On coenzyme Q orientation in membranes: A linear dichroism study of ubiquinones in a model bilayer

Summary A general approach is developed to interpret linear dichroism (LD) spectra of ubiquinones (Q _n) in host bilayers. Information is reported in terms of guest-host mutual orientation and localization. The overall orientational anisotropy of guest ubiquinone molecules is described by a basic set of limiting orientation/localization modes. Assignments of the UV transitions of the ubiquinone chromophore were obtained by the liquid crystal-linear dichroism technique and molecular orbital (CNDO/S) calculations. The LD spectra of Q _n in the bilayers provided by the lyotropic nematic mesophase exhibited by water solutions of potassium laurate and decanol were interpreted on the basis of the above assignments. The resulting experimental evidence showed a multisite distribution in the host bilayer for the aromatic heads of all the investigated Q _n derivatives except Q₀. The orientational distribution suggested by the LD spectra fits the solubilization model recently proposed by G. Lenaz [J. Membrane Biol. (1988) 104:193–209] for ubiquinone in lipid membranes. Within this model Q _n molecules are located in the midplane and their headgroups oscillate transversally across the membrane. Q ₀ instead has a single site location, close to the polar bilayer interface. Experimental evidence that the headgroup carbonyls tend to grasp the polar interface of the host bilayer was also obtained. Orientation and location distributions of Q _n guest molecules are therefore likely to result from the tendency of their aromatic heads to grasp the polar heads of the host bilayer and from the concurrent tendency of their chains to settle into the hydrocarbon host interior.abbreviations AA average absorption - OD, OD optical densities for plane polarized radiations parallel () and perpendicular () to the sample optical axis - OD OD — OD - EPR electron paramagnetic resonance - LC-LD liquid crystal-linear dichroism - LD linear dichroism - LD _r reduced linear dichroism. - MO molecular orbital - N nematic - NMR nuclear magnetic resonance - S _jj order parameters of the directions j of the transition moments of the guest chromophore - S _ii order parameters of the orientational axes i of the guest molecule with respect to the magnetic field - S _ii order parameters of the axes i of the guest molecules with respect to the bilayer axis a - S _a order parameters of the host bilayer axis a with respect to the orienting magnetic field - _j,i deflection angles between the directions j and the axes i - O _i optical factors of the i axis see Eq. (A4)] - Q_n ubiquinone whose isoprenoid chain contains n isoprenoid units Dr. A. Rossi is gratefully acknowledged for the t.e.m. reduction of the spectra. Ubiquinone homologs were kind gifts from Eisai Co., Tokyo, Japan. This work was supported by M.U.R.S.T., and C.N.R. Target Project on Biotechnology and Bioinstrumentation, Rome, Italy.     

The relationship between ETS (electron transport system) activity and oxygen consumption in lake plankton: a cross-system calibration

The ETS (electron transport system) assay to measure respirationof aquatic organisms has been widely applied in studies of marinemetabolism, but its use in freshwaters has been much more limited.This method calculates oxygen consumption from the measuredETS activity using an empirical conversion factor. This factorhas been calculated for various marine organisms, and for naturalplankton communities, but calibrations for freshwater organismsare lacking. The aim of this paper was to determine the relationshipbetween lake plankton respiration and ETS activity, based onmeasured epilimnetic plankton oxygen uptake and ELS activityin 20 southern Quebéc lakes. The relationship betweenplankton oxygen consumption and ETS varies significantly bothwithin lakes over the growing season, and among lakes. The magnitudeof the error associated with calculating respiration from ETSis, however, similar to the error in other standard limnologicalprocedures used in plankton carbon flow studies. Oxygen consumptionis not a linear function of ETS across the range of lakes, butis rather a power function. The respiration:ETS ratio for lakeplankton therefore not constant: it is high in oligotrophicand colored lakes, and declines with trophy. These results areconsistent with the changes expected in the structure of theplankton along trophic gradients.     

The effects of elongational stress exposure on the activation and aggregation of blood platelets.

Hemodynamic shear is known to stimulate blood and endothelial cells and induce platelet activation. Many studies of shear-induced platelet stimulation have employed rotational viscometers in which secondary flow effects are assumed to be negligible. Shear induced platelet activation occurs at elevated shear rates where secondary flows may contribute a significant percentage of the total hydrodynamic force experienced by the sample. Elongational stress, one component of this secondary flow, has been shown to alter transmembrane ion flux in intact cell and the permeability of synthetic membrane preparations. Elongational flow also occurs in the vasculature at sites of elevated shear stress. Secondary flow components may contribute to platelet activation induced during shear stress application in rotational viscometry. A unique 'constrained convergence' elongational flow chamber was designed and fabricated to study platelet response to elongational stress exposure. The elongational flow chamber was capable of producing an elongation rate of 2.1 s-1 with a corresponding volume averaged shear rate of 58.33 s-1. Significant changes were observed in the total platelet volume distribution and measured response to added chemical antagonists after elongational stress exposure. The total platelet volume histogram shifted toward larger particle sizes, suggesting the formation of large aggregates as a result of elongational stress exposure. Platelets exposed to elongational stress demonstrated a dose dependent decrease in added ADP-induced aggregation rate and extent of aggregation.     

