Über die ursache der hemmwirkung der humusstoffe auf den populationszuwachs vonoscillatoria sancta

Algologically pure strain ofOscillatoria sancta (KÜTZING) GOMONT was cultivated in a phototermostat using a modified medium of Chu-Gerloff. The inhibiting effect of Na-humate on the numbers of cells and dry weight of this blue-green alga was investigated in dependence on polyvalent cations (Ca²⁺, Mg²⁺, Fe³⁺) concentration, nature of associated anions, and pH of the nutrient solution. Moreover the absorption of light due to humate colour was taken into consideration. Humate restricted the uptake of polyvalent cations (especially calcium); its unfavourable effect on the growth of the investigated organism decreased at the optimum pH.     

Precipitation of Metallic Cations by the Acidic Exopolysaccharides from Bradyrhizobium japonicum and Bradyrhizobium (Chamaecytisus) Strain BGA-1

The interaction between the acidic exopolysaccharides produced by two Bradyrhizobium strains and several metal cations has been studied. Aqueous solutions in the millimolar range of Fe³⁺ but not of Fe²⁺ precipitated the exopolysaccharides from Bradyrhizobium (Chamaecytisus) strain BGA-1 and, to a lesser extent, Bradyrhizobium japonicum USDA 110. The precipitation was pH dependent, with a maximum around pH 3. The precipitate was redissolved by changing the pH and by Fe³⁺ reduction or chelation. Deacetylation of B. japonicum polysaccharide increased its precipitation by Fe³⁺. At pH near neutrality, the polysaccharide from Bradyrhizobium (Chamaecytisus) strain BGA-1 stabilized Fe³⁺ solutions, despite the insolubility of Fe(OH)₃. Aluminum precipitated Bradyrhizobium (Chamaecytisus) polysaccharide but not the polysaccharide produced by B. japonicum. The precipitation showed a maximum at about pH 4.8, and the precipitate was redissolved after Al³⁺ chelation with EDTA. Precipitation was inhibited by increases in the ionic strength over 10 mM. Bradyrhizobium (Chamaecytisus) polysaccharide was also precipitated by Th⁴⁺, Sn²⁺, Mn²⁺, and Co²⁺. The presence of Fe³⁺ increased the exopolysaccharide precipitation by aluminum. No precipitation, gelation, or increase in turbidity of polysaccharide solutions occurred when K⁺, Na⁺, Ca²⁺, Mg²⁺, Cu²⁺, Cd²⁺, Pb²⁺, Zn²⁺, Hg²⁺, or U⁶⁺ was added at several pH values. The results suggest that the precipitation is based on the interaction between carboxylate groups from different polysaccharide chains and the partially hydrolyzed aquoions of Fe³⁺, Al³⁺, Th⁴⁺, and Sn²⁺.     

Interaction of the Circadian Cycle with the Cell Cycle in Pyrocystis fusiformis

Dividing pairs or single cells of the large dinoflagellate, Pyrocystis fusiformis Murray, were isolated in capillary tubes and their morphology was observed over a number of days, either in a light-dark cycle or in constant darkness. Morphological stages were correlated with the first growth stage, G₁, DNA synthesis, S, the second growth stage, G₂, mitosis, M, and cytokinesis, C, segments of the cell division cycle. The S phase was identified by measuring the nuclear DNA content of cells of different morphologies by the fluorescence of 4′, 6-diamidino-2-phenylindole dichloride.

Cells changed from one morphological stage to the next only during the night phase of the circadian cycle, both under light-dark conditions and in continuous darkness. Cells in all segments of the cell division cycle displayed a circadian rhythm in bioluminescence. These findings are incompatible with a mechanism for circadian oscillations that invokes cycling in G_q, an hypothesized side loop from G₁. All morphological stages, not only division, appear to be phased by the circadian clock.

