Glucose excretion by the symbiotic Chlorella of Spongilla fluviatilis

Chlorella sorokiniana strain 211-40c, a symbiotic Chlorella isolated from a freshwater sponge, excreted between 3% and 5% of assimilated ¹⁴CO₂ as glucose in the light, with a pH optimum around 5. This percentage increased when the illuminance was lowered (to 15% at 20 lx). Release of [¹⁴C]glucose continued in the dark and could be inhibited by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Net efflux of glucose occurred even at a concentration ratio of extracellular/intracellular glucose of 4. This, together with the sensitivity to FCCP, is taken as evidence for active transport. Exogenous [¹⁴C]glucose was taken up by the cells under conditions of net glucose efflux, showing uptake and excretion to take place simultaneously.Abbreviations FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - p.c. packed cells     

Soluble and membrane-bound ferrisiderophore reductases of Escherichia coli K-12

After uptake of microbial ferrisiderophores, iron is assumed to be released by reduction. Two ferrisiderophore-reductase activities were identified in Escherichia coli K-12. They differed in cellular location, susceptibility to amytal, and competition between oxygen and ferrichrome-iron(III) reduction. The ferrisiderophore reductase associated with the 40,000×g sediment (membrane-bound enzyme) was inhibited by 10 mM amytal in contrast to the ferrisiderophore reductase present in the 100,000×g supernatant (soluble enzyme). Reduction by the membrane-bound enzyme followed sigmoid kinetics, but was biphasic in the case of the soluble enzyme. The soluble reductase could be assigned to a protein consisting of a single polypeptide of M _r 26000. Reduction of iron(III) by the purified enzyme depended on the addition of NADH or NADPH which were equally active reductants. The cofactor FMN and to a lesser degree FAD stimulated the reaction. Substrate specificity of the soluble reductase was low. In addition to the hydroxamate siderophores arthrobactin, schizokinen, fusigen, aerobactin, ferrichrome, ferrioxamine B, coprogen, and ferrichrome A, the iron(III) complexes of synthetic catecholates, dihydroxy benzoic acid, and dicitrate, as well as carrier-free iron(III) were accepted as substrates. Both ferrisiderophore reductases were not controlled by the fur regulatory system and were not suppressed by anaerobic growth.Abbreviations DHB dihydroxybenzoic acid - MECAM 1,3,5-N,N,N-tris-(2,3-dihydroxybenzoyl)-triamino-methylbenzene - MECAMS 2,3-dihydroxy-5-sulfonyl-derivative of MECAM     

Nickel requirement for the formation of active urease in purple sulfur bacteria (Chromatiaceae)

Nickel was found to be required for expression of urease activity in batch cultures of Thiocapsa roseopersicina strain 6311, Chromatium vinosum strain 1611 and Thiocystis violacea strain 2311, grown photolithotrophically with NH₄Cl as nitrogen source. In a growth medium originally free of added nickel and EDTA, the addition of 0.1–10 M nickel chloride caused an increase in urease activity, while addition of EDTA (0.01–2 mM) caused a strong reduction. Variation of the nitrogen source had no pronounced influence on the level of urease activity in T. roseopersicina grown with 0.1 M nickel in the absence of EDTA. Only nickel, of several heavy metal ions tested, could reverse suppression of urease activity by EDTA. Nickel, however, did not stimulate and EDTA did not inhibit the enzyme in vitro. When nickel was added to cultures already growing in a nickel-deficient, EDTA-containing medium, urease activity showed a rapid increase which was not inhibited by chloramphenicol. It is concluded that the (inactive) urease apoprotein may be synthesized in the absence of nickel and can be activated in vivo without de novo protein synthesis by insertion of nickel into the pre-formed enzyme protein.     

Forces in the spider orb web

Summary Pretensile forces were measured in individual threads of intact spider webs. In the orb web of Araneus diadematus forces decrease from mooring threads to frame threads and radii, a typical ratio being 1071. The smaller number of radii in the upper than in the lower half of the orb is paralleled by force ratios of 21 to 31. A similar difference between radii built first during web construction and radii added after completion of the frame underlines the importance of the former as part of the scaffolding. High tensions in the auxiliary spiral stabilize the radii in addition to providing a pathway for the spider when inserting the sticky spiral. Radial pretension (F) changes with spider mass (m). F/m is similar for different animals indicating an adaptation of radial forces to those resulting from spider mass. Several observations suggest tension control by the spider. When forced to anchor its web to thin flexible rods tension in the threads remains in the normal range. Tension values are similar in the webs of A. diadematus, Zygiella x-notata, Nuctenea umbratica, and Nephila clavipes indicating independence from details of web geometry. Only the mooring threads of Nephila show unusually large forces suggesting a narrower working range of tensions for the catching area than for the scaffolding.     

