Subtype-selectivity of metal-dependent methionine aminopeptidase inhibitors

Inhibitors of methionine aminopeptidases (MetAPs) are treatment options for various pathological conditions. Several inhibitor classes have been described previously, but only few data on the subtype selectivity, which is of crucial importance for these enzymes, is available. We present a systematic study on the subtype- and species-selectivity of MetAP inhibitors that require the binding of an auxiliary metal ion. This includes, in particular, compounds based on the benzimidazole pharmacophore, but also hydroxyquinoline and picolinic acid derivatives. Our data indicates that a significant degree of selectivity can be attained with metal-dependent MetAP inhibitors.     

Effects of suboptimal root zone temperatures and shoot demand on net translocation of micronutrients from the roots to the shoot of maize

The effects of suboptimal root zone temperatures (RZTs) on net translocation rates from the roots to the shoots and the concentrations of Fe, Mn, Zn, and Cu were examined in maize grown in nutrient solution or soil. Plants were grown at 12 °C, 18 °C and 24 °C RZT. At each RZT, the growth-related shoot demand for nutrients was varied by independently modifying the temperature of the shoot base (SBT) including the apical shoot meristem. The net translocation rates of Mn and Zn from the roots to the shoots were reduced at low RZTs, irrespective of the SBT and of the substrate (soil or nutrient solution). Obviously, the net translocation rates of Mn and Zn at low RZT were mainly regulated by temperature effects on the roots and not by the chemical nutrient availability in the rhizosphere or by shoot growth rate as controlled by SBTs. When both RZT and SBT were reduced, the decrease in net translocation rates of Mn and Zn was similar to the decline in the shoot growth rate and concentrations of Mn and Zn in the shoot fresh matter were not greatly affected or were even increased by low RZT. However, at high SBT and low RZT in nutrient solution, the depressed net translocation rates of Mn and Zn combined with the increased shoot growth resulted in significantly decreased concentrations of Mn and Zn in the shoot, indicating that Mn and Zn may become deficient even at high chemical availability. By contrast to Mn and Zn, the net translocation rates of Fe and Cu at all RZTs were markedly enhanced by increased SBTs. Accordingly, the concentrations of Fe and Cu in the shoot fresh matter were not greatly affected by RZTs, irrespective of the SBTs. These results indicate that the ability of roots to supply Fe and Cu to the shoot was internally regulated by the growth related shoot demand per unit of roots. Deceased 21 September 1996 Deceased 21 September 1996     

Root colonization and iron nutritional status of a Pseudomonas fluorescens in different plant species

Root colonization and induction of an iron stress regulated promoter for siderophore production by Pseudomonas fluorescens 2-79RLI was studied in vitro and in the rhizosphere of different plant species. P. fluorescens 2-79RLI was previously genetically modified with an iron regulated ice nucleation reporter, which allowed calibration of ice nucleation activity with siderophore production. Initial experiments examined ice nucleation activity and siderophore production under different growth conditions in vitro. These studies demonstrated that P. fluorescens 2-79RLI could utilize both Fe-citrate and Fe-phytosiderophore as iron sources, suggesting that production of these compounds by plants would increase iron availability for P. fluorescens 2-79RLI in the rhizosphere. Fe demand and Fe stress were further shown to be a function of nutrient availability and were reduced when carbon was limiting for growth. Subsequent experiments extended these observations to rhizosphere cells. Cells were sampled from the rhizosphere and the rhizoplane. Results of a soil microcosm experiment showed that Fe stress was reduced for P. fluorescens 2-79RLI in the barley rhizosphere as compared to the cells in the rhizosphere.of lupin. In lupin, relative Fe stress of P. fluorescens 2-79RLI was greater at the root tip than in the lateral root zone. In a second experiment comparing zucchini and bean, iron stress was greater for P. fluorescens 2-79RLI associated with zucchini than with bean. In a third experiment with rape plants under P deficient conditions, addition of soluble P was shown to increase Fe stress for P. fluorescens 2-79RLI located at the root tip, but not in the lateral root zone. This study showed that Fe stress of P. fluorescens 2-79RLI in the rhizosphere may be influenced by plant species, P source, root zone and localization of the cells within the rhizosphere.     

Extension of the phosphorus depletion zone in VA-mycorrhizal white clover in a calcareous soil

To examine the influence of vesicular-arbuscular (VA) mycorrhizal fungi on phosphorus (P) depletion in the rhizosphere, mycorrhizal and non-mycorrhizal white clover (Trifolium repens L.) were grown for seven weeks in a sterilized calcareous soil in pots with three compartments, a central one for root growth and two outer ones for hyphae growth. Compartmentation was accomplished by a 30-μm nylon net. The root compartment received a uniform level of P (50 mg kg⁻¹ soil) in combination with low or high levels of P (50 or 150 mg kg⁻¹ soil) in the hyphal compartments. Plants were inoculated withGlomus mosseae (Nicol. & Gerd.) Gerd. & Trappe or remained uninfected. Mycorrhizal inoculation doubled P concentration in shoot and root, and increased dry weight, especially of the shoot, irrespective of P levels. Mycorrhizal contribution accounted for 76% of total P uptake at the low P level and 79% at the high P level, and almost all of this P was delivered by the hyphae from the outer compartment. In the non-mycorrhizal plants