Novel ultramicrobacterial isolates from a deep Greenland ice core represent a proposed new species, Chryseobacterium greenlandense sp. nov.

Three novel orange, ultramicrobacterial isolates, UMB10, UMB14, and UMB34^T were isolated from enrichment cultures inoculated with a melted 3,043 m deep Greenland ice core sample. Phylogenetic analysis of the 16S rRNA gene sequences indicated that the isolates belonged to a single species within the genus Chryseobacterium. They were most closely related to Chryseobacterium aquaticum (99.3%), Chryseobacterium soli (97.1%), and Chryseobacterium soldanellicola (96.9%). Genomic hybridization showed low levels of relatedness between UMB34^T and C. aquaticum and C. soldanellicola (19–30%) and C. soli and Chryseobacterium jejuense (45–56%). Comparative genomic fingerprinting analysis using the enterobacterial repetitive intergenic consensus (ERIC) sequence showed nearly identical banding patterns for the three isolates and these patterns were distinct from those of C. aquaticum, C. soldanellicola, C. soli, and C. jejuense. The cells were short rods, lacked flagella, had cell volumes of <0.1 μm³, formed buds and smaller protrusions (blebs), produced copious extracellular material and a flexirubin type pigment. UMB34^T produced acids from carbohydrates and utilized glucose and maltose although it did not assimilate mannose. The DNA G + C was 39.6–41.6 mol%. Based on the differences from validly named Chryseobacterium species, it was concluded that these isolates represent a new species for which the name, Chryseobacterium greenlandense is proposed. The type strain is UMB34^T (=CIP 110007T = NRRL B-59357).     

The application of a biotin-anti-biotin gold technique providing a significant signal intensification in electron microscopic immunocytochemistry: a comparison with the ultrasmall immunogold silver staining procedure

 A three-step biotin-anti-biotin gold-detection system (method A) has been applied for ultraimmunocytochemistry using ultrasmall colloidal gold (0.8 nm) linked to anti-biotin antibodies which were visualized and enhanced by silver reduction. The reactivity for glucagon in human pancreatic islets and for cytochrome-c oxidase in heart mitochondria has been compared to a two-step ultrasmall immunogold technique (method B). For both antigens, method A provided significantly higher labelling indices (P<0.001): the labelling density for cytochrome-c oxidase was 223/μm² using method A and 78/μm² using method B. For glucagon, the labelling density was 1455/μm² with method A and 322/μm² with method B. The results demonstrate that the silver-intensified biotin-anti-biotin gold-detection system is a valuable immunocytochemical method for signal enhancement. The method utilizes biotinylated antibodies from different species, allowing its broad application at the electron microscopic level. Accepted: 24 June 1997     

Ultrastructural organization and development cycle of soil ultramicrobacteria belonging to the class <Emphasis Type="Italic">Alphaproteobacteria</Emphasis>

Gram-negative chemoorganotrophic soil ultramicrobacteria (UMB), strains NF1 and NF3, have been isolated. In their development cycle, the strains formed small coccoid cells of 400–800 nm and ultrasmall cells of 200–300 nm. Phylogenetically, the strains NF1 and NF3 belong to Alphaproteobacteria and are close to the type strain of the recently described species Kaistia adipata. The ultrastructure of UMB cells has been studied using ultrathin sections and freeze-fracturing. It has been shown that the structure of UMB cell walls is of the gram-negative type; the outer membrane and peptidoglycan layer are well differentiated. The cell surface has numerous protrusions (prosthecae) of conical or spherical shape filled with the contents of the periplasm. The formation of unusual cellular structures (not occurring in known free-living bacteria) is a feature of UMB; these include the following: (a) piles of rod-like subunits, ca. 30 Å in diameter and 150–250 Å in length; (b) long bunches (up to 300–400 Å) comprised of filamentous subunits; and (c) large electron-dense spherical bodies (up to 200–300 Å in diameter) localized in the periplasm. A distinctive feature of UMB is their ability to grow as facultative parasites on living cyanobacterial (CB) cells. In this case, three types of interaction between UMB and CB have been revealed: (1) adsorption of UMB cells on the surface of CB cells; (2) penetration of UMB into polysaccharide sheathes; and (3) penetration of UMB into CB cytoplasm. UMB cells have been shown to reproduce by budding, with buds (up to 2–3) located directly on the mother cell, without formation of intermediate hyphae.     