     

Der chemotropische Effekt einiger Kationen bei den Wurzeln vonSinapis alba L. mit Berücksichtigung mancher Faktoren

Chemotropic effects exerted by metal cations have been investigated simultaneously with their influence on the increments ofSinapis alba roots cultivated in dark and light. The effects of Na-EDTA and Na-humate and its fractions, as well as the interaction of these substances with cations have also been studied. Chemotropics have been administered in agar-agar to the ends of roots growing on glass plates. Chemotropic and growth effects were exerted by Cu²⁺, Fe²⁺, Fe³⁺ and Ca²⁺ cations. The light strongly changed the effects of all but Cu²⁺ cations. Both Na-EDTA and humante appeared to be active chemotropically and generally abolished the effects of cations, although in some cases Na-EDTA made this influence more intense. Of the humate fractions, only those characterized by the ability to form complex compounds appeared to be active. No correlation has been found between chemotropic effects and the influence on the growth of roots, except for the Cu²⁺ cations, which always caused positive chemotropism and inhibited the growth of roots.     

Trigger factor assists the refolding of heterodimeric but not monomeric luciferases

The refolding of thermally inactivated protein by ATP-independent trigger factor (TF) and ATP-dependent DnaKJE chaperones was comparatively analyzed. Heterodimeric (αβ) bacterial luciferases of Aliivibrio fischeri, Photobacterium leiognathi, and Vibrio harveyi as well as monomeric luciferases of Vibrio harveyi and Luciola mingrelica (firefly) were used as substrates. In the presence of TF, thermally inactivated heterodimeric bacterial luciferases refold, while monomeric luciferases do not refold. These observations were made both in vivo (Escherichia coli ΔdnaKJ containing plasmids with tig gene) and in vitro (purified TF). Unlike TF, the DnaKJE chaperone system refolds both monomeric and heterodimeric luciferases with equal efficiency.     

Drosophila TRPML Forms PI(3,5)P2-activated Cation Channels in Both Endolysosomes and Plasma Membrane

Transient Receptor Potential mucolipin (TRPML) channels are implicated in endolysosomal trafficking, lysosomal Ca²⁺ and Fe²⁺ release, lysosomal biogenesis, and autophagy. Mutations in human TRPML1 cause the lysosome storage disease, mucolipidosis type IV (MLIV). Unlike vertebrates, which express three TRPML genes, TRPML1–3, the Drosophila genome encodes a single trpml gene. Although the trpml-deficient flies exhibit cellular defects similar to those in mammalian TRPML1 mutants, the biophysical properties of Drosophila TRPML channel remained uncharacterized. Here, we show that transgenic expression of human TRPML1 in the neurons of Drosophila trpml mutants partially suppressed the pupal lethality phenotype. When expressed in HEK293 cells, Drosophila TRPML was localized in both endolysosomes and plasma membrane and was activated by phosphatidylinositol 3,5-bisphosphate (PI(3,5)P₂) applied to the cytoplasmic side in whole lysosomes and inside-out patches excised from plasma membrane. The PI(3,5)P₂-evoked currents were blocked by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂), but not other phosphoinositides. Using TRPML A487P, which mimics the varitint-waddler (Va) mutant of mouse TRPML3 with constitutive whole-cell currents, we show that TRPML is biphasically regulated by extracytosolic pH, with an optimal pH about 0.6 pH unit higher than that of human TRPML1. In addition to monovalent cations, TRPML exhibits high permeability to Ca²⁺, Mn²⁺, and Fe²⁺, but not Fe³⁺. The TRPML currents were inhibited by trivalent cations Fe³⁺, La³⁺, and Gd³⁺. These features resemble more closely to mammalian TRPML1 than TRPML2 and TRPML3, but with some obvious differences. Together, our data support the use of Drosophila for assessing functional significance of TRPML1 in cell physiology.     

Functional artificial luciferases as an optical readout for bioassays

This study elucidates functional artificial luciferases (ALucs) wholly synthesized for bioassays and molecular imaging. The ALucs bearing epitopes were newly created by amending the sequences of our previously reported ALucs in light of a multi-sequence alignment and hydrophobicity search. The synthesized ALucs are survived in live cells and stable in culture media for 25 days after secretion. The epitopes in ALucs are exposed during the secretion process and indeed valid for column purification and immunological assays. The ALucs exerted a 9400-times stronger optical intensity with a coelenterazine derivative (CTZ i), when compared with Renilla reniformis luciferase 8.6–535. A supersecondary structure of ALuc30 was predicted with respect to the X-ray crystallographic information of the coelenterazine-binding protein (CBP). The structure revealed that ALuc30 has a room for accommodating the iodide of CTZ i. This study guides on how to create functional artificial luciferases and predicts the structural details with the current bioinformatics technologies.     

