, the serotonin content of platelets at 20° C was twice that of resting platelets. In comparison, moderate heat shock conditions (42° 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° C storage of platelets for transfusion and the in vivo hemostatic failure after hypothermia.

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

K-Tocopheryl succinate (K-TS) is a potent inhibitor of tumor cell proliferation. The goal of the present study was to investigate whether and to what extent K-TS associates with plasma lipoproteins and if K-TS-enriched lipoproteins inhibit breast cancer cell growth in a manner comparable to the free drug. In vitro enrichment of human plasma revealed that K-TS readily associated with the main lipoprotein classes, findings confirmed in vivo in mice. At the highest K-TS concentrations, lipoproteins carrying 50 000 (VLDL), 5000 (LDL) and 700 (HDL) K-TS molecules per lipoprotein particle were generated. KTS enrichment generated lipoprotein particles with slightly decreased density and increased particle radius. To study whether the level of LDL-receptor (LDL-R) expression affects K-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) HDL3-associated K-TS was nearly identical for both cell lines. In contrast, uptake of LDL-associated K-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 K-TS inhibited proliferation most effectively at the highest concentration of K-TS used (100% inhibition of MCF-7 growth with 20 Wg/ml of lipoprotein-associated K-TS). However, also K-TS-free VLDL and LDL inhibited HBL-100 cell proliferation up to 55%. In both cell lines, K-TS-enriched HDL3 inhibited cell growth by 40^60%. Incubation of both cell lines in the presence of free or lipoprotein-associated K-TS resulted in DNA fragmentation indicative of apoptosis. Collectively, the present findings demonstrate that: (1) K-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 K-TS was apparently dependent on the level of LDL-R expression ; and (4) lipoproteins were efficient K-TS carriers inducing reduced cell proliferation rates and apoptosis in human breast cancer cells as observed for the free drug. fl 2000 Elsevier Science B.V. All rights reserved.

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

Abstract 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 α-helical content of ParD as determined by CD-spectrometry was 35%. The protein exhibited high thermostability with a TM of 64°C and ΔH of 25 kcal/mol as shown by differential scanning calorimetry. Upon complex formation the TM increased by 10°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 CDspectroscopy.

The Essential Transfer Protein TraM Binds to DNA as a Tetramer

The TraM proteins encoded by F-like plasmids are sequence specific DNA binding proteins that are essential for conjugative DNA transfer. We investigated the quarternary structure and the DNA binding properties of the TraM wild-type protein of the resistance plasmid R1 and two mutant forms thereof. Size-exclusion chromatography and differential scanning calorimetry showed that purified TraM protein (amino acids 2–127) forms stable tetramers in solution. A truncated version of the protein termed TraMM26 (amino acids 2–56) forms dimers. Thus, the dimerization and tetramerization domains can be assigned to the N-terminal and C-terminal domains of TraM, respectively. Further analyses using chemical cross-linking and light scattering corroborated the preferentially tetrameric nature of the protein but also suggest that TraM has a tendency to form higher aggregates. Band-shift and fluorescence spectroscopy investigations of TraM-DNA complexes revealed that the TraM protein is also tetrameric when bound to its minimal DNA binding site. The deduced binding constant in the range of 108 m −1 demonstrated a very strong binding of TraM to its preferred DNA sequence. Secondary structure analysis based on CD measurements showed that TraM is mainly α-helical with a significant increase in α-helicity (48 to 58%) upon DNA-binding, indicating an induced fit mechanism.

Determination of Absolute Intensity and Molecular Weight of Small Colloidal Particles in the Presence of Some Large Aggregates. A Combined Study Using Static and Dynamic Light Scattering

For the purpose of absolute intensity calibration in static light scattering experiments, a reference standard (e.g. toluene or water), from which the absolute scattering is known, can be used. The calculation of the molecular weight and/or the aggregation number of particles is done by determining the mean scattering intensity in the forward direction of the particles under consideration. Having small particles compared with the wavelength, e.g. proteins or micelles mixed with a small amount of large aggregates, one is confronted with the problem that these impurities normally contain only a very small part of the sample mass, but contribute strongly to the measured signal. To separate the intensity scattered from the large impurities or aggregates we use the additional information available from a dynamic light scattering measurement. The areas in the intensity distribution function, which can be calculated by inverse Laplace transformation of the field correlation function, are directly related to the intensity of the particular populations. The measured intensity of the whole sample is then reduced by the peak area of the large aggregates or impurities. This intensity correction procedure is the central part of this contribution. Simulations of bimodal size distributions and application to experimental data for bimodal latex mixtures prove that this intensity correction procedure gives very good agreement between theoretical predictions and measured data over a wide range of relative intensities and sizes.