Correlations between the thermal stability of chloroplast (thylakoid) membranes and the composition and fluidity of their polar lipids upon acclimation of the higher plant,Nerium oleander,to growth temperature

The thermal stability of excitation transfer from pigment proteins to the Photosystem II reaction center of Nerium oleander adjusts by 10 Celsius degrees when cloned plants grown at 20°C/15°C, day/night growth temperatures are shifted to 45°C/32°C growth temperature or vice versa. Concomitant with this adjustment is a decrease in the fluidity of thylakoid membrane polar lipids as determined by spin labeling. The results are consistent with the hypothesis that there is a limiting maximum fluidity compatible with maintenance of native membrane structure and function. This limiting fluidity was about the same as for a number of other species which exhibit a range of thermal stabilities. Inversely correlated shifts in lipid fluidity and thermal stability occurred during the time course of acclimation of N. oleander to new growth temperatures. Thus, the temperature at which the limiting fluidity was reached changed during acclimation while the limiting fluidity remained constant. Although the relative proportion of the major classes of membrane polar lipids remained constant during adjustments in fluidity, large changes occured in the abundance of specific fatty acids. These changes were different for the phospho- and galacto-lipids suggesting that the fatty acid composition of these two lipid classes is regulated by different mechanisms. Comparisons between membrane lipid fluidity and fatty acid composition indicate that fluidity is not a simple linear function of fatty acid composition.     

Ubiquinol-cytochrome c oxidoreductase of higher plants. Isolation and characterization of the bc1 complex from potato tuber mitochondria.

A procedure is described for isolation of active ubiquinol-cytochrome c oxidoreductase (bc1 complex) from potato tuber mitochondria using dodecyl maltoside extraction and ion exchange chromatography. The same procedure works well with mitochondria from red beet and sweet potato. The potato complex has at least 10 subunits resolvable by gel electrophoresis in the presence of dodecyl sulfate. The fifth subunit carries covalently bound heme. The two largest ("core") subunits either show heterogeneity or include a third subunit. The purified complex contains about 4 mumol of cytochrome c1, 8 mumol of cytochrome b, and 20 mumol of iron/g of protein. The complex is highly delipidated, with 1-6 mol of phospholipid and about 0.2 mol of ubiquinone/mol of cytochrome c1. Nonetheless it catalyzes electron transfer from a short chain ubiquinol analog to equine cytochrome c with a turnover number of 50-170 mol of cytochrome c reduced per mol of cytochrome c1 per s, as compared with approximately 220 in whole mitochondria. The enzymatic activity is stable for weeks at 4 degrees C in phosphate buffer and for months at -20 degrees C in 50% glycerol. The activity is inhibited by antimycin, myxothiazol, and funiculosin. The complex is more resistant to funiculosin and diuron than the beef heart enzyme. The optical difference spectra of the cytochromes were resolved by analysis of full-spectrum redox titrations. The alpha-band absorption maxima are 552 nm (cytochrome c1), 560 nm (cytochrome b-560), and 557.5 + 565.5 nm (cytochrome b-566, which has a split alpha-band). Extinction coefficients appropriate for the potato cytochromes are estimated. Despite the low lipid and ubiquinone content of the purified complex, the midpoint potentials of the cytochromes (257, 51, and -77 mV for cytochromes c1, b-560, and b-566, respectively) are not very different from values reported for whole mitochondria. EPR spectroscopy shows the presence of a Rieske-type iron sulfur center, and the absence of centers associated with succinate and NADH dehydrogenases. The complex shows characteristics associated with a Q-cycle mechanism of redox-driven proton translocation, including two pathways for reduction of b cytochromes by quinols and oxidant-induced reduction of b cytochromes in the presence of antimycin.     

Effects of stable flies (Diptera: Muscidae) and heat stress on weight gain and feed efficiency of feeder cattle.

Cattle respond to the feeding of stable flies, Stomoxys calcitrans (L.), by bunching to protect their front legs. This bunching can increase heat stress which indirectly accounts for much of the reduction in cattle weight gains. We used fly-screened, self-contained feedlot pens which allowed regulation of fly populations feeding on cattle. The indirect fly effects (bunching and heat stress) accounted for 71.5% of the reduced weight gain. The direct effect of the biting flies and energy loss involved in fighting flies accounted for 28.5% of the reduced weight gain.     