Acquisition of phosphorus and copper by VA-mycorrhizal hyphae and root-to-shoot transport in white clover

White clover (Trifolium repens L.) plants were grown in a calcareous soil in pots with three compartments, a central one for root growth and two outer ones for growth of vesicular-arbuscular (VA) mycorrhizal (Glomus mosseae [Nicol. & Gerd.] Gerdemann & Trappe) hyphae (hyphal compartments). Phosphorus (P) was applied at three levels (0, 20 and 50 mg kg⁻¹ soil) in the outer compartments in mycorrhizal treatments. Root and shoot dry weight were increased in mycorrhizal plants with hyphal access to outer compartments. Growth of the mycorrhizal hyphae in the outer compartments was not significantly affected by variation in P level in these compartments. However, both concentration and amount of P in roots and shoots sharply increased with increasing P supply in the outer (hyphal) compartments. With increasing P levels the calculated delivery of P by the hyphae from the outer compartments increased from 34% to 90% of total P uptake. Hyphal access to the outer compartments also significantly increased both concentration and quantity of Cu in the plants. The calculated delivery of Cu by the hyphae from the outer compartments ranged from 53% to 62% of total Cu uptake, irrespective of the P levels and the amounts of P taken up and transported by the hyphae. However, the distribution of Cu over roots and shoots was largely dependent on P levels. With increase in P level in the outer compartments the calculated hyphal contribution to the total amount of Cu in the shoots increased from 12% to 58%, but decreased in the roots from 75% to 46%. In conclusion, uptake and transport by VA-mycorrhizal hyphae may contribute substantially not only to P nutrition, but also to Cu nutrition of the host.     

Stages of tubulin assembly and disassembly studied by time-resolved synchrotron X-ray scattering

The structural transitions occurring during the assembly and disassembly of pig brain microtubule protein were investigated by time-resolved X-ray scattering using synchrotron radiation. The reactions were introduced by a slow temperature scan (2 deg.C/min) from 0 °C to 37 °C and back. Several structurally distinct states could be resolved during one cycle of assembly/disassembly. During the temperature rise, one observes four main phases: prenucleation events, microtubule nucleation, growth, and postassembly events.Heating from 0 °C to 22 °C results in a biphasic breakdown of rings and other aggregates, while the apparent mean diameter increases from 38 to 41 nm. Parallel time-resolved electron microscopic observations suggest that the initial solution contains several types of aggregates, mostly double concentric and single rings, but also rod-like particles, clusters of rings and other aggregates. All of these tend to break down with increasing temperature. Double concentric rings seem to dissociate into large and small single rings before both types of rings break down into protofilament fragments and tubulin subunits. From the breakdown products, associations of several protofilament fragments are formed, which are important for initiating microtubule nucleation. Assembly of nuclei begins around 22 °C. Microtubule elongation takes place between 25 and 30 °C. They grow mainly by addition of tubulin subunits but not via rings.During the reverse temperature scan, microtubules shorten by the release of subunits and/or small protofilament fragments from their ends. The degree of disassembly is strongly increased below 22 °C. Below about 10 °C rings are reformed, probably from the fragments, but their final number is much less than initially.Conditions that prevent microtubule nucleation such as GDP or Ca²⁺ also stabilize rings, even at 37 °C. Thus, rings are viewed as storage aggregates of tubulin and microtubule associated proteins, whose breakdown is a prerequisite for microtubule formation, and whose reformation is independent of microtubule breakdown.The midpoints of microtubule growth and breakdown differ by about 12 deg.C so that the system shows hysteresis-like behavior. It is dependent on microtubule formation and is not seen when the temperature is cycled below that required for nucleation. Thus, even during a slow temperature scan, microtubule assembly is kinetically limited by nucleation. By contrast, depolymerization proceeds close to equilibrium.The radius of gyration of the tubulin heterodimers is 3.1 nm. The weight average diameter of rings in cold solutions is 38 nm, that of microtubules is 24.5 nm.At radiation dose rates of about 100 rad/s. radiation damage is of minor importance, as judged by the criterion of polymerizability. Total doses of up to 500,000 rad can be applied.Some concepts of analyzing time-resolved X-ray scattering data are presented. They make use of the fact that the scattering intensities vary continuously both with scattering angle and time. Cross-correlation of different regions of the pattern, and comparison of their temperature derivatives, reveals structural transitions not seen by other techniques.     