Trigger factor-dependent refolding of bacterial luciferases in Escherichia coli: Kinetics, efficiency, and effect of bichaperone system

The main parameters of the trigger-factor-dependent refolding process of thermally inactivated bacterial luciferases were determined. It has been demonstrated that TF-dependent refolding is less efficient and more time consuming than DnaKJE-dependent refolding. An increase in the cellular concentration of TF was found to result in a dramatic decrease in the maximum level of refolding of thermally inactivated bacterial luciferases. Additionally, the efficiency of TF-dependent refolding was shown to decrease with an increase in the thermal stability of the substrate, that is, the level of TF-dependent refolding is significantly higher for thermolabile luciferases than for thermostable luciferases. For example, the maximum TF-dependent refolding level was determined as 30–40% for thermolabile luciferases from Aliivibrio fischeri and Photobacterium leiognathi, 10% in the case of luciferase from Vibrio harveyi, which is characterized by an average thermal stability, and finally 0.5% in the case of highly stable at high temperatures luciferase from Photorhabdus luminescens. An effect of the DnaKJE-ClpB bichaperone system on the efficiency of TF-dependent refolding was investigated. The ClpB component of the bichaperone system was shown to negatively affect the process efficiency, that is, TF-dependent refolding of bacterial luciferases was found to be far more efficient in E. coli clpB::kan cell strains than in E. coli clpB⁺ strains.     

CIRCADIAN REGULATION OF BIOLUMINESCENCE IN THE DINOFLAGELLATE PYROCYSTIS LUNULA1

In the unicellular algae Pyrocystis lunula Schütt and Gonyaulax polyedra Stein, bioluminescence and its circadian regulation are similar in several respects, but there are also several important differences. As in G. polyedra, P. lunula emits light both as bright flashes and as a low intensity glow. At 20° C, the individual flashes are considerably brighter than in G. polyedra, and their durations are typically less than 500 ms. Both species show a circadian rhythm in the frequency of spontaneous flashes, which peaks in the night-phase under light–dark cycles and continues in both continuous light and dark. However, compared to G. polyedra, the circadian system in P. lunula is more sensitive to light: 10 min exposures (500 μmol · m^–2· s^–1 white light) can shift the phase of the rhythm by more than 8 h, and rhythmicity is completely suppressed at an irradiance above 20 μmol · m^–2· s^–1, where the G. polyedra rhythym persists for weeks. Like G. polyedra, period length increases with increasing irradiance of continuous red light but decreases with increasing intensity of continuous blue light. The glow in P. lunula differs markedly from that in G. polyedra in that it occurs at about the same intensity at all times during the circadian cycle; thus, it is not under circadian control but may fluctuate 5–10-fold in intensity within a time frame of seconds. This suggests that the glow may differ in its physiological basis in the two organisms. The results also indicate that the circadian regulation of luciferase activity differs in the two species. In G. polyedra, the organelle responsible for bioluminescence and luciferase is lost and then reformed on a daily basis; in P. lunula, the luciferase is conserved and localized elsewhere during the nonbioluminescent phase of the cycle.     

Stimulable and Spontaneous Bioluminescence in the Marine Dinoflagellates, Pyrodinium bahamense, Gonyaulax polyedra, and Pyrocystis lunula

P. bahamense, G. polyedra, and P. lunula exhibit interspecies differences in stimulable and spontaneous bioluminescence. For each species the total number of photons that can be emitted upon mechanical stimulation is a constant, regardless of the time during scotophase at which stimulation occurs. Ratios of stimulable bioluminescence per organism during scotophase and photophase are as high as 950:1 for laboratory cultures and have been observed as high as 4000: 1 for natural populations of P. bahamense. Spontaneous emission in darkness shows flashing as well as low-level continuous emission. Natural populations of P. bahamense, placed in darkness during natural photophase, exhibit a dual character to their stimulable bioluminescence. Mechanical stimulation techniques are described for rapid and reproducible stimulation of bioluminescence.     