Carrier-like behaviour from a static but electrically responsive model pore.

Because of the low dielectric constant of most proteins and lipids, the electric field of an ion passing through a narrow pore is long range and will interact with neighbouring ionizable residues of the channel protein. The electrical structure of the channel may thus change transiently in response to an ion passing through the pore. Model calculations then reveal that the ratio of the unidirectional ion fluxes may approach 1 as expected for a carrier or shuttling ionophore rather than the Ussing ratio expected for a pore. Saturation behaviour also becomes carrier-like. Computer simulation is reported showing a continuous variation between pore-like and carrier-like behaviour as the parameters of the system are allowed to change smoothly.     

A hybrid protein of urokinase growth-factor domain and plasminogen-activator inhibitor type 2 inhibits urokinase activity and binds to the urokinase receptor.

The binding of urokinase-type plasminogen activator (uPA) to its specific cell-surface receptor (uPAR) localises the proteolytic cascade initiated by uPA to the pericellular environment. Inhibition of uPA activity or prevention of uPA binding to uPAR might have a beneficial effect on disease states wherein this activity is deregulated, e.g. cancer and some inflammatory diseases. To this end, a bifunctional hybrid molecule consisting of the uPAR-binding growth-factor domain of uPA (amino acids 1-47; GFuPA) at the N-terminus of plasminogen-activator inhibitor type 2 (PAI-2) was produced in Saccharomyces cerevisiae. The purified protein inhibited uPA with kinetics similar to placental or recombinant PAI-2 and was also found to bind to U937 cells and to FL amnion cells. GFuPA-PAI-2 competed with uPA, the N-terminal fragment of uPA and a proteolytic fragment of uPA (amino acids 4-43) in cell binding experiments, indicating that the molecule bound to the cells via uPAR. Hence, both the uPA-inhibitory and uPAR-binding domains of the hybrid molecule were functional, demonstrating the feasibility of the novel concept of introducing an unrelated, functional domain onto a member of the serine-protease-inhibitor superfamily.     

Binding of purified Bacillus sphaericus binary toxin and its deletion derivatives to Culex quinquefasciatus gut: elucidation of functional binding domains.

Highly larvicidal strains of Bacillus sphaericus produce a binary toxin composed of 51 and 42 kDa proteins which binds to sharply delineated regions of the gastric caecum and posterior midgut of susceptible larvae of the mosquito Culex quinquefasciatus. To investigate the role of the individual subunits and the organization of functional binding regions within the toxin, plasmids were constructed for the expression in Escherichia coli of the toxin proteins and their NH2- and COOH-terminal deletion derivatives as fusions with glutathione S-transferase (GST). Toxin proteins were purified by affinity chromatography followed by cleavage from the GST carrier with thrombin. The LC50 values for the purified toxin proteins and their deletion derivatives were determined. The binding patterns of fluorescently labelled toxin suggested that the 51 kDa protein is the primary binding component of the toxin and mediates the regional binding and internalization of the 42 kDa protein. Examination of the toxin deletion derivatives revealed that the NH2-terminal region of the 51 kDa protein was required for binding to the larval gut, whilst the COOH-terminal region was responsible for interacting with the 42 kDa protein. Toxicity was strongly correlated with the subsequent internalization of the toxin, probably by endocytosis.     

Modulation of inwardly rectifying Na+-K+ channels by serotonin and cyclic nucleotides in salivary gland cells of the leech,Haementeria

