Staining kinetics in single cells. Part II. Diffusion processes inside the cell

Applying hydrodynamic conditions, which certify a negligible influence of convective diffusion, the time-dependent uptake of thionin in lymphocytes, monkey kidney cells, and their separated nuclei was measured spectroscopically. Using fixed cell material the dye transport inside the cell is not hindered due to plasma membrane and cytoplasm. The staining rate depends on the dye concentration, the pretreatment of the cell, and on the electrolyte concentration of the dye solution. The mechanism of dye migration inside the cell is in accordance with a porous matrix model. The diffusion process takes place inside the pores and channels filled with liquid and is modified by adsorption of dye molecules on the walls of the pores. A dynamic reversible equilibrium exists between migrating dye molecules and the binding sites on the pore walls described by the Freundlich adsorption isotherm. The proposed model explains the observed order of reaction of the staining kinetics.     

Staining kinetics in single cells

Summary Applying hydrodynamic conditions, which certify a negligible influence of convective diffusion, the time-dependent uptake of thionin in lymphocytes, monkey kidney cells, and their separated nuclei was measured spectroscopically. Using fixed cell material the dye transport inside the cell is not hindered due to plasma membrane and cytoplasm. The staining rate depends on the dye concentration, the pretreatment of the cell, and on the electrolyte concentration of the dye solution. The mechanism of dye migration inside the cell is in accordance with a porous matrix model. The diffusion process takes place inside the pores and channels filled with liquid and is modified by adsorption of dye molecules on the walls of the pores. A dynamic reversible equilibrium exists between migrating dye molecules and the binding sites on the pore walls described by the Freundlich adsorption isotherm. The proposed model explains the observed order of reaction of the staining kinetics.     

Protective mechanisms in photosynthesis of Anabaena cylindrica

The role of O₂ photoreduction was studied in intact cells of normal and photobleached Anabaena cylindrica Lemm. strain PCC 7122. We found that O₂ photoreduction represents a protective mechanism against over-reduction of the photosyn-thetic electron transport chain only in normal Anabaena cells. This protective mechanism was not functioning in photobleached cells in spite of the increased rate of photosynthetic electron flow. A new electron acceptor, the induced reversible hydrogenase, is suggested to be operating in photobleached Anabaena cylindrica .     

Molecular cloning of the cyanobacterial adenylate cyclase gene from the filamentous cyanobacterium Anabaena cylindrica.

Molecular cloning of the structural gene for adenylate cyclase (cya) of the cyanobacterium Anabaena cylindrica was carried out by complementation of an Escherichia coli strain defective in the cya gene. The cya-defective strain produced significant amounts of cyclic AMP when it was transformed with the cya gene isolated from A. cylindrica. This gene encodes a polypeptide consisting of 502 amino acid residues (molecular weight, 55,300). The deduced primary protein structure showed that the carboxyl-terminal region of the adenylate cyclase of A. cylindrica shows strong structural similarity to the conserved regions of the adenylate cyclases of various eukaryotes. No similarity was found between the amino acid sequences of the cya gene of A. cylindrica and that of E. coli. A hydropathy plot suggests that this protein has two hydrophobic regions, a transmembrane span and a signal peptide. An antiserum specific to this adenylate cyclase was prepared by immunizing a rabbit with a glutathione S-transferase-adenylate cyclase fusion protein expressed in E. coli. This antiserum recognized a 55-kDa protein in Anabaena cell lysates. Subcellular fractionation analysis showed that A. cylindrica adenylate cyclase localized in the thylakoid membrane.     

The intracellular localisation of malate dehydrogenase in Anacystis nidulans

Abstract Malate dehydrogenase has been reported to be active as a Krebs cycle enzyme in Anabaena cylindrica and Anacystis nidulans [1,2] and as an enzyme of the glycollate pathway in Anabaena cylindrica [1,3]. This enzyme was also reported in Oscillatoria spp. [4] and in Nostoc muscorum [5]. The isoenzyme of eukaryotic organisms was known to participate in various metabolic pathways and to be localized in different subcellular organelles [6–9]. Kovatcheva and Bergman [5] have purified the enzyme from the reddish-brown 20 000 × g × 20 min supernatant. We have determined the intracellular distribution of malate dehydrogenese of Anacystis nidulans and present evidence that it is largely associated with the thylakoids. The significance of this study is discussed in terms of the dual role of cyanobacterial thylakoids in photosynthesis and respiration.     

