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     

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.     

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.     

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.     

Vertically stratified frugivore community composition and interaction frequency in a liana fruiting across forest strata

Vertical stratification is a key feature of tropical forests and structures plant–frugivore interactions. However, it is unclear whether vertical differences in plant-frugivore interactions are due to differences among strata in plant community composition or inherent preferences of frugivores for specific strata. To test this, we observed fruit removal of a diverse frugivore community on the liana Marcgravia longifolia in a Peruvian rain forest. Unlike most other plants, Marcgravia longifolia produces fruits across forest strata. This enabled us to study effects of vertical stratification on fruit removal without confounding effects of plant species and stratum. We found a high number of visits of a few frugivore species in the understorey and a low number of visits of many different frugivores in the canopy and midstorey. Whereas partial and opportunistic frugivores foraged across strata with differing frequencies, obligate frugivores were only found eating fruits in the higher strata. Avian frugivores foraging in the canopy were mainly large species with pointed wings, whereas under- and midstorey avian foragers were smaller with rounded wings. Our findings suggest a continuous shift in the frugivore community composition along the vertical gradient, from a few generalized frugivores in the understorey to a diverse set of specialized frugivores in the canopy. This shift in the frugivore community leads to correlated, reciprocal changes from specialized to generalized plant-frugivore interactions. Thus, we conclude that vertical niche differentiation between species in tropical forests persists even when food resources are available across strata. This highlights its role for promoting biodiversity and ecosystem functioning.     

Retention of xenobiotics along the phloem path

Detached leaves of Cyclamen persicum Mill. can be used as a simple source-sink system. Phloem transport in the excised material was monitored by the noninvasive ¹¹C-technique. Assimilate movement stopped immediately when the petiole was cut off. However, within 20 min a recovery of transport was observed. The translocation rate in the detached leaf was only 13% of that in the intact plant. ¹⁴C-Xenobiotics and [³H]sucrose were injected into the upper petiole parenchyma (source). They moved downstream by a symplastic route. The stump of the petiole was inserted into a buffer solution containing ethylenediaminetetraacetic acid (sink). After 3 h, the distribution of sucrose and xenobiotics was determined in five subsequent segments of the petiole (path). The retention coefficient (r) was calculated from the ratio of radioactivity in the vascular bundle to that in the petiole parenchyma. The distribution along the vascular path was given by a geometric progression, whereas its constant was the transport coefficient (q). Values of r and q corresponded with the degree of phloem mobility and ambimobility. Four groups of compounds were classified: (i) acidic substances with log K_ow = — 2 to — 2.4 (K_ow is the partition coefficient octanol/water) at pH 8 (pH of sieve tube sap), retained by ion trapping and exhibiting small lateral efflux (q0.7; maleic hydrazide, dalapon); (ii) acidic substances with log K_ow = — 0.7 to — 0.8 at pH 8, retained by ion trapping and subjected to a moderate lateral efflux (0.7>q> 0.5; 2,4-dichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid, bromoxynil); (iii) nonionised substances retained by optimum permeability, exhibiting a considerable lateral leakage (q<0.5; glyphosate, amitrole); (iv) substances without basipetal transport in the phloem (atrazine, diuron). Retention of sucrose corresponded quantitatively with that shown in group (i). This classification was also supported by results of uptake and efflux experiments using the isolated conducting tissue. Theoretical translocation profiles were calculated from the determined transport coefficients (q).Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - K_ow partition coefficient octanol/water - MCPA 2-methyl-4-chloro-phenoxyacetic acid - q transport coefficient in the vascular bundle - r retention coefficient in the vascular bundle The authors gratefully acknowledge the assistance of H. Fiedler and M. Neugebauer. We are particularly grateful to K. Dutschka, G. Hudepokl, and Dr. J. Knust for producing ¹¹CO₂.     

Actions and interactions of growth hormone and insulin-like growth factor-II: body and organ growth of transgenic mice

To characterize long-term actions and interactions of growth hormone (GH) and insulin-like growth factor-II (IGF-II) on postnatal body and organ growth, hemizygous phosphoenolpyruvate carboxykinase (PEPCK)-human IGF-II transgenic mice were crossed with hemizygous PEPCK-bovine GH transgenic mice. The latter are characterized by two-fold increased serum levels of IGF-I and exhibit markedly increased body, skeletal and organ growth. Four different genetic groups were obtained: mice harbouring the IGF-II transgene (I), the bGH transgene (B), or both transgenes (IB), and non- transgenic controls (C). These groups of mice have previously been studied for circulating IGF-I levels (Wolf et al., 1995a), whereas the present study deals with body and organ growth. Growth curves (week 3 to 12) were estimated by regression with linear and quadratic components of age on body weight and exhibited significantly (p < 0.001) greater linear coefficients in B and IB than in I and C mice. The linear coefficients of male I and C mice were significantly (p < 0.001) greater than those of their female counterparts, whereas this sex-related difference was absent in the bGH transgenic groups. The weights of internal organs as well as the weights of abdominal fat, skin and carcass were recorded from 3.5- to 8- month-old mice. In addition, organ weight-to-body weight-ratios (relative organ weights) were calculated. Except for the weight of abdominal fat, absolute organ weights were as a rule significantly greater in B and IB than in I and C mice. IGF-II overproduction as a tendency increased the weights of kidneys, adrenal glands, pancreas and uterus both in the absence and presence of the bGH transgene. Analysis of relative organ weights demonstrated significant (p < 0.05) effects of elevated IGF- II on the relative growth of kidneys (males and females) and adrenal glands (females), confirming our previous report on organ growth of PEPCK-IGF-II transgenic mice. In females, IGF-II and GH overproduction were additive in stimulating the growth of spleen and uterus, providing evidence for tissue-specific postnatal growth promoting effects by IGF-II in the presence of elevated IGF-I     

Cell wall-lytic activity in Chlorella fusca

The soluble fraction of homogenates of synchronous Chlorella fusca was tested for carbohydrate-lyzing activities. With isolated cell walls and -1,4-mannan or carboxymethyl cellulose as substrates, a sharp increase in activity occurred shortly before release of the daughter cells followed by a decline during release. The lytic activities were partially purified by ammonium sulphate precipitation and analyzed by gel filtration on a calibrated column. Apparent molecular weights were 27,000 for cell wall autolysin(s) and -1,4-mannanase, 36,000 for carboxymethyl cellulase and 70,000 for another -1,4-mannanase. Incubation of isolated cell walls with an enzyme preparation purified by ammonium sulphate precipitation resulted in release of up to 70% of the cell wall carbohydrate as monosaccharide, predominantly mannose and glucose. The carbohydrate released in vivo into the culture medium shortly before and during liberation of the daughter cells consisted largely of polymeric material with rhamnose, fucose and mannose as main constitutents. Upon poisoning the cells with NaN₃ or carbonyl cyanide p-trifluoromethoxy-phenylhydrazone, however, a monosaccharide fraction consisting of mannose and glucose was predominant in the medium. It is suggested that the major products of cell wall lysis in vivo are monosaccharides which are rapidly taken up and metabolized by the developing daughter cells in an energy-dependent manner.