Summary The electrically excitable salivary cells of the giant Amazon leech, Haementeria, display a time-dependent inward rectification. Under voltage clamp, hyperpolarizing steps to membrane potentials negative to about –70 mV were associated with the activation of a slow inward current (I _h) which showed no inactivation with time. The time course of activation of I _hwas described by a single-exponential function and was strongly voltage dependent. The activation curve of_hranged from –72 to –118 mV, with half-activation occurring at –100 mV. Ion-substitution experiments indicated that I _his carried by both Na⁺ and K⁺ ions. 5-Hydroxytryptamine (5-HT) increased the amplitude of I _hand its rale of activation. It also produced a positive shift of the activation curve of the conductance underlying I _h G_hwithout altering the slope factor, thus indicating that the voltage dependence of I _hwas modulated by 5-HT. Cs⁺ blocked both I _hand the 5-HT-polentiated current in a voltage-independent manner, whereas Ba²⁺ had little effect. It is concluded that 5-HT increases I _hby modulating the inwardly rectifying Na⁺-K⁺ channels in the salivary cells. The effect of 5-HT may be mediated by an increase in adenylate cyclase activity since I _hwas increased by 8-bromocyclic AMP and by the phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthine. In contrast, I _hwas reduced by 8-bromo-cyclic GMPand by zaprinast (an inhibitor of cyclic GMP-scnsitive phosphodieslerase). Cyclic GMP itself also reduced I _h, and the effect was specific to the 3,5 form; 2,3-cyclic GMP was inactive. The results suggest that the inward-rectifier channel may be modulated in opposite directions by cyclic AMP and cyclic GMPThis work was supported by a grant from the Science and Engineering Research Council (no. GR/F/17087). We are grateful to the SmithKline (1982) Foundation for provision of a pulse generator     

Measurements of the Engagement of Cyanide-Resistant Respiration in the Crassulacean Acid Metabolism Plant Kalanchoë daigremontiana with the Use of On-Line Oxygen Isotope Discrimination

Discrimination against ¹⁸O during dark respiration in tissues of Kalanchoë daigremontiana, Medicago sativa, and Glycine max was measured using an on-line system that enabled direct measurements of the oxygen fractionation of samples in a gas-phase leaf disk electrode unit. Discrimination factors for cytochrome pathway respiration were 18.6 to 19.8%_o for all tissues. However, discrimination in cyanide-resistant respiration was significantly higher in green tissues (30.4-31.2%_o) compared with nongreen tissues (25.3-25.9%_o). Using these discrimination factors, the partitioning of electron transport to these pathways was calculated from measurements of discrimination in the absence of inhibitors. Changes in flux through the alternative pathway were measured during the light and dark phases of Crassulacean acid metabolism in leaf disks of K. daigremontiana. The flux of electrons through the alternative pathway was higher during deacidification than during the other phases of Crassulacean acid metabolism. The increase in alternative pathway electron flux accounted for all of the increased respiration in the light phase. Despite this increase, simultaneous measurements of malate concentration and respiratory flux confirm that only a small proportion of the total malate decarboxylation occurs in the mitochondria.     

Preinfection cell wall formation in roots and developing nodules ofAlnus rubra Bong

Summary The establishment of actinorhizal root nodules involves penetration of host cell walls and intracellular colonization by the nitrogen-fixing endosymbiont,Frankia (Actinomycetales). In the early stages of the infection process inAlnus, unusual cell walls with undulate profiles were observed in root tip meristematic derivatives, and in early (preinfection) derivatives of the nodule lobe meristem, inFrankia-inoculated plants. The irregular cell walls attached obliquely to preexisting walls, but were not discontinuous. Serial sections revealed that the unusual walls divided two daughter cells. Microtubules in bundled arrays were abundant near the undulate walls, and radiated in several planes. In the root tips, the anomalous cell walls were observed within one day of inoculation withFrankia.     

Assessment of inter- and intra-population variation in quinoline alkaloid profile of juvenile shoot cultures of Cinchona ledgeriana: effect of method of nutrient delivery on alkaloid profile

Intra-population quinoline alkaloid profiles surveying quinine, quinidine, cinchonine and cinchonidine were determined for each of five populations of Cinchona ledgeriana grown as shoot-culture for 125 days. No significant difference in respect of mean alkaloid content between populations was detected. In contrast, there was considerable between-seedling variation in alkaloid content within each population. When nutrients were delivered to shoot-cultures in droplet form by means of an aerosol spray (as compared to the supply of nutrients direct from agar-or liquid-based reservoirs) alkaloid profile was greatly perturbed; most notable in this respect was a four-fold increase in the production of cinchonidine concomitant with a four-fold decrease in the production of cinchonine. These data are discussed with reference to the optimisation of quinoline alkaloid production by juvenile shoot-cultures of Cinchona ledgeriana.