Removal of lead from aqueous solutions on palm shell activated carbon

The performance of a commercially available palm shell based activated carbon to remove lead ions from aqueous solutions by adsorption was evaluated. The adsorption experiments were carried out at pH 3.0 and 5.0. The effect of malonic and boric acid presence on the adsorption of lead ions was also studied. Palm shell activated carbon showed high adsorption capacity for lead ions, especially at pH 5 with an ultimate uptake of 95.2mg/g. This high uptake showed palm shell activated carbon as amongst the best adsorbents for lead ions. Boric acid presence did not affect significantly lead uptake, whereas malonic acid decreased it. The diffuse layer surface complexation model was applied to predict the extent of adsorption. The model prediction was found to be in concordance with the experimental values.     

The kinetics and mechanism of lead (II) biosorptionby powderized Rhizopus oligosporus

The kinetics and mechanism of lead biosorption by powderized Rhizopus oligosporus were studied using shake flask experiment. The optimum biomass concentration and initial solution pH for lead sorption at initial lead concentrations ranging from 50–200 mg/l was obtained at 0.5 g/l and pH5, respectively. In term of the ratio of initial lead concentration to biomass concentration ratio, the highest lead adsorption was obtained at 750 mg/g which gave the maximum lead uptake capacity of 126 mg/g. The experimental data of lead sorption by R.oligosporus fitted well to the Langmuir sorption isotherm model, indicating that the sorption was similar to that for an ion-exchange resin. This means that the sorption is a single layer metal adsorption that occurred as a molecular surface coverage. This assumption was confirmed by the examination of lead sorption using transmission electron microscope and energy dispersive X-ray analysis, which showed that during sorption most of the lead was adsorbed on the surface of cell.     

Effects of long-term treatment with acetylene on nitrogen-fixing microorganisms.

Long periods of experimental incubation with acetylene led to a multifold enhancement of acetylene-reducing activity in Anabaena cylindrica, Anabaenopsis circularis, Rhodospirillum rubrum, and Azotobacter vinelandii. Rates of acetylene reduction showed a gradual increase and reached a peak after 2 to 6 h of continuous incubation under acetylene. Thereafter, enzyme activity rapidly declined. A similar enhancement of ethylene production was observed when pretreatment with acetylene was interrupted periodically by a brief exposure to ambient (or oxygen-free) atmosphere without acetylene although the decline of acetylene-reducing activity was less rapid. Pretreatment with acetylene depressed photosynthetic 14CO2 fixation and 15N2 incorporation in Anabaena cylindrica. It is concluded that assessments based on long-term experimental incubation with acetylene may grossly overestimate the actual quantities of fixed nitrogen in the field.     

Effects of long-term treatment with acetylene on nitrogen-fixing microorganisms.

Long periods of experimental incubation with acetylene led to a multifold enhancement of acetylene-reducing activity in Anabaena cylindrica, Anabaenopsis circularis, Rhodospirillum rubrum, and Azotobacter vinelandii. Rates of acetylene reduction showed a gradual increase and reached a peak after 2 to 6 h of continuous incubation under acetylene. Thereafter, enzyme activity rapidly declined. A similar enhancement of ethylene production was observed when pretreatment with acetylene was interrupted periodically by a brief exposure to ambient (or oxygen-free) atmosphere without acetylene although the decline of acetylene-reducing activity was less rapid. Pretreatment with acetylene depressed photosynthetic 14CO2 fixation and 15N2 incorporation in Anabaena cylindrica. It is concluded that assessments based on long-term experimental incubation with acetylene may grossly overestimate the actual quantities of fixed nitrogen in the field.     

The isocitrate dehydrogenase from cyanobacteria

The present communication describes the properties of isocitrate dehydrogenase in crude extracts from the unicellular Anacystis nidulans and from heterocysts and vegetative cells of Nostoc muscorum and Anabaena cylindrica. The activity levels of this enzyme are much higher in heterocysts than in vegetative cells of N. muscorum and A. cylindrica. Isocitrate dehydrogenase is virtually inactive in vegetative cells of A. cylindrica. The enzyme is negatively regulated by the reduction charge and scarcely affected by oxoglutarate in the three cyanobacteria. The inhibition by ATP and ADP is competitive with respect to isocitrate and NADP+ in A. cylindrica and N. muscorum and noncompetitive in A. nidulans. Isocitrate dehydrogenase from the three cyanobacteria seems to be a hysteretic enzyme. All the experimental data suggest that the major physiological role of isocitrate and the isocitrate dehydrogenase in heterocysts is not to generate reducing equivalents for N2-fixation. Oxoglutarate formed by the enzyme reaction is likely required for the biosynthesis of glutamate inside the heterocysts. Thioredoxin preparations from spinach chloroplasts or from A. cylindrica activate isocitrate dehydrogenase from either heterocysts or vegetative cells of A. cylindrica. Activation is completed within seconds and requires dithiothreitol besides thioredoxin. The thioredoxin preparation which activates isocitrate dehydrogenase also activates NADP+-dependent malate dehydrogenase from spinach chloroplasts or heterocysts of A. cylindrica. Isocitrate dehydrogenase from A. cylindrica is deactivated by oxidized glutathione. It is speculated that isocitrate dehydrogenase and thioredoxin play a role in the differentiation of vegetative cells to heterocysts.     