Introduction to beetle luciferases and their applications

All beetle luciferases have evolved from a common ancestor: they all use ATP, O₂, and a common luciferin as substrates. The most studied of these luciferases is that derived from the firefly Photinus pyralis, a beetle in the superfamily of Cantharoidea. The sensitivity with which the activity of this enzyme can be assayed has made it useful in the measurement of minute concentrations of ATP. With the cloning of the cDNA coding this luciferase, it has also found wide application in molecular biology as a reporter gene. We have recently cloned other cDNAs that code for luciferases from the bioluminescent click beetle, Pyrophorus plagiophthalamus, in the superfamily Elateroidea. These newly acquired luciferases are of at least four different types, distinguishable by their ability to emit different colours of bioluminescence ranging from green to orange. Unique properties of these luciferases, especially their emission of multiple colours, may make them additionally useful in applications.     

SPECIES OF OCEANIC DINOFLAGELLATES IN THE GENERA DISSODINIUM AND PYROCYSTIS: INTERCLONAL AND INTERSPECIFIC COMPARISONS OF THE COLOR AND PHOTON YIELD OF BIOLUMINESCENCE1

We have examined aspects of the bioluminescence of 5 clones of Dissodinium, 1 clone of Pyrocystis acuta, 4 clones of Pyrocystis fusiformis, and 5 clones of Pyrocystis noctiluca. All clones produced the same color bioluminescence with an intensity peak near 474 nm. The in vivo emission spectra of these clones agreed with those previously determined, for 4 other species of marine dinoflagellates. The amount of light emitted by the dinoflagellates in scotophase when mechanically stimulated to exhaustion was determined for most of the clones. The largest species, P. noctiluca and P. fusiformis, emitted 37–89 × 10⁹ photons cell^?1 and 23–62 × 10⁹ photons cell^?1, respectively, about a thousand, times as much light as Gonyaulax species. Pyrocystis acuta emitted 3–6 × 10⁹ photons cell^?1. Three of the 5 clones of Dissodinium were bioluminescent. The range for 3 clones was 5–13 × 10⁹ photons cell^?1. All 5 clones of Dissodinium are morphologically distinct. Both the clones of Dissodinium and Pyrocystis produced much higher numbers of photons per cell nitrogen (ca. 7–50 times) than Gonyaulax polyedra or Pyrodinium bahamense. The data suggested that enzyme turnover occurred in the reactions producing light during mechanical stimulation of Dissodinium and Pyrocystis species.     

The Activity of Ribulose Diphosphate Carboxylase in Extracts of Gonyaulax polyedra in the Day and the Night Phases of the Circadian Rhythm of Photosynthesis

The ribulose 1,5-diphosphate carboxylase from Gonyaulax polyedra Stein. has a half-life of about four hours in buffer, but can be stabilized by the addition of 50% glycerol. The optimum pH is 7.8 to 8.0 and the optimum Mg²⁺ concentration is 3 mm. Heavy metal ions (Cu²⁺, Hg²⁺, Ni²⁺, Zn²⁺), EDTA, pyrophosphate, and adenosine triphosphate were strongly inhibitory. Ribulose 1,5-diphosphate carboxylase from Gonyaulax was not cold-sensitive or activated by light activation factor from tomato or Gonyaulax. No difference in the activity of this enzyme was detected when extracts prepared at the maximum and the minimum of the circadian rhythm of photosynthesis were compared. The Km of HCO₃⁻ was also the same (16 to 19 mm).     

The adnosine 3',5'-monophosphate and guanosine 3',5'-monophosphate phosphodiesterases from human lung tissue.

