Characterization of chicken cystatin binding to rat renal brush-border membranes

Chicken cystatin, a homologue of human cystatin C, like other low-molecular-weight proteins is metabolized by renal proximal tubule cells. However, the precise mechanism(s) of this process has not been elucidated yet. To characterize chicken cystatin binding to renal brush-border membranes, the incubation of fluorescein labelled protein with rat cortical homogenate was performed. Saturation-dependent and reversible binding with low affinity (K_d = 3.67–4.07 μM) and high capacity (B_max = 2.32–2.79 nmol/mg) was observed. Bovine albumin was the most potent competitor (K_i = 0.7 μM) among other megalin/cubilin ligands tested. The presence of Ca^+ 2 ions was necessary to effective cystatin binding by brush-border membranes. Obtained data strongly support the hypothesis that chicken cystatin is a novel ligand for megalin/cubilin receptors tandem on proximal tubular cells.     

Production of C<Subscript>35</Subscript> isoprenoids depends on H<Subscript>2</Subscript> availability during cultivation of the hyperthermophile<Emphasis Type="Italic"> Methanococcus jannaschii</Emphasis>

A series of five progressively saturated C₃₅ isoprenoids has been identified in cell-free extracts of the deep-sea methanogen Methanococcus jannaschii. Production and relative abundance of the isoprenoids were dependent on culture conditions; significant production occurred in a 16-l fermentor (12-l working volume) and a 2.5-l fermentor (2-l working volume) but could not be duplicated in serum bottles. Several factors were investigated and shown not to account for the different production levels, including medium composition, pH, and temperature. However, the interphase mass transfer rate was shown to significantly affect the production of C₃₅ isoprenoids in a fermentor. The structures of the novel isoprenoids were confirmed by hydrogenation reactions and mass spectra of the isoprenoids. Indirect evidence based on genomics and mass spectrometry data implicates head-to-head condensation of farnesyl pyrophosphate (C₁₅) with geranylgeranyl pyrophosphate (C₂₀) as the mechanism for C₃₅ synthesis.Communicated by J. WiegelB.P. Manquin and J.A. Morgan contributed equally to this work.     

Effect of thyroid hormone on the distribution and activity of Na, K-ATPase in ventricular myocardium

Employing detergent-free sucrose-density gradient fractionation method we isolated cholesterol-rich lighter membrane fractions containing ∼10% of protein, ∼30% of cholesterol in membranes of ventricular myocardium. Cholesterol-rich lighter membrane fractions contain >70% of Na, K-ATPase and caveolins 1 and 3 and <10% of β-actin. Treatment of hypothyroid rats with T₃ increased the relative abundance of both α1 and β1 Na, K-ATPase subunits in total membranes by 4- to 5-fold (with no change in caveolin-3), and resulted in 1.9-fold increase in enzyme activity. T₃-induced Na, K-ATPase subunits were preferentially distributed to the lighter fractions (#s 4, 5 and 6); and increased abundance of α1 and β1 were 34-70% and 43-68%, respectively. We conclude that the activity of Na, K-ATPase is not uniform in cardiac membranes, and while a significant amount of Na, K-ATPase is present in cardiac cholesterol-rich membrane fractions, the intrinsic activity is significantly less than the enzyme present in relatively cholesterol-poor membranes.     

Lipid membrane domain formation and alamethicin aggregation studied by calorimetry, sound velocity measurements, and atomic force microscopy

An experimental study of phosphocholine membranes made from one lipid, from mixtures of DPPC and DLPC, and also from lipids and small amounts of alamethicin is presented. We used atomic force microscopy to investigate the spatial organization and structure of lipid domains and also of the defects induced by the peptide. Alamethicin was found to alter the state of lipids in the gel state in a way that domains of fluid lipids are formed close to the defects. Differential calorimetry revealed phase characteristics of the lipid mixtures and the effect of small amounts of alamethicin on the phase behavior. It was also shown that the sound velocity profiles of the membranes suspensions can be well calculated from the heat capacity traces of the samples. This result confirms the correlation between the mechanical properties and the specific heat of membrane systems.     

Characterization of nascent HDL particles and microparticles formed by ABCA1-mediated efflux of cellular lipids to apoA-I

The nascent HDL created by ABCA1-mediated efflux of cellular phospholipid (PL) and free (unesterified) cholesterol (FC) to apolipoprotein A-I (apoA-I) has not been defined. To address this issue, we characterized the lipid particles released when J774 mouse macrophages and human skin fibroblasts in which ABCA1 is activated are incubated with human apoA-I. In both cases, three types of nascent HDL containing two, three, or four molecules of apoA-I per particle are formed. With J774 cells, the predominant species have hydrodynamic diameters of approximately 9 and 12 nm. These discoidal HDL particles have different FC contents and PL compositions, and the presence of acidic PL causes them to exhibit alpha-electrophoretic mobility. These results are consistent with ABCA1 located in more than one membrane microenvironment being responsible for the production of the heterogeneous HDL. Activation of ABCA1 also leads to the release of apoA-I-free plasma membrane vesicles (microparticles). These larger, spherical particles released from J774 cells have the same PL composition as the 12 nm HDL and contain CD14 and ganglioside, consistent with their origin being plasma membrane raft domains. The various HDL particles and microparticles are created concurrently, and there is no precursor-product relationship between them. Importantly, a large fraction of the cellular FC effluxed from these cells by ABCA1 is located in microparticles. Collectively, these results show that the products of the apoA-I/ABCA1 interaction include discoidal HDL particles containing different numbers of apoA-I molecules. The cellular PLs and cholesterol incorporated into these nascent HDL particles originate from different cell membrane domains.     