Evidence for direct repression of nitrogenase by ammonia in the cyanobacterium Anabaena cylindrica

The nitrogenase activity of the cyanobacterium Anabaena cylindrica was repressed upon addition of ammonium salts after preincubation in the presence of a concentration of L-methionine-DL-sulfoximine sufficient to totally inhibit glutamine synthetase. Repression was also observed when urea was added to cells in the presence of the glutamine synthetase inhibitor. Measurements of ammonia concentrations were made in each case and provided evidence that ammonia itself is a primary regulator of nitrogenase levels in A. cylindrica.     

几种固氮蓝藻的固氮酶活性及其某些特性

对三种固氮蓝藻:固氮鱼腥藻(水生686)、柱孢鱼腥藻和鱼腥藻7120的整细胞及无细胞抽提液的固氮酶活性,进行了比较研究。水生686的整细胞酶活虽然不低(51.9毫米乙烯峰高/光密度/30分),仅次于柱孢鱼腥藻,但其无细胞抽提液的酶活却最低。这可能与它含有大量藻胶有关。研究了Mn++、Fe++对蓝藻固氮酶的作用,以及测定其在不同酶浓度下的反应动力学表明:柱孢鱼腥藻中不存在象深红螺菌中所看到的那种激活因子。用甲苯-乙醇溶液处理藻细胞,对固氮酶作原位测定,探索了它的氧损伤及氧保护机理。       

Siderophore-mediated iron uptake in different strains of Anabaena sp.

Anabaena sp. strain 6411, which produces the dihydroxamate siderophore schizokinen to facilitate iron uptake, is also capable of using the related siderophore aerobactin. The two siderophores compete for the same iron transport system, but there is a markedly higher affinity for ferric schizokinen than for ferric aerobactin. The trihydroxamate siderophore ferrioxamine B is far less effective as an iron donor in this organism. Anabaena sp. strain 7120 appears to be closely related to strain 6411. It synthesizes schizokinen as its major siderophore and shows rates of iron uptake from ferric schizokinen, ferric aerobactin, and ferrioxamine B which are similar to those observed with strain 6411. Anabaena cylindrica Lemm. 7122 and 1611, on the other hand, differ from strain 6411. In contrast to schizokinen, the hydroxamate which they produce in response to iron starvation cannot be extracted with water from the organic layer and does not support the growth of the siderophore auxotroph Arthrobacter flavescens JG-9. Strain 7122 can use its endogenous siderophore or schizokinen to promote iron uptake, but at 50-fold-lower rates than are observed with Anabaena sp. strain 6411 or 7120.     

APPLICATION OF GAS-LIQUID CHROMATOGRAPHY TO STUDY OF THE ENVELOPE LIPIDS OF HETEROCYSTS12

Gas-liquid chromatography of algal extracts provides a sensitive assay for their content of 1-(O-α-d -glycopyranosyl)-3, 25-hexacosanediol, the principal glycolipid from the envelope of heterocysts of Anabaena cylindrica. Different patterns of lipids were found in the heterocysts of 5 species of Anabaena and Nostoc. Lipids with comparable chromatographic properties were detected in Gloeocapsa.     

Modes of reduction of nitrogen in heterocysts isolated from Anabaena species.

N2 fixation (acetylene reduction) has been studied with heterocysts isolated from Anabaena cylindrica and Anabaena 7120. In the presence of ATP and at very low concentrations of sodium dithionite, reducing equivalents for activity of nitrogenase in these cells can be derived from several compounds. In the dark, D-glucose 6-phosphate, 6-phosphogluconate and DL-isocitrate support acetylene reduction via NADPH. In the light, reductant can be generated by Photosystem I.     