Effects of cell culture density on phagocytosis parameters in IC-21 macrophages

Cell culture density is shown to alter the parameters characterizing phagocytic activity of cells in vitro. Phagocytosis index (PI, mean number of beads per cell in the bead-containing population) and phagocytosis percent (PP, percentage of bead-containing cells in cell population under study) for IC-21 macrophages incubated in the presence of non-opsonized 2-μm fluorescent latex beads were determined using fluorescent microscopy and ImageJ software specially adapted for the purpose. Under control conditions (DMEM without serum), increase in cell culture density was accompanied with a decrease of both parameters of the phagocytic activity. At a mean density of 4 cells/10⁵ μm² (9 cells per a viewfield) PI was 7.1 ± 0.2 beads/cell and at 20 cells/10⁵ μm² (40 cells per a viewfield) PI dropped to 4.6 ± 0.1 beads/cell. PP was less sensitive, varied in the range of 95–100% but also decreased as the cell density grew. At any density, PI was 1.5–2 times higher than the expected value (number of beads per μm² × cell contour area); apparently this divergence can be accounted for by cell locomotion and capture of a larger number of beads than could drop onto a motionless cell with a constant contour area. Increase in cell density was also accompanied by a decrease of the cell contour area (S _c), which amounted to 750 ± 16 μm² at a density of 4 cells/10⁵ μm² and 346 ± 4 μm² at a density of 20 cells/10⁵ μm². As the bead concentration was the same in all experiments, density-dependent decrease in PI and PP may be related with the observed decrease in cell contour area. Yet, the bead number per cell area unit (PI/S _c) was bigger at higher density and PI/S _c was higher in cells with smaller S _c. Thus, individual (specific) activity of the cells did not lessen with an increase of the cell culture density in the range studied (4–20 cells/10⁵ μm²). Reduction of the cell contour area may reflect alteration in cell adhesion to the substrate as well as competitive relations between adhesion and phagocytic processes. The data obtained imply that cell culture density has to be controlled as a factor notably altering the phagocytic activity parameters. The effects of serum, methyl-β-cyclodextrin, and carbenoxolon reported earlier [Golovkina et al. 2009. Biol membrany. 26 (5), 379–386] are re-evaluated and confirmed here.     

Novel ultramicrobacteria,strains NF4 and NF5, of the genus <Emphasis Type="Italic">Chryseobacterium</Emphasis>: Facultative epibionts of <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Two strains, NF4 and NF5, of a yellow-colored gram-negative bacterium were isolated from sediments of Lake Baikal and from old oil sludge of the Nizhnekamsk oil-processing plant. The cells of the strains are ultrasmall coccoids or short rods, measuring 0.2–0.4 × 0.2–0.5 μm; the average cell volume ranges from 0.004 to 0.04 μm³. A considerable proportion (30–60%) of cells have nanometer dimensions (180–300 nm in diameter and 0.004–0.02 μm³ in volume). The new isolates are thus among the smallest representatives of presently known free-living ultramicrobacteria. The two studied isolates are gram-negative nonmotile cells possessing a pronounced outer membrane. The cells do not have flagella and are not capable of gliding motility. They divide by constriction, budding, and multiple septation. The multiplicity of reproduction mechanisms results in a high degree of cell polymorphism. The isolates are chemoorganotrophic, aerobic, psychrotolerant, oxidase- and catalase-positive. Their characteristic trait is the absence of extracellular hydrolytic enzymes, such as proteases, lipases, pectinases, and cellulases. Menaquinone MK-6 is the main respiratory quinone; the flexirubin pigment was not detected. The G + C contents of the DNA of strains NF4 and NF5 are 40.8 and 40.5 mol %, respectively. The DNA-DNA hybridization level of strains NF4 and NF5 was close to 100%. Analysis of the 16S rRNA gene sequences and the fatty acid compositions showed that the isolates are most closely related to certain representatives of the genus Chryseobacterium (C. solincola, C. antarcticum, and C. jeonii). However, the differences in the 16S rRNA gene sequences, as well as in the phenotypic properties, such as formation of ultrasmall cells, the absence of extracellular hydrolases, oligotrophy, and the capacity for epibiosis on bacterial cells, suggest that the studied strains belong to a new species of the genus Chryseobacterium. The capacity for epibiosis, i.e., the ability to exist in a tightly adhered state on the surfaces of host Bacillus subtilis cells, is a peculiar trait of the studied isolates. It is assumed that adhesion of the cells of strains NF4 and NF5 (members of the phylum Bacteroidetes) occurs via by the same unique mechanism as the mechanism that we previously described for representatives of Alphaproteobacteria (Kaistia sp., NF1, and NF3), which use polysaccharide chains equipped with sticky granules as trapping and constricting cords.     