Lysis of autologous human melanoma cells by in vitro allosensitized peripheral blood lymphocytes

Summary Peripheral blood lymphocytes (PBL) of melanoma patients were sensitized in vitro with lymphocytes of a single donor or with a pool of lymphocytes of 5–20 different donors. After 6–7 days, the cytotoxic activity of the sensitized PBL was tested against cultured autologous tumor cells and lymphocytes in a ⁵¹Cr-release assay. Tumor lysis was observed in 13 of 16 cases in which patients' PBL (Pt-PBL) were stimulated by a pool of allogeneic lymphocytes and in five out of seven cases when single sensitization was performed. In no case was lysis of autologous normal lymphocytes or blasts seen. Cultivation of Pt-PBL with irradiated autologous tumor cells never led to the induction of lymphocytes cytotoxic to melanoma cells. Lysability by pool-activated autologous Pt-PBL of fresh cryopreserved tumor cells was compared to that of short-term cultured tumor cells, and no significant differences were observed. Cold-target inhibition experiments indicated that the cytotoxicity of Pt-PBL was tumor-restricted since only autologous melanoma cells but not lymphocytes were able to inhibit the reaction. These results indicate that activation of Pt-PBL is necessary in order to elicit or amplify their antitumor activity.     

Proton-induced grana formation in chloroplasts. Distribution of chlorophyll-protein complexes and Photosystem II photochemistry

Lowering the pH of the incubation medium to pH 5.4 leads to grana formation morphologically similar to that induced by metal cations. The same phenomenon is observed in EDTA-washed chloroplasts, indicating that it is not due in part to electrostatic ‘masking’ by residual cations associated with the membranes. Digitonin fractionation studies have indicated that the distribution of the major chlorophyll-protein complexes between granal and stromal membrane regions is similar at pH 5.4 in the absence of Mg²⁺, and at pH 7.4 in the presence of Mg²⁺. Chlorophyll fluorescence induction studies have indicated that the primary photochemistry of Photosystem II (PS II) is stimulated by lowering the pH to 5.4, just as it is upon metal cation addition at higher pH values. The failure to observe such an increase at pH 5.4 by measuring electron transport to ferricyanide is attributed to a combination of an inhibition by this pH of electron transport at a site after Q reduction and an increase in the number of PS II centres detached from the plastoquinone pool. We conclude that the stacked configuration of chloroplast membranes leads to increased PS II primary photochemistry, which is most simply explained in terms of a redistribution of excitation energy towards PS II.     

Congenital coronary artery-left heart fistulas: Report of three cases

Of 59 patients who underwent operative correction of congenital coronary artery fistulas from May 1956 through May 1980 at our institution, three had fistulas that arose from the coronary artery and terminated in the left heart. The chief indication for surgical correction in such patients is the presence of symptoms or the development of complications, which include rupture, endocarditis, and congestive heart failure. The principal objective of repair is closure or obliteration of the fistulous communication and preservation of distal myocardial perfusion. Because symptoms and complications tend to occur with age, elective ligation is warranted during childhood, even in asymptomatic patients. The three cases described here, as well as the reviewed series of left heart fistulas, substantiate this fact. All three patients were symptomatic before operation and asymptomatic afterward.     

Na+-dependent,potential-sensitive l-ascorbate transport across brush border membrane vesicles from kidney cortex

l-Ascorbate is taken up into brush border vesicles from kidney cortex of rat, rabbit and guinea pig by an efficient, Na⁺-dependent and potential-sensitive transport process. This uptake shows saturation ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>:0.1–0.3\; mM</mtext>}}$) and is strongly stimulated by low concentrations of N₃^?. Erythorbate (d-isoascorbate) seems to be another, but poorer, substrate of the same transporter.     

Interrelationship between oxygen consumption,superoxide anion and hydrogen peroxide formation in phagocytosing guinea pig polymorphonuclear leucocytes

The paper presents an experimental procedure for a simultaneous assay of oxygen consumption, O2- release and H2O2 accumulation at a very early stage of the respiratory burst that is induced by phagocytosis in guinea pig polymorphonuclear leucocytes. The main findings are as follows: (a) The oxygen consumption that is measurable does not correspond to all oxygen that is reduced. The relationship between the actual oxygen consumed and the amount that is reduced depends on the fate of the intermediate products O2- and H2O2. (b) O2- is measurable extracellularly by the reduction of cytochrome c. When cytochrome c oxidizes the extracellular O2-, molecular oxygen is formed. This fact is shown by a decrease of oxygen consumption. The molar ratio between the O2- detected and the oxygen given back is 1. (c) The amount of O2- released from the cells accounts for only a small part of oxygen actually reduced. (d) H2O2 is detectable only in the presence of NaN3. In this condition almost all oxygen consumed is recovered in the form of H2O2. The molar ratio O2/H2O2 is near unity. The amount of H2O2 derived from dismutation of O2- released is only an aliquot of the total H2O2 accumulated. Thus, most of H2O2 is derived from intracellular sources. (e) In the absence of inhibitors of H2O2 degrading reactions, no detectable accumulation of peroxide occurs. Under these conditions, the main part of H2O2 formed is degraded in almost equal amount by catalase and myeloperoxidase, while only a small aliquot is degraded by NaN3 insensitive reactions.