Chain length-dependent association of distamycin-type oligopeptides with A·T and G·C pairs in polydeoxynucleotide duplexes

Different binding affinities of various distamycin analogs including the deformylated derivative with poly(dA-dC)·poly(dG-dT) were investigated using CD measurements. The inhibitory effect of distamycins on the DNAase I cleavage activity of DNA duplexes strongly supports the binding data. The base specificity of the ligand interaction with duplex DNA depends on the chain length of distamycin analogs. Netropsin, distamycin-2 and the deformylated distamycin-3 show no binding to dG·dC containing sequences at moderate ionic strength and are classified as highly dA·dT specific. In contrast distamycin having three, four or five methylpyrrolecarboxamide groups also forms more or less stable complexes with dG·dC-containing duplexes. These ligands possess a lower basepair specificity. The correlation between binding behavior and oligopeptide structure shows that presence of the number of hydrogen acceptor and donor sites determines the basepair and sequence specificity. The additional interaction with dG·dC pairs becomes essential when the number of hydrogen acceptor sites exceeds n = 3.     

Maltose excretion by the symbiotic Chlorella of the heliozoan Acanthocystis turfacea

Chlorella sp. strain 3.83, a symbiotic Chlorella isolated from the heliozoan Acanthocystis turfacea, excreted between 8% and 16% of assimilated ¹⁴CO₂ as maltose in the light (15000 lx), with a pH optimum around 4.8. This percentage increased when the illuminance was lowered (36% at 1700 lx). Release of [¹⁴C]maltose continued in darkness and could be inhibited by the uncoupler carbonyl cyanide p-trifluoro-methoxyphenylhydrazone and by diethylstilbestrol. Net efflux of maltose was observed even at a concentration ratio of extracellular/intracellular maltose of 7.8. Exogenous [¹⁴C]maltose (5 mM) was taken up by the cells with a rate <2% of that of simultaneous maltose release, indicating a practically unidirectional transport. It is concluded that maltose excretion is an active-transport process.Abbreviations DES diethylstilbestrol - FCCP carbonyl cyanide p-trifluoromethoxyphenyl hydrazone - p.c. packed cells This work was supported by the Deutsche Forschungsgemeinschaft. Thanks are due to Doris Meindl for skillful experimental help.     

Reactions of wild tamarins,Saguinus mystax andSaguinus fuscicollis to avian predators

During a field study in northeastern Peru, the reactions of a group ofSaguinus mystax on Padre Isla and of a mixed-species troop ofS. mystax andSaguinus fuscicollis at the Río Blanco to raptorial and other birds were observed. Alarms that are specific to flying stimuli were elicited by birds of prey, but other birds that do not represent a threat to the tamarins also caused alarm calls. Alarm events (i.e., instances when one or more alarm calls were given) were observed at rates of 0.3/hr (Padre Isla) and 0.5/hr (Río Blanco). Rates of alarm events significantly increased after the birth of an infant in the Padre Isla group and after the attack of an ornate hawk-eagle (Spizaetus ornatus) on the Río Blanco group. Reactions to alarming stimuli/alarm calls ranged from looking up to falling down from trees. The modification of the tamarins' behavior by the potential presence of aerial predators indicates that raptors represent an important predatory threat to tamarins.     

Efficient generation of chimaeric mice using embryonic stem cells after long-term culture in the presence of ciliary neurotrophic factor

The aim of our study was to evaluate whether ciliary neurotrophic factor (CNTF) can substitute for leukaemia inhibitory factor (LIF) in maintaining pluripotential embryonic stem (ES) cells in culture. Two subclones of D3 ES cells were used to assess cell proliferation and differentiation in the presence of CNTF, LIF or Buffalo rat liver (BRL) cell-conditioned medium, or in the absence of exogenous differentiation inhibiting factors. ES cells maintained in medium supplemented with CNTF for up to four weeks were injected into blastocysts to investigate theirin vivo pluripotency in terms of chimaera formation. CNTF inhibited ES cell differentiation in a dose-dependent manner. The most effective concentration was 10 ng CNTF per ml of medium. The effects of CNTF on ES cell differentiation and proliferation were comparable to those of LIF at the same concentration. BRL cell-conditioned medium was less effective at preventing ES cell differentiation but induced their proliferation very markedly. Both ES cell clones efficiently formed chimaeras after long-term culture with CNTF as the only differentiation inhibiting agent. The ability of these ES cells to colonize the germ-line is the ultimate proof that CNTF can preserve the pluripotency of ES cells.