The influence of growth conditions on the cell dry weight per unit biovolume of <Emphasis Type="Italic">Klebsormidium flaccidum</Emphasis> (Charophyta), a typical ubiquitous soil alga

The dry weight per unit biovolume of 810 single, living cells of the ubiquitous soil algae Klebsormidium flaccidum from 80 experiments were determined using a Mach–Zehnder double-beam interference microscope. Different substrates such as agarized nutrient solution, different soils, and slag heap material, different pH values, temperatures and light intensities were used and cells from both growth and stationary phase were measured. The total possible range of dry weight with respect to carbon per unit biovolume (C/ubv) values was 93–226 fg μm⁻³. The mean value of all data was 147 fg μm⁻³, which concurs with the average value as taken from literature data of several planktonic algal species and groups within the respective size range (cell volume 300–1,000 μm³). We could show that C/ubv is suitable to quantify environmental stress conditions: C/ubv values ≤140 fg μm⁻³ are characteristic of cells grown under optimum conditions, and values ≥160 fg μm⁻³ reflect quantitatively graded stress situations. We propose integrating the microscope interferometric method using K. flaccidum as a test organism into a soil test system to determine the prevailing environmental conditions.     

Sodium-dependent and -independent Choline Uptake by Type II Epithelial Cells from Rat Lung

The uptake of ³H-labeled choline by a suspension of isolated type II epithelial cells from rat lung has been studied in a Ringer medium. Uptake was linear for 4 min at both 0.1 μm and 5.0 μm medium choline; at 5 μm, only 10% of the label was recovered in a lipid fraction. Further experiments were conducted at the low concentration (0.1 μm), permitting characterization of the properties of high-affinity systems. Three fractions of choline uptake were detected: (i) a sodium-dependent system that was totally inhibited by hemicholinium-3 (HC-3); (ii) a sodium-independent uptake, when Na⁺ was replaced by Li⁺, K⁺ or Mg²⁺, inhibited by HC-3; (iii) a residual portion persisting in the absence of Na⁺ and unaffected by HC-3. Choline uptake was sigmoidally related to the medium Na⁺ concentration. Kinetic properties of the uptake of 0.1 μm ³H-choline in the presence and absence of medium Na⁺ were examined in two ways. (a) Inhibition by increasing concentrations of unlabeled choline (0.5–100 μm) was consistent with the presence of two Michaelis-Menten-type systems in the presence of Na⁺; a Na⁺-dependent portion (a mean of 0.52 of the total) had a K _m for choline of 1.5 μm while K _m in the absence of Na⁺ (Li⁺ substituting) was 18.6 μm. (b) Inhibition by HC-3 (0.3–300 μm) gave K_i values of 1.7 μm and 5.0 μm HC-3 for the Na⁺-dependent and -independent fractions. The apparent K _m of the Na⁺-dependent uptake is lower than that reported previously for lung-derived cells and is in the range of the K _m values reported for high-affinity, Na⁺-dependent choline uptake by neuronal cells. Received: 18 February 1997/Revised: 7 December 1997     