Rhodopsin and other proteins in artificial lipid membranes

Some basic aspects of incorporation of hydrophobic peptides and proteins in artificial lipid membranes are discussed. As examples valinomycin as a carrier model and gramicidin A as a channel former in lipid vesicles and in planar lipid membranes are presented.In the second part of the lecture some examples of incorporation of membrane proteins into lipid vesicles and planar lipid membranes are reported. The interaction with artificial lipid membranes of the Ca⁺ ATPase from the sarcoplasmic reticulum, of Rhodopsin, and of Bacteriorhodopsin is presented.Presented at the EMBO-Workshop on Transduction Mechanism of Photoreceptors, Jülich, Germany, October 4–8, 1976     

An unusual ultrastructural association of smooth membranes and glycogen particles: The glycogen body

Summary An electron microscope study of the epithelium of rabbit fallopian tube demonstrated a rarely described intracytoplasmic structure consisting of an array of smooth membranes associated with glycogen particles. This organelle is seen exclusively in the ciliated cells. A three-dimensional reconstruction of these glycogen bodies has been made from serial sections. The peripheral localization of the rough-surfaced membranes in continuity with intra-corpuscular smooth membranes, which have lost their granules, suggests a possible role for the rough membranes in the genesis of the smooth membranes of these glycogen bodies. The role of both the smooth and the rough membranes in glycogenesis and glycogenolysis is discussed.This investigation was made in part in the Laboratoire d'Hormonologie et de Cytologie Expérimentale, Hôpital Broca, Paris.     

Protein recovery from cell debris using rotary and tangential crossflow microfiltration

Protein recovery from a bacterial lysate was accomplished using microfiltration membranes in a flat crossflow filter and in a cylindrical rotary filter. Severe membrane fouling yielded relatively low long-term permeate flux values of 10(-4)-10(-3) cm/s (where I cm/s = 3.6 x 10(4) L/m(2) - h). The permeate flux was found to be nearly independent of transmembrane pressure and to increase with increasing shear rate and decreasing solids concentration. The flux increased with shear to approximately the one-third power or greater for the flat filter and the one-half power or greater for the rotary filter; the stronger dependence for the rotary filter is thought to result from Taylor vortices enhancing the back transport of debris carried to the membrane surface by the permeate flow. The average protein transmission or sieving coefficient was measured at approximately 0.6, but considerable scatter in the transmission data was observed. The largest sieving coefficients were obtained for dilute suspensions at high shear rate. The rotary filter provided higher fluxes than did the flat filter for dilute suspensions, but not for concentrated suspensions. (c) 1995 John Wiley & Sons, Inc.     

Spectral reflectance and integrity of cell membranes and chlorophyll relative to the concentration of airborne mineral elements in a lichen

To determine the environmental impact of oil-combustion pollutants and soil dust on a lichen, we examined the spectral reflectance of thalli of the epiphytic fruticose lichen, Ramalina duriaei , expressed as values of NDVI (the normalized difference vegetation index). We analyzed electrolyte leakage caused by degradation of cell membranes in terms of electric conductivity of water, apart from chlorophyll degradation, the latter expressed as changes in the A435 nm/A415 nm ratio to indicate the physiological status of the lichen. The concentrations of Al, Cr, Fe, K, Ni, P, sulfate-S, Ti and V in the lichen thallus were measured to quantify the degree of pollution. Thalli of R. duriaei , growing in a nature reserve on the periphery of a 40-year-old industrial town, Ashdod, in southwest Israel were compared with thalli of R. duriaei from an unpolluted forest in the northeastern part of the country transplanted to the polluted areas in and around the town. After an exposure for 10 months, many transplants exhibited lower NDVI values, higher electric conductivity values as well as a lower A435 nm/A415 nm ratio. The three physical/physiological parameters thus reflected severe injury in the lichen transplants. The concentrations of Al, Cr, Fe, Ni, sulfate-S, Ti and V in the lichen transplants were found to correlate inversely with the NDVI values, whereas the concentrations of Fe, Ni, Ti and V were found to correlate with electric conductivity. The decrease in the A435 nm/A415 nm ratio was found to correlate with high concentrations of Al, Fe, Ni, sulfate-S, Ti and V in the lichen transplants, whereas the concentration of K and P correlated with both the NDVI value and the A435 nm/A415 nm ratio. It is concluded that in situ thalli of R. duriaei , the only indigenous fruticose lichen growing in the region of Ashdod, are endangered by the presence of pollutants and by acid rain due to the combustion of heavy fuel oil.