Influence of cytosolic pH changes on the organisation of the endoplasmic reticulum in epidermal cells of onion bulb scales: Acidification by loading with weak organic acids

Summary The anastomosing ER system of epidermal cells of onion bulb scales is composed of three modifications: lamellar and tubular elements, located in the cell periphery, and long tubular stands located deeper in the cytoplasm. Cytoplasmic acidification of epidermal cells by loading with weak organic acids like acetic or propionic acid causes the decay of the lamellar elements and the disappearance of long tubular strands. Organelle movement is also inhibited. The effects depend on the pH of the incubation medium and on the administered acid concentration, and are characterized by a distinct lag phase of about 7 min. The induced ER changes are transient with adaptation starting after about 50min. Buffer components alone have little influence on the cellular ER organization within a pH-range of 4.0–8.0. However, the pH of the medium strongly affects the time course of the effects as well as recovery after omitting the administered acid. Both modulation and recovery occur more rapidly at neutral or slightly alkaline pH. Actin filaments, which play a major role in ER organization and organelle movement, are not affected by cytosolic acidification.Dedicated to the memory of Professor Oswald Kiermayer     

The stroma-vascular fraction of rat inguinal and epididymal adipose tissue and the adipoconversion of fat cell precursors in primary culture

The stroma-vascular fraction (SVF) of inguinal and epididymal fat pads of 4 week-old rats was studied by electron microscopy. Among the various cell types, endothelial cells and preadipocytes were found in both SVF, while mesothelial cells were only detected in the epididymal SVF. The resulting heterogeneity of primary culture and the adipoconversion of the fat cell precursors were studied in a serum-supplemented medium enriched with insulin (14.5 nM) and exogenous triglycerides. Despite the heterogeneity of the inoculum, the primary cultures were rather homogeneous, fat cell precursors being the main cell type. Distinctive contaminant fibroblast-like cells were observed in both cultures, whereas epithelial-like cells, which correspond most probably to mesothelial cells, were only found in epididymal cultures. Differentiation of fat cell precursors was assessed by the appearance of lipoprotein lipase (LPL) and glycerol-3-phosphate dehydrogenase (GPDH). LPL activity was found in the same level in cells of both deposits while GPDH activity was elevated in inguinal vs epididymal derived stroma-vascular cells. The different adipose conversion pattern of both cultures was confirmed by morphological quantification: the maturation of epididymal fat cell precursors was faster but less extensive. These differences could be related mainly to regional localization rather than to different maturation of the two fat deposits.     

Responses to water stress of apoplastic water fraction and bulk modulus of elasticity in sunflower (Helianthus annuus L.) genotypes of contrasting capacity for osmotic adjustment

The responses to water stress of the bulk modulus of elasticity () and the apoplastic water fraction were examined using six sunflower cultivars of differing capacity for osmotic adjustment (OA). Water stress did not affect the partitioning of water between apoplastic (ca. 20%) and symplastic fractions in leaves which expanded during the exposure to stress in any genotype. Hence, no genotype-linked effects on either the buffering of cell water status during stress or on the estimates of bulk leaf osmotic potential could be expected. Genotypes differed in the degree of change in (estimated from pressure/volume [P/V] curves) and OA (estimated using both ln RWC/ ln o plots and P/V curves) induced by exposure to stress. In three genotypes increased significantly (p=0.05) as a consequence of stress, in another three change were small. OA was the only attribute of the three examined that could have contributed to turgor maintenance under stress. There was a strong negative association between leaf expansion and degree of OA across genotypes (r=–0.91) and a strong positive one between OA and (r=0.94). However all genotypes evidenced some degree of OA. These results are consistent with part of the genotype differences in OA being attributable to variations in leaf expansion during exposure to stress.     

Enhancement of NMDA-mediated responses by cyanide

The effect of cyanide on NMDA-activated ion current and MK801 binding was studied in cultured rat hippocampal neurons. In microfluorometric analysis using fura-2, removal of extracellular Mg²⁺ resulted in a five-fold increase in NMDA-induced peak of [Ca²⁺]_i. One mM NaCN enhanced the peak NMDA responses in the presence, but not in the absence of extracellular Mg²⁺. Cyanide enhanced the immediate rise in [Ca²⁺]_i produced by NMDA, followed over a 1–5 min period by a gradual increase of [Ca²⁺]_i. Similar results were obtained in whole-cell patch clamp recordings from hippocampal neurons. One mM KCN enhanced the NMDA-activated current in the presence, but not in the absence of extracellular Mg²⁺. This effect was independent of cyanide-mediated metabolic inhibition since the recording pipette contained ATP (2 mM). In binding assays NaCN (1 mM) increased the binding affinity of [³H]MK-801 to rat forebrain membranes in the presence of Mg²⁺, whereas in the absence of Mg²⁺, NaCN did not influence binding. These results indicate that cyanide enhances NMDA-mediated Ca²⁺ influx and inward current by interacting with the Mg²⁺ block of the NMDA receptor. The effect of cyanide can be explained by an initial interaction with the Mg²⁺ block of the NMDA receptor/ionophore which appears to be energy-independent, followed by a gradual increase in Ca²⁺ influx resulting from cellular energy reserve depletion.Abbreviations NMDA N-Methyl-D-Aspartate - EAA excitatory amino acid - MK-801 (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohept-5,10-imine maleate     

Halide permeation through three types of epithelial anion channels after reconstitution into giant liposomes