Aeration volume and photosynthetic photon flux affect cell growth and secondary metabolite contents in bioreactor cultures ofMorinda citrifolia

To improve their growth and secondary metabolite production, we culturedMorinda citrifolia leaf cells for 3 weeks in bioreactors with different aeration volumes (0.05, 0.1, 0.2, or 0.3 vvm; or 0.05/0.1/0.2/0.3 vvm, as increased at 5-d interval), and photosynthetic photon fluxes (PPF; 0, 15, 30, or 45 μ,moL m^-2 s^-1). Cell growth was greatest (15.6 g L^-1 dry weight) at 0.3 vvm whereas the accumulation of secondary metabolites (total anthraquinones, phenolics, and flavonoids) was maximized at 0.1 vvm. A PPF of 15 μmoLm^-2 s^-1 accelerated the accumulation of both cell biomass and metabolites. Dark-culturing suppressed cell growth, while a high PPF (45 μmoLm^-2 s^-1) inhibited metabolite biosynthesis. Further studies are required to understand the reason for differences in the effect of light on cell growth and secondary metabolite contents inM. citrifolia cell cultures.     

Ultrastructural organization and development cycle of soil ultramicrobacteria belonging to the class Alphaproteobacteria

Gram-negative chemoorganotrophic soil ultramicrobacteria (UMB), strains NF1 and NF3, have been isolated. In their development cycle, the strains formed small coccoid cells of 400-800 nm and ultrasmall cells of 200-300 nm. Phylogenetically, the strains NF1 and NF3 belong to Alphaproteobacteria and are close to the type strain of the recently described species Kaistia adipata. The ultrastructure of UMB cells has been studied using ultrathin sections and freeze-fracturing. It has been shown that the structure of UMB cell walls is of the gram-negative type; the outer membrane and peptidoglycan layer are well differentiated. The cell surface has numerous protrusions (prosthecae) of conical or spherical shape filled with the contents of the periplasm. The formation of unusual cellular structures (not occurring in known free-living bacteria) is a feature of UMB: these include the following: (a) piles of rod-like subunits, ca. 30 A in diameter and 150-250 angstroms in length: (b) long bunches (up to 300-400 angstroms) comprised of filamentous subunits; and (c) large electron-dense spherical bodies (up to 200-300 angstroms in diameter) localized in the periplasm. A distinctive feature of UMB is their ability to grow as facultative parasites on living cyanobacterial (CB) cells. In this case, three types of interaction between UMB and CB have been revealed: (1) adsorption of UMB cells on the surface of CB cells; (2) penetration of UMB into polysaccharide sheathes; and (3) penetration of UMB into CB eytoplasm. UMB cells have been shown to reproduce by budding, with buds (up to 2-3) located directly on the mother cell, without formation of intennediate hyphae.     

Generation and maintenance of suspension cultures from cotyledons and their organogenic potential of two mangrove species, Sonneratia alba and S. caseolaris

Liquid cultures were successfully generated from cotyledons of two Sonneratia species, S. alba and S. caseolaris in Murashige and Skoog (MS) medium containing 0.1 μmol L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Adventitious roots differentiated from cotyledons of S. alba. Proliferated cells were subcultured and a large volume of suspension cells was subsequently established in 100-mL flasks. All the cytokinins tested inhibited cell proliferation. After three years of culture, the potential to differentiate was tested as indicated by greening of the cells. Greening occurred when suspension cells were transferred to solid MS medium with and without 0.1 μmol L⁻¹ 2,4-D. Greening was stimulated by low concentrations of the weak auxins indolebutyric acid (IBA) and naphthaleneacetic acid (NAA) while 2,4-D stimulated late-stage greening. Abscisic acid (ABA) inhibited greening. Gibberellic acid (GA₃) at 1.0 μmol L⁻¹ stimulated callus greening and was not inhibitory even when tested at high concentrations. Cytokinins were inhibitory in combination with 0.1 μmol L⁻¹ of either IBA or NAA. The cause of different effects of plant hormones on growth and differentiation was discussed. Small-scale liquid media and 24-well culture plates of solid media methods developed in this paper are suitable for the optimization of hormonal conditions for cell proliferation and differentiation.     

