Effects of dark- and light-induced proton gradients in thylakoids on the Q and B thermoluminescence bands

Thermoluminescence experiments have been carried out to study the effect of a transmembrane proton gradient on the recombination properties of the S₂ and S₃ states of the oxygen evolving complex with Q_A ^- and Q_B ^-, the reduced electron acceptors of Photosystem II. We first determined the properties of the S₂Q_A ^- (Q band), S₂Q_B ^- and S₃Q_B ^- (B bands) recombinations in the pH range 5.5 to 9.0, using uncoupled thylakoids. The, a proton gradient was created in the dark, using the ATP-hydrolase function of ATPases, in coupled unfrozen thylakoids. A shift towards low temperature of both Q and B bands was observed to increase with the magnitude of the proton gradient measured by the fluorescence quenching of 9-aminoacridine. This downshift was larger for S₃Q_B ^- than for S₂Q_B ^- and it was suppressed by nigericin, but not by valinomycin. Similar results were obtained when a proton gradient was formed by photosystem I photochemistry. When Photosystem II electron transfer was induced by a flash sequence, the reduction of the plastoquinone pool also contributed to the downshift in the absence of an electron acceptor. In leaves submitted to a flash sequence above 0°C, a downshift was also observed, which was supressed by nigericin infiltration. Thus, thermoluminescence provides direct evidence on the enhancing effect of lumen acidification on the S₃S₂ and S₂S₁ reverse-transitions. Both reduction of the plastoquinone pool and lumen acidification induce a shift of the Q and B bands to lower temperature, with a predominance of lumen acidification in non-freezing, moderate light conditions.Abbreviations 9-AA 9-aminoacridine - E_A activation energy - F₀ constant fluorescence level - F_M maximum fluorescence, when all PS-II centers are closed - F_V variable fluorescence (F_M–F₀) - PS I, PS II Photosystem I, photosystem II - PQ plastoquinone - TL thermoluminescence     

Phalaenopsis bellina (Rchb.f.) Christenson,a segregate from P. violacea Witte (Orchidaceae: Aeridinae)

A reevaluation of floral and vegetative morphology together with data from analysis of floral fragrance and flavonoid co-pigment chemistry suggest that specific status is warranted for the two kinds ofPhalaenopsis violacea Witte known informally as the “Bornean type” and the “Malayan type.”Phalaenopsis violacea var.bellina Rchb.f. is elevated to specific rank, providing a name for the “Bornean type” plants.     

Intrasynaptosomal Free Mg2+ Concentration Measured with the Fluorescent Indicator Mag-Fura-2: Modulation by Na+ Gradient and by Extrasynaptosomal ATP

Abstract: Synaptosomes can be loaded with mag-fura-2 without significant perturbation of their ATP content by incubation for 10 min at 37°C with 10 µM mag-fura-2 acetoxymethyl ester in Hanks'-HEPES buffer (pH 7.45). The intrasynaptosomal free Mg²⁺ concentration ([Mg²⁺]_i) was found to be dependent on external Mg²⁺ concentration, increasing from 0.8 to 1.25 mM when the concentration of Mg²⁺ in the incubation medium increased from 1 to 8 mM. Dissipation of the Na⁺ gradient across the plasma membrane of synaptosomes by treatment with the Na⁺ ionophore monensin (0.2 mM) or with veratridine (0.2 mM) and ouabain (0.6 mM) produced a moderate increase of [Mg²⁺]_i, from 1.0 to 1.2–1.3 mM in an incubation medium containing 5 mM Mg²⁺. Plasma membrane depolarization by incubation of synaptosomes in a medium containing 68 mM KCl and 68 mM NaCl had no effect on [Mg²⁺]_i. Reversal of the Na⁺ gradient by incubation of synaptosomes in a medium in which external Na⁺ was replaced by choline increased [Mg²⁺]_i up to 1.6 and 2.2 mM for extrasynaptosomal Mg²⁺ concentrations of 1 and 8 mM, respectively. We conclude that a Na⁺/Mg²⁺ exchange operates in the plasma membrane of synaptosomes. In the presence of Mg²⁺ in the incubation medium, extrasynaptosomal ATP, but not ADP or adenosine, increased [Mg²⁺]_i from 1.1 ± 0.1 up to 1.6 ± 0.1 mM. The nonhydrolyzable ATP analogue adenosine 5′-(βγ-imido)triphosphate antagonized the effect of ATP, but had no effect by itself on [Mg²⁺]_i. It is concluded that Mg²⁺ transport across the plasma membrane of synaptosomes is modulated by the activity of an ecto-ATPase or an ecto-protein kinase.     