Theoretical study on temperature dependence of cellular uptake of QDs nanoparticles

Cellular uptake kinetics of nanoparticles is one of the key issues determining the design and application of the particles. Models describing nanoparticles intrusion into the cell mostly take the endocytosis process into consideration, and the influences of electrical charges, sizes, concentrations of the particles have been investigated. In this paper, the temperature effect on the cellular uptake of Quantum Dots (QDs) is studied experimentally. QDs are incubated with the SPCA-1 human lung tumor cells, and the nanoparticles on the cell membrane and inside the cell are quantified according to the fluorescence intensities recorded. It is found that the amounts of nanoparticles attached onto the cell membrane and inside the cell both increase with temperature. Based on the experimental results, a model is proposed to describe the cellular uptake dynamic process of nanoparticles. The process consists of two steps: nanoparticles adsorption onto the cell membrane and the internalization. The dynamic parameters are obtained through curve fitting. The simulated results show that the internalization process can be categorized into different phases. The temperature dependent internalization rate constant is very small when below 14?°C. It increases distinctly when temperature rises from 14?°C to 22?°C, but there is no evident increase as temperature further increases above 22?°C. Results show that by incorporating a temperature-independent internalization factor, the model predictions well fit the experimental results.     

Purification and characterization of thioredoxin from the N2-fixing cyanobacterium Anabaena cylindrica

Thioredoxin has been purified to homogeneity from the cyanobacterium Anabaena cylindrica. The protein consists of a single polypeptide chain with a relative molecular mass of about 11 680 which has two cysteine residues (residues 31 and 34) in the sequence-Cys-Gly-Pro-Cys- and an isoelectric point at pH 4.55. The N-terminal amino acid sequence of 39 residues shows distinct homologies with the sequences of Escherichia coli and Corynebacterium nephridii thioredoxins. Anti-(A. cylindrica thioredoxin) antiserum was used to quantify the thioredoxin which constituted about 0.22% of the soluble protein in cell-free extracts of N2-fixing, NO3- -grown or NH4+-grown A. cylindrica. Activation of fructose-1,6-bisphosphatase of A. cylindrica, activation of glutamine synthetase and NADP+-dependent malate dehydrogenase of the green alga Scenedesmus obliquus but not of A. cylindrica, and deactivation of glucose-6-P dehydrogenase of the cyanobacterium Anabaena variabilis were all achieved using the same thioredoxin species. No other thioredoxin species were detected in extracts of A. cylindrica when examined for the activation of these enzymes.     

Copper adsorption kinetics of cultured algal cells and freshwater phytoplankton with emphasis on cell surface characteristics

Comparative studies were carried out on the adsorption of copper by a range of laboratory-cultured algae and freshwater phytoplankton samples. The level of surface mucilage associated with the cultured algae ranged from high (Anabaena spiroides, Eudorina elegans) to moderate (Anabaena cylindrica, Microcystis aeruginosa) to complete absence (Chlorella vulgaris, Asterionella formosa, Aulacoseira varians, Ceratium hirundinella). With laboratory cultures, the rapid uptake, EDTA release and quantitative similarity between living and dead (glutaraldehyde-fixed) algae were consistent with physical binding of Cu at the cell surface. The higher Cu adsorption per unit surface area and longer adsorption time of mucilaginous algae in the time-course study, and the relatively high level of Cu bound to mucilage found by X-ray microanalysis suggest that mucilage played an important role in metal binding. For all species examined, Cu adsorption kinetics (external Cu concentrations 1 to 1000 mg L⁻¹) showed a clear fit to the Freundlich, but not the Langmuir isotherm, indicating a monolayer adsorption model with heterogenous binding sites. The Freundlich adsorption capacity constant (K_f) was higher in mucilaginous (3.96–12.62) compared to nonmucilaginous (0.36–3.63) species, but binding intensity (Freundlich constant 1/n) did not differ between the two cell types. The results suggest that mucilaginous algal species may have potential as biosorbents for treatment of industrial effluents containing heavy metals. Investigation of the Cu adsorption behavior of four mixed phytoplankton samples also revealed a good fit to the Freundlich, but not the Langmuir, isotherm. Freundlich constants were in the range 2.3–3.2 for samples dominated by Chlorophyta, Bacillariophyta and Cyanophyta, but recorded a value of 7.4 in the sample dominated by Dinophyta. Comparison with data from laboratory monocultures suggested that the adsorption kinetics of mixed environmental phytoplankton samples cannot be predicted simply in terms of the major algal species.     

Movement of Carbon from Vegetative Cells to Heterocysts in Anabaena cylindrica

Radioactive carbon assimilated by vegetative cells of Anabaena cylindrica in the light passed via an intrafilamentous route into heterocysts in the dark. After several hours, label per heterocyst approximated label per vegetative cell. Much of the label entering heterocysts was not available for diffusional exchange back into vegetative cells.