Evaluation of antigenotoxicity effects of umbelliprenin on human peripheral lymphocytes exposed to oxidative stress

The protective properties of a prenylated coumarin, umbelliprenin (UMB), on the human lymphocytes DNA lesions were tested. Lymphocytes were isolated from blood samples taken from healthy volunteers. DNA breaks and resistance to H₂O₂-induced damage were measured using a single-cell microgel electrophoresis technique under alkaline conditions (comet assay). Human lymphocytes were incubated in UMB (10, 25, 50, 100, 200, and 400 μM) alone or a combination of different concentrations of UMB (10, 25, 50, 100, 200, and 400 μM) and 25 μM H₂O₂. Untreated cells, ascorbic acid (AA; 25, 50, 100, 200, and 400 μM) and H₂O₂ (25 μM) were considered as negative control, positive control, and the standard antioxidant agent for our study, respectively. Single cells were analyzed with “TriTek Cometscore version 1.5” software. The DNA damage was expressed as percent tail DNA. UMB exhibited a concentration-dependent increase in protection activity against DNA damage induced by 25 μM H₂O₂ (from 67.28% to 39.17%). The antigenotoxic activity of AA, in the range 0–50 μM, was greater than that of UMB. However, no significant difference (p > 0.05) in the protective activity was found between UMB and AA at concentrations of approximately higher than 50 μM.     

The modulation of growth of normal rat liver epithelial cells in calcium-poor medium by epidermal growth factor,phenobarbital, phorbol ester,and retinoic acid

Summary The ability of a normal rat liver epithelial cell line with phenotypic characteristics of “oval” cells to grow in calcium-poor medium has been investigated. The growth of these cells could be arrested in medium containing 0.03 mM Ca²⁺, a concentration below which cell necrosis began to occur 24 h postexposure. With increasing calcium concentration, progressive cell proliferation was observed. Epithelial growth factor (EGF) (10 ng/ml) increased the survival and proliferation of cells in calcium-poor medium and the response was inversely correlated with the extracellular calcium concentration. In contrast, phenobarbital (0.2 to 2 mM), 12-0-tetradecanoylphorbol-13-acetate (0.01 to 1 μg/ml), or retinoic acid (0.001 to 0.1 μg/ml) depressed growth of cells in calcium-poor medium. The results confirm the ability of EGF to lower the calcium requirement for proliferation of normal cells, but such an effect does not seem to be a universal property of tumor promoters. This research was supported by National Institutes of Health Grant CA 29323.     

Amphotericin B Kills Unicellular Leishmanias by Forming Aqueous Pores Permeable to Small Cations and Anions