Developmental asynchrony caused by steep temperature gradients does not impair pattern formation in the wasp,Pimpla turionellae L.

Generating developmental gradients by temperature gradients established within a developing organism is an easy, non-invasive technique to study physiological interdependencies between locally separated subsystems. A linear temperature gradient of about 10° C/mm was maintained up to 5 h in either direction along the long axis of a long-germ-type hymenopteran egg, which was simultaneously filmed by the 16 mm timelapse technique. The result was a dramatic desynchronization of development, which between the egg poles could reach up to 9.3 h relative to normal development. Within the same egg, up to seven mitotic waves (i.e. eight different nuclear generations) were observed at the same time, and the subsequent cellularization process was extremely asynchronous. The initial regions of the mitotic waves, the fountain flow of the ooplasm, and the gastrulation process were shifted towards the egg pole kept at higher temperatures. Developmental processes occurring successively in normal development now took place simultaneously, with either direction of the temperature gradient. For instance, while gastrulation had started in the warm region, midblastula transition and cellularization were in progress in the middle of the egg, and intravitelline nuclear multiplication occurred at the cold pole, by rapid and still biphasic cell cycles. In some respects, development resembled that of a short-germ-type insect egg. Nevertheless, the developmental processes were resynchronized after the temperature gradient was switched off. Surprisingly, the extreme desynchronization during early development did not affect the segment pattern of the resulting embryos. The technique of inducing well-defined developmental asynchronies might be applied in Drosophila to analyse the subtle interplay between maternal and zygotic gene activities described in this species.     

A link between transport and plasma membrane redox system(s) in carrot cells

Carrot (Daucus carota L.) cells grown in suspension culture oxidized exogeneous NADH. The NADH oxidation was able to stimulate K⁺ (⁸⁶Rb⁺) transport into cells, but it did not affect sucrose transport.N,N'-Dicyclohexyl-carbodiimide, diethylstilbestrol, and oligomycin, which only partially inhibited NADH oxidation, almost completely collapsed the K⁺ (⁸⁶Rb⁺) transport. Vanadate, which is less effective as an ion transport inhibitor, was less effective in inhibiting the NADH-driven transport of K⁺ (⁸⁶Rb⁺).p-Fluormethoxycarbonylcyanide phenylhydrazone inhibits the K⁺ transport over 90% including that induced by NADH. The results are interpreted as evidence that a plasma membrane redox system in root cells is closely associated with the ATPase which can drive K⁺ transport. Because of the inhibitor effects, it appears that membrane components common to the redox system and ATPase function in the transport of K⁺.     

Potassium accumulation in growing Methanobacterium thermoautotrophicum and its relation to the electrochemical proton gradient

Cultures of Methanobacterium thermoautotrophicum (Marburg) growing on media low in potassium accumulated the cation up to a maximal concentration gradient ([K⁺]_{intracellular}/[K⁺]_{extracellular}) of approximately 50,000-fold. Under these conditions, the membrane potential was determined by measuring the equilibrium distribution of the lipophilic cation (¹⁴C) tetraphenylphosphonium (TPP⁺). This cation was accumulated by the cells 350-to 1,000-fold corresponding to a membrane potential (inside negative) of 170–200 mV. The pH gradient, as measured by equilibrium distribution of the weak acid, benzoic acid, was found to be lower than 0.1 pH units (extracellular pH=6.8). The addition of valinomycin (0.5–1 nmol/mg cells) to the culture reduced the maximal concentration gradient of potassium from 50,000-to approximately 500-fold, without changing the membrane potential. After dissipation of the membrane potential by the addition of ¹²C-TTP⁺ (2 mol/mg cells) or tetrachlorosalicylanilide (3 nmol/mg cells), a rapid and complete efflux of potassium was observed.These data indicate that potassium accumulation in the absence of valinomycin is not in equilibrium with the membrane potential. It is concluded that at low extracellular K⁺ concentrations potassium is not accumulated by M. thermoautotrophicum via an electrogenic uniport mechanism.Non-common abbreviations TPP⁺ Tetra phenylphosphonium bromide - DTE Dithioerythritol - TCS 3,5,3,4-Tetrachlorosalycylanilide     

Magnetic separation in biotechnology

New developments in magnetic labelling techniques for cells and microspheres have extended the useful range of magnetic separation, particularly high gradient magnetic separation, into biotechnical areas. The basic magnetic principles involved are reviewed and representative samples of labelling techniques and results drawn from the past three years are presented. Illustrative examples of large scale operation in other industries are also presented, demonstrating the potential of the biotechnological applications.     