Human lung tissue contains phosphodiesterase enzymes capable of hydrolyzing both adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP). The cyclic AMP enzyme exhibits three distinct binding affinities for its substrate (apparent Km = 0.4μM, 3μM, and 40μM) while the cyclic GMP enzyme reveals only two affinities (Km = 5μM and 40μM). The pH optima for the cyclic AMP and cyclic GMP phosphodiesterase are similar (pH 7.6–7.8). Both are inhibited by known inhibitors of phosphodiesterase activity (aminophylline, caffeine, and 3-isobutyl-1-methylxanthine). The divalent cations Mg²⁺ and Mn²⁺ stimulate cyclic AMP phosphodiesterase activity (in the absence of Mg²⁺) while Ca²⁺, Ni²⁺, and Cu²⁺ inhibit the enzyme. Histamine and imidazole slightly stimulate cyclic AMP hydrolytic activity. Thus, human lung tissue does contain multiple forms of both the cyclic AMP and cyclic GMP phosphodiesterase which are influenced by a variety of effectors.     

Characterization of Mg2+-ATPase from sheep kidney medulla: purification.

Magnesium-dependent adenosine triphosphatase has been purified from sheep kidney medulla plasma membranes. The purification, which is based on treatment of a kidney plasma membrane fraction with 0.5% digitonin in 3 mm MgCl₂, effectively separates the Mg²⁺-ATPase from (Na⁺ + K⁺)-ATPase present in the same tissue and yields the Mg²⁺-ATPase in soluble form. The purified enzyme is activated by a variety of divalent cations and trivalent cations, including Mg²⁺, Mn²⁺, Ca²⁺, Co²⁺, Fe²⁺, Zn²⁺, Eu³⁺, Gd³⁺, and VO²⁺. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme shows two bands with R_f values corresponding to molecular weights of 150,000 and 77,000. The larger peptide is phosphorylated by [γ-³²P]ATP, suggesting that this peptide may contain the active site of the Mg²⁺-ATPase. The Mg²⁺-ATPase activity is unaffected by the specific (Na⁺ + K⁺)-ATPase inhibitor ouabain.     

EFFECTS OF LIGHT INTENSITY ON DIVISION RATE,STIMULABLE BIOLUMINESCENCE AND CELL SIZE OF THE OCEANIC DINOFLAGELLATES DISSODINIUM LUNULA,PYROCYSTIS FUSIFORMIS AND P. NOCTILUCA12

Preadapted cultures were grown in a 12:12 LD cycle at a series of light intensities under cool-white, fluorescent lamps. Pyrocystis fusiformis Murray maintained high division rates at low light intensities at the expense of cell size. In contrast, Dissodinium lunula (Schuett) Taylor had relatively lower division rates at low light intensities with little concomitant decrease in size. The response of P. noctiluca Murray was intermediate between these two species. For all three, cell numbers did not increase above an intensity of 5–10 μEin·m^?2·sec^?1 and division rate was saturated at ca. 30, 60, and 60μEin·m^?2·sec^?1 for P. fusiformis, P. noctiluca, and D. lunula, respectively. The capacity for stimulable bioluminescence was saturated at light intensities of 0.15 μEin·m^?2·day in short-term (2-day) experiments. In cultures of P. fusiformis and P. noctiluca, maintained for at least one month at lower intensities than needed to saturate division rate, a decrease in the capacity for stimulable bioluminescence was accompanied by a reduction in cell size. Our results suggest that cell size and bioluminescent capacity may prove to be a potentially useful indication of the history of exposure of natural populations of Pyrocystis spp. to ambient intensities.     

Combined effects of waterlogging and salinity on electrochemistry, water-soluble cations and water dispersible clay in soils with various salinity levels