Anion-selective channels from apical membranes of cultured CFPAC-1 cells were isolated and incorporated into giant liposomes for patch clamp recording. Liposomes were formed from L--lecithin by a dehydration-hydration method. Ion channels were characterized using the excised inside-out patch clamp configuration. The most commonly observed anion channels were similar to those observed in native epithelial tissues. The linear 20 pS Cl channel had the halide permeability sequence Cl^– > I^– Br^– > F^–, and showed anomalous mole-fraction behavior in solutions containing different proportions of Cl^– and F^–, ions. The autwardly rectifying Cl^– channel had the halide permeability sequence I^– > Br^– > Cl^– > F^–, and also showed anomalous molefraction behavior, indicating that both these channels probably contain multi-ion pores. The third, voltage-dependent anion channel showed at least five different substrates, had a conductance of 390 pS in the main state, and showed two types of kinetics, fast (openings and closings < 1 ms), and slow (openings and closings > 1 s). The channel was seen more frequently after reconstitution into giant liposomes than in intact cells. It was not selective amongst the halides, and there was no deviation from a linear dependence of relative current on molar fractions, indicating relatively simple permeation through the pore. Differences in halide permeabilities suggest that different anion channels may be related to different membrane proteins. Comparison with the chloride channel proteins isolated biochemically from epithelial cell membranes is discussed. Correspondence to: M. Duszyk     

Tetracyclines modulate cytosolic Ca2+ responses in the osteoclast associated with “Ca2+ receptor” activation

We report the effects of tetracycline analogues on cytosolic Ca²⁺ transients resulting from application of ionic nickel (Ni²⁺), a potent surrogate agonist of the osteoclast Ca²⁺ receptor. Preincubation with minocycline (1 mg/l) or a chemically modified tetracycline, 4-dedimethyl-aminotetracycline (CMT-1) (1 or 10 mg/l), resulted in a significant attenuation of the magnitude of the cytosolic [Ca²⁺] response to an application of 5 mM-[Ni²⁺]. Preincubation with doxycycline (1 or 10 mg/l) failed to produce similar results. In addition, application of minocycline alone (0.1–100 mg/l) resulted in a 3.5-fold elevation of cytosolic [Ca²⁺]. The results suggest a novel action of tetracyclines on the osteoclast Ca²⁺ receptor.     

Ion-selective Microelectrodes: Their Use and Importance in Modern Plant Cell Biology

The exact ion gradients across cellular membranes and their changes due to metabolic or transport processes can be best studied with the use of ion-selective microelectrodes. The last decade of research using ion-selective microelectrodes in intact cells has proven this technique to be indispensable for the investigation of a variety of physiological questions of regulatory processes, membrane transport, cellular signalling, developmental biology and plant nutrition. Their application to selected problems has led to numerous exciting observations, many of which have changed our view concerning cellular responses to environmental stimuli and in many instances have led to a new understanding of plant cell physiology. Since, with these electrodes, intracellular as well as extracellular free ion concentrations can be simultaneously detected with electrical transport parameters such as membrane potential and membrane conductance, they can be powerful tools in the hands of many plant cell biologists.     

Titles of related papers in other sections

Previous results have shown that the autoantibody eluted from the glomeruli of rats with active Heymann nephritis contain a population of antibodies not only to the putative autoantigen of the disease, gp330, but alos to plasminogen. Since gp330 has been shown to serve as a receptor for plasminogen, we have analyzed the effects of autoantibody on plasminogen-binding to gp330 and activation of plasminogen to plasmin by urokinase. Autoantibody does not inhibit the binding of plasminogen to gp330. The change in the conformation of plasminogen when its lysine-binding sites are occupied or after conversion to plasmin results in a significant decrease in autoantibody-binding. The most significant effect of autoantibody on this system is the inhibition of plasminogen activation to plasmin by urokinase. The binding of autoantibody to plasminogen acts as a competitive inhibitor of the reaction by apparently blocking access of urokinase to plasminogen's activation site. These results indicate that autoantibody obtained from the immune deposits in the glomeruli of rats with active Heyman nephritis does not inhibit the binding of plasminogen to gp330 but does significantly alter the urokinase catalyzed activation of plasminogen to plasmin.     

Root traits of perennial C4 grasses contribute to cultivar variations in soil chemistry and species patterns in particulate and mineral-associated carbon pool formation

Recent studies have indicated that the C₄ perennial bioenergy crops switchgrass (Panicum virgatum) and big bluestem (Andropogon gerardii) accumulate significant amounts of soil carbon (C) owing to their extensive root systems. Soil C accumulation is likely driven by inter- and intraspecific variability in plant traits, but the mechanisms that underpin this variability remain unresolved. In this study we evaluated how inter- and intraspecific variation in root traits of cultivars from switchgrass (Cave-in-Rock, Kanlow, Southlow) and big bluestem (Bonanza, Southlow, Suther) affected the associations of soil C accumulation across soil fractions using stable isotope techniques. Our experimental field site was established in June 2008 at Fermilab in Batavia, IL. In 2018, soil cores were collected (30 cm depth) from all cultivars. We measured root biomass, root diameter, specific root length, bulk soil C, C associated with coarse particulate organic matter (CPOM) and fine particulate organic matter plus silt- and clay-sized fractions, and characterized organic matter chemical class composition in soil using high-resolution Fourier-transform ion cyclotron resonance mass spectrometry. C₄ species were established on soils that supported C₃ grassland for 36 years before planting, which allowed us to use differences in the natural abundance of stable C isotopes to quantify C₄ plant-derived C. We found that big bluestem had 36.9% higher C₄ plant-derived C compared to switchgrass in the CPOM fraction in the 0–10 cm depth, while switchgrass had 60.7% higher C₄ plant-derived C compared to big bluestem in the clay fraction in the 10–20 cm depth. Our findings suggest that the large root system in big bluestem helps increase POM-C formation quickly, while switchgrass root structure and chemistry build a mineral-bound clay C pool through time. Thus, both species and cultivar selection can help improve bioenergy management to maximize soil carbon gains and lower CO₂ emissions.