The polyene antibiotic amphotericin B (AmB) is known to form two types of ionic channels across sterol-containing liposomes, depending on its concentration and time after mixing (Cohen, 1992). In the present study, it is shown that AmB only kills unicellular Leishmania promastigotes (LPs) when aqueous pores permeable to small cations and anions are formed. Changes of membrane potential across ergosterol-containing liposomes and LPs were followed by fluorescence changes of 3,3′ dipropylthiadicarbocyanine (DiSC₃(5)). In KCl-loaded liposomes suspended in an iso-osmotic sucrose solution, low AmB concentrations (≤0.1 μm) induced a polarization potential, indicating K⁺ leakage, but no movement of cations and anions was allowed until AmB concentrations greater than 0.1 μm were added. In agreement with these data, it was found that AmB altered the negative membrane potential held across LPs in a manner consistent with the differential cation/anion selectivity exhibited by the channels formed in liposomes. Thus, LPs suspended in an iso-osmotic sucrose solution did not exhibit any AmB-induced membrane depolarization effect brought about by efflux of anions until 0.1 μm or higher AmB concentrations were added. By contrast, LPs suspended in an iso-osmotic NaCl solution and exposed to 0.05 μm AmB exhibited a nearly total collapse of the negative membrane potential, indicating Na⁺ entry into the cells. The concentration dependence of the AmB-induced permeability to different salts was also measured across vesicles derived from the plasma membrane of leishmanias (LMVs), by using a rapid mixing technique. At concentrations above 0.1 μm, AmB induced the formation of aqueous pores across LMVs with a positive cooperativity, yielding Hill coefficients between 2 to 3. Measured anion selectivity across such aqueous pores followed the sequence: SCN > NO3 > Cl > I > Br > acetate (SO²⁻ ₄ being impermeable). Cell killing by AmB was followed by fluorescence changes of the DNA-binding compound ethidium bromide (EB). At low concentrations (≤0.1 μm), AmB was found to be nonlethal against LPs but, above this concentration, leishmanias were rapidly killed. The rate and extent of such an effect were found to be dependent on the type of cation and anion present in the external aqueous solution. For both NH⁺ ₄ and Na⁺ salts, the measured rank order of AmB cell killing followed the same sequence that was determined for AmB-induced salt permeation across LMVs. Further, replacement of either extracellular Na⁺ by choline or Cl⁻ by SO²⁻ ₄, or its partial substitution by sucrose, in iso-osmotic conditions, led to a complete inhibition of the killing effect exerted by otherwise lethal AmB concentrations. Finally, it was shown that tetraethylammonium (TEA⁺), an organic cation that is known to block AmB-induced salt permeation across LMVs was able to retard the time lag observed for EB incorporation across LPs, indicating that this parameter can be taken to represent the time taken for salt accumulation inside the parasites. The present results thus indicate clearly that low AmB concentrations (≤0.1 μm) were able to form across LPs, cation channels that collapsed the parasite membrane potential but are not lytic. At high concentrations (<≥0.1 μm), a salt influx via the aqueous pores formed by the antibiotic was followed by osmotic changes leading to cell lysis. This last stage is supported by electron microscopy observations of the changes of parasite morphology immediately upon addition of AmB, which indicated that the typical elongated promastigote cell forms became rounded and the flagella swells and round up. The present work is the first demonstration of the in vitro sensitivity of Leishmania promastigotes to osmotic lysis by AmB. Received: 25 September 1995/Revised: 11 March 1996     

The relative importance of different ciliate taxa in the pelagic food web of lake constance

Abundance, biovolume, and species composition of pelagic ciliates in Lake Constance were recorded over two annual cycles (1987/88). Production was estimated from mean annual biovolumes and size-specific growth rates obtained from the literature. Cell concentrations and biovolumes ranged from 0.1 to 120 cells ml^–1 and from 3 to 1,200 mm³ m^–3, respectively. Mean annual values were, respectively, 6.8 cells ml^–1 and 94 mm³ m^–3 in 1987, and 12.0 cells ml^–1 and 130 mm³ m^–3 in 1988. In both years, prostome nanociliates (<20m) dominated numerically, while strobiliids in the size range 20–35m contributed most significantly to ciliate production. Ciliate community production, according to a crude calculation, yielded approximately 10–15 g C m^–2 year^–1.     

Quantitative comparison of food niches in some freshwater zooplankton

Summary The abilities of some zooplankton (rotifers, cladocerans, copepods) to ingest different sizes and kinds of food cells were quantified by determining the relative efficiencies with which they ingested nine tracer-cell types, ranging from a coccoid bacterium (0.45 m³) to the alga Cryptomonas erosa (800–920 m³). These efficiencies were obtained by dividing the clearance rate of each zooplankton group (species population, developmental stage or size class of a species population) on each ³²P-labeled cell type by that of a simultaneously-offered, ³³P-labeled, standard cell type — Chlamydomonas reinhardtii. Similarities of efficiency patterns on these cell types (food niches) between all possible pairs of the 17 zooplankton groups from 4 ecosystems were determined by calculating correlation coefficients. Although the utilization of the tested cell types may vary greatly within a species, three feeding guilds could be distinguished — based primarily on the efficiencies with which the smallest cell types were ingested. Guild I (Poyarthra vulgaris, Keratella crassa, Diaptomus minutus nauplii) ate the smallest cells (<4 m diameter) (bacterium, Synechococcus, Nannochloris) and Ankistrodesmus very ineffifently but the three Cryptomonas species very efficiently. Guild II (Bosmina longirostris, D. minutus copepodites and Adults) had higher efficiencies on Synechococcus, Nannochloris, Ankistrodesmus, Stichococcus, and Stephanodiscus than guild I but similarly low ones on the bacterium and high ones on the Cryptomonas species. Guild III (Conochilus inicornis, Keratella cochlearis, Ceriodaphnia quadrangula, Diaphanosoma leuchtembergianum) differed from guilds I and II in having uniformly high efficiencies on all the small cells as well ad the larger ones. Principal component analysis of the matrix of correlation coefficients provided objective confirmation of the three guilds and provided a visual representation of the food niches of the 17 zooplankton groups in 3-dimensional space.     

Isoform-Specific Function and Distribution of Na/K Pumps in the Frog Lens Epithelium

Epithelial cells from the anterior and equatorial surfaces of the frog lens were isolated and used the same day for studies of the Na/K ATPase. RNase protection assays showed that all cells express α₁- and α₂-isoforms of the Na/K pump but not the α₃-isoform, however the α₂-isoform dominates in anterior cells whereas the α₁-isoform dominates in equatorial cells. The whole cell patch-clamp technique was used to record functional properties of the Na/K pump current (I _P ), defined as the current specifically inhibited by dihydro-ouabain (DHO). DHO-I _P blockade data indicate the α₁-isoform has a dissociation constant of 100 μm DHO whereas for the α₂-isoform it is 0.75 μm DHO. Both α₁- and α₂-isoforms are half maximally activated at an intracellular Na⁺-concentration of 9 mm. The α₁-isoform is half maximally activated at an extracellular K⁺-concentration of 3.9 mm whereas for the α₂-isoform, half maximal activation occurs at 0.4 mm. Lastly, transport by the α₁-isoform is inhibited by a drop in extracellular pH, which does not affect transport by the α₂-isoform. Under normal physiological conditions, I _P in equatorial cells is approximately 0.23 μA/μF, and in anterior cells it is about 0.14 μA/μF. These current densities refer to the area of cell membrane assuming a capacitance of around 1 μF/cm². Because cell size and geometry are different at the equatorial vs. anterior surface of the intact lens, we estimate Na/K pump current density per area of lens surface to be around 10 μA/cm² at the equator vs. 0.5 μA/cm² at the anterior pole. Received: 17 May 2000/Revised: 11 August 2000     

<Emphasis Type="Italic">In vitro</Emphasis> selection of NaCl-tolerant callus lines and regeneration of plantlets in a bamboo (<Emphasis Type="Italic">Dendrocalamus strictus</Emphasis> Nees)

Summary Sodium chloride-tolerant plantlets of Dendrocalamus strictus were regenerated successfully from NaCl-tolerant embryogenic callus via somatic embryogenesis. The selection of embryogenic callus tolerant to 100 mM NaCl was made by exposing the callus to increasing (0–200 mM) concentrations of NaCl in Murashige and Skoog medium having 3% (w/v) sucrose, 0.8% (w/v) agar, 3.0 mg l⁻¹ (13.6 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.5mg l⁻¹ (2.3μM) kinetin (callus initiation medium). The tolerance of the selected embryogenic callus to 100 mM NaCl was stable through three successive transfers on NaCl-free callus initiation medium. The tolerant embryogenic callus had high levels of Na⁺, sugar, free amino acids, and proline but a slight decline was recorded in K⁺ level. The stable 100 mM NaCl-tolerant embryogenic callus differentiated somatic embryos on maintenance medium [MS medium +3% sucrose +0.8% agar +2.0 mg l⁻¹ (9.0 μM) 2,4-D+0.5 mg l⁻¹ (2.3 μM) kinetin] supplemented with different (0–200 mM) concentrations of NaCl. About 39% of mature somatic embryos tolerant to 100 mM NaCl germinated and converted into plantlets in germination medium [half-strength MS+2% sucrose+0.02 mg l⁻¹ (0.1 μM) α-naphthaleneacetic acid +0.1 mg l⁻¹ (0.49 μM) indole-3-butyric acid] containing 100 mM NaCl. Of these plantlets about 31% established well on transplantation into a garden soil and sand (1:1) mixture containing 0.2% (w/w) NaCl.     

Photosynthetic rates in relation to leaf phosphorus content in pioneer versus climax tropical rainforest trees

In Guyana dense rainforest occurs on intensely weathered acid soils, low in soil phosphorus. To investigate whether low P availability limits photosynthesis of trees growing on these soils more than N does, leaf P and N content, and their relationship with the photosynthetic capacity (A _sat, mol CO₂ m^-2 s^-1) were studied for nine pioneer and climax tree species in a range of light climates. Light environment was described using hemispherical photographs. For both pioneer and climax species, leaf P content (r ²=0.71 and 0.23, respectively) is a more important determinant of A _sat than leaf N content (r ²=0.54 and 0.12, respectively). Pioneer species have a higher leaf P and N content than climax species. At similar P or N content, pioneers have a higher A _sat than climax species. The saplings studied had a relatively high A _sat, considering their low P concentration (15–30 mol P g^-1). All species studied had a constant leaf P and N concentration and photosynthetic capacity across light climates, because specific leaf mass (g m^-2) increased similarly with light availability. This acclimation to a change in light environment makes a possible limitation of A _sat by P or N independent of light environment.     

Hyperaccumulation of nickel by two Alyssum species from the serpentine soils of Iran

Serpentine soils, which contain relatively high concentrations of nickel and some other metals, are the preferred substrate for some plants, especially those that accumulate Ni in their tissues. In temperate regions more Ni-hyperaccumulator plants are found in Alyssum than in any other genus. In this study, serpentine soils of two areas (Marivan and Dizaj) in the west/northwest of Iran and also perennial Alyssum plants growing on these soils were analyzed for Ni and some other metals. The highest concentrations of total metals in the soils of these areas for Ni, Cr, Co and Mn were 1,350, 265, 94 and 1,150 μg g⁻¹, respectively, while concentrations of Fe, Mg and Ca reached 3.55%, 16.8% and 0.585% respectively. The concentration of exchangeable Ni in these soils is up to 4.5 μg g⁻¹. In this study two Alyssum species, A. inflatum and A. longistylum, have been collected from Marivan and Dizaj, respectively. Analysis of leaf dry matter shows that they can contain up to 3,700 and 8,100 μg Ni g⁻¹, respectively. This is the first time that such high Ni concentrations have been found in these species. The concentrations of other metals determined in these species were in the normal range for serpentine plants, except for Ca, which was higher, up to 5.3% and 3.5%, respectively     

Phytoextraction of selenium from soils irrigated with selenium-laden effluent

This two-part study compared the efficacy of different plant species to extract Se from soils irrigated with Se-laden effluent. The species used were: Brassica napus L. (canola), Brassica juncea Czern L. and Coss (Indian mustard), and Hordeum vulgare L. (barley). In Study 1 we irrigated the plants with a saline effluent containing 0.150 mg Se L^–1, while in Study 2, the same species were planted in a saline soil selenized with 2 mg Se L^–1. Plants were simultaneously harvested 120 days after planting. In Study 1, there were only slight effects of treatment on dry matter (DM) yield. Plant Se concentrations averaged 21 g Se g^–1DM for the Brassica species, and 4.0 g Se g^–1 DM for barley. Total Se added to soils via effluent decreased by 40% for Brassica species and by 20% for barley. In Study 2, total DM decreased for all species grown in saline soils containing Se. Plant Se concentrations averaged 75 g g^–1 DM for Brassica species and 12 g Se g^–1 DM for barley. Total Se added to soils prior to planting decreased by 40% for Brassica species and up to 12% for barley. In both studies, plant accumulation of Se accounted for at least 50% of the Se removed in soils planted to Brassica and up to 20% in soils planted to barley. Results show that although the tested Brassica species led to a significant reduction in Se added to soil via use of Se-laden effluent, additional plantings are necessary to further decrease Se content in the soil.