Floral development of Bougainvillea spectabilis Willd., Boerhaavia diffusa L. and Mirabilis jalapa L. (Nyctaginaceae)

Three morphological problems were investigated in three species of the Nyctaginaceae: epiphylly, phyllotaxis and placentation. Epiphylly, which occurs in Bougainvillea spectabilis , is the result of ontogenetic displacement resulting from the activity of an intercalary meristem at the base of the floral bract and the floral bud. Floral development of Bougainvillea spectabilis was compared with that of Boerhaavia diffusa and Mirabilis jalapa . Considerable variation occurs with regard to the number and arrangement of stamens. Five stamens are initiated simultaneously, alternate to the petals, in Mirabilis . In Bougainvillea , eight stamens arise sequentially at divergence angles suggestive of a 3/8 spiral. No developmental evidence was found to support the derivation of the eight stamens from a two whorled pentamerous androecium. Boerhaavia normally has only two stamens which most frequently are initiated toward opposite sides of the floral apex, but may also be formed in a 2/5 to 3/8 divergence. In some flowers only one or three stamens are formed. The gynoecium is formed in the same way in all three species: growth occurs in a crescent-shaped zone at the periphery of the floral apex thus producing the gynoecial wall. The single ovule, which is basal in the mature gynoecium, is formed from the gradual upgrowth and transformation of the floral apex and is developmentally terminal. Even the two-layered tunica is maintained as the floral apex is transformed into the ovule primordium. If 'carpel' is defined traditionally as a folded megasporophyll which bears and encloses ovule(s) then carpels are not present in the gynoecia of the three species studied. If 'carpel' is re-defined as an appendage which encloses ovule(s), then the gynoecia of the Nyctaginaceae are carpellate. However, the ovules remain cauline regardless of which definition is adopted.     

Identification of polypeptides encoded by the replication of resistance factor R100

Several polypeptides encoded by the resistance factor R100 were synthesized in a DNA-dependent protein synthesis system using a miniplasmid derived from R100 as a template. Nine polypeptides were detected. The locations of the genes for these polypeptides were investigated by using DNA restriction fragments as templates, and also by examining the effect of restriction endonuclease digestion of these templates on the synthesis of the polypeptides. The genes for seven of the polypeptides were identified or located by comparing the results with the known nucleotide sequence and restriction map of this region. Three of the polypeptides appeared to be encoded by the repA1, repA2 and repA3 genes, which are located in the region required for the replication of R100 and the expression of incompatibility. Four of the polypeptides were encoded by regions that are not required for the autonomous replication of R100 in Escherichia coli. One is the gene product of finO, which regulates the expression of the tra genes on R100.The miniplasmid used for these experiments carried one ISI sequence that has three potential genes. However, no polypeptide was detected that could be clearly demonstrated to be encoded by ISI.     

Characterization of prolamellar bodies and prothylakoids fractionated from wheat etioplasts

Prolamellar bodies and prothylakoids from etioplasts of wheat ( Triticum aestivum L. cv. Starke II, Weibull) were separated by sucrose density gradient centrifugation. Top-loaded and bottom-loaded sucrose gradients were compared. As a consequence of avoiding long time exposure of the membranes to low sucrose concentrations, separation in bottom-loaded gradients, as compared to separation in top-loaded gradients, resulted in a sharper and more narrow band of prothylakoids, and in better preservation of phototransformable protochlorophyllide, especially in the prothylakoids. In bottom-loaded gradients, the prothylakoids were found concentrated in a band at a density of 1.20 g'ml⁻¹. The prolamellar bodies were found at a density of 1.17 g'ml⁻¹. In top-loaded gradients the prothylakoids were found at a lower density than the prolamellar bodies. The prothylakoid fraction contained about 60% of the recovered protochlorophyllide and about 85% of the recovered protein. Absorption and fluorescence emission spectra revealed a higher amount of phototransformable protochlorophyllide, in relation to non-phototransformable, in the prolamellar body fraction than in the prothylakoid fraction. Polyacrylamide gel electrophoresis indicated a high proportion of protochlorophyllide reductase in the prolamellar bodies. Chloroplast ATPase (CF₁) was found predominantly in the prothylakoid fraction. Thus, our results strongly indicate the presence of phototransformable protochlorophyllide in the prolamellar bodies proper, while the main bulk of proteins are located in the prothylakoids.