The combination effects of waterlogging and salinity on redox potential (Eh), pH, electric conductivity (EC), water-soluble cations (NH₄ ⁺, K⁺, Na⁺, Ca²⁺, Mg²⁺, Fe²⁺, and Mn²⁺) and water-dispersible clay (WDC) were studied in six soils collected near salt lakes in western Australia. The soils with various salinity levels were incubated under a waterlogged condition at 30 °C for 12 weeks. The Eh, pH, EC, and cations of soil solutions were monitored over the waterlogged period. The Eh values generally dropped to the lowest point within 12 days of waterlogging, then increased slightly, and reached equilibrium after 4 weeks of waterlogging. Increasing salinity levels increased soil Eh. While waterlogging increased soil pH in the first 3–4 weeks, increasing salinity level decreased soil pH during the entire waterlogging period. Waterlogging increased the EC values in the first 2 weeks, partly due to dissolution of insoluble salts. The concentrations of water-soluble NH₄ ⁺ were significantly increased with salinity level and waterlogging, and reached maximum values at week 2, and then declined to the initial level. Waterlogging and salinity increased the concentrations of water-soluble K⁺, Ca²⁺, Mg²⁺, Fe²⁺, and Mn²⁺ ions, but the magnitudes of changes were greatly affected by soil properties. Increases in water-soluble K⁺, Ca²⁺ and Mg²⁺ were attributed to increased solubility of insoluble salts, and increased competition for the adsorption sites of the soil exchange complex due to elevated concentrations of Na⁺, Fe²⁺ and Mn²⁺. Increases in water-soluble Fe²⁺ and Mn²⁺ induced by waterlogging were attributed to the dissolution of Fe and Mn oxides under reduced conditions. Waterlogging increased, but salinity decreased, the amounts of water-dispersible clay in the soils of low EC value. The higher salinity level can counteract the adverse effect of waterlogging on clay flocculation.     

Schistosoma mansoni: effect of some cations on the proteolytic enzymes of cercariae

The effects of various salts on the proteolytic activity of extracts from Schistosoma mansoni cercariae were tested. Using an Azocoll substrate, stimulation (2 to 2.5-fold) of activity by the monovalent cations Na⁺ and K⁺ was demonstrated, with maximum stimulation at 20–40 mM concentrations. The divalent cations Mg²⁺ and Ca²⁺ stimulated proteolytic activity at low concentrations (between 0 and 10 mM) but inhibited activity at higher concentrations. The divalent cations Zn²⁺, Cu²⁺, Fe²⁺, and Co²⁺ were inhibitory even at very low concentrations. The results presented here are discussed in relation to previously described ion effects on cercarial infectivity.     

The structural origin and biological function of pH-sensitivity in firefly luciferases.

Firefly luciferases are called pH-sensitive because their bioluminescence spectra display a typical red-shift at acidic pH, higher temperatures, and in the presence of heavy metal cations, whereas other beetle luciferases (click beetles and railroadworms) do not, and for this reason they are called pH-insensitive. Despite many studies on firefly luciferases, the origin of pH-sensitivity is far from being understood. This subject is revised in view of recent results. Some substitutions of amino-acid residues influencing pH-sensitivity in firefly luciferases have been identified. Sequence comparison, site-directed mutagenesis and modeling studies have shown a set of residues differing between pH-sensitive and pH-insensitive luciferases which affect bioluminescence colors. Some substitutions dramatically affecting bioluminescence colors in both groups of luciferases are clustered in the loop between residues 223-235 (Photinus pyralis sequence). A network of hydrogen bonds and salt bridges involving the residues N229-S284-E311-R337 was found to be important for affecting bioluminescence colors. It is suggested that these structural elements may affect the benzothiazolyl side of the luciferin-binding site affecting bioluminescence colors. Experimental evidence suggest that the residual red light emission in pH-sensitive luciferases could be a vestige that may have biological importance in some firefly species. Furthermore, the potential utility of pH-sensitivity for intracellular biosensing applications is considered.     

Biosynthesis of polyuronides inMucor rouxii: Partial characterization of fucosyl transferase

Most of the fucosyl transferase activity fromMucor rouxii was detected in a crude membrane fraction. The enzyme transferredl-fucose from GDP-fucose to endogenous and exogenous acceptors. When crude membrane fractions were treated with neutral detergents such as Trition X-100 or Brij 36 T enzyme activity became dependent on exogenous acceptors such as mucoric acid or mucoran. Brij-treated membrane fractions showed maximum fucosyl transferase activity at pH 6.5, and at a temperature between 22 and 28°C. The cations Mn²⁺, Mg²⁺, Co²⁺, Zn²⁺, Fe²⁺, and Ca²⁺ activated the enzyme about twofold. The former was slightly more stimulatory at 4 mM. Km for GDP-fucose was 10 μM. Evidence was obtained that mucoric acid serves as acceptor for fucosyl moieties. Acid hydrolysis of the product synthesized from GDP-fuc by Brij-treated membrane fractions revealed fucose as the major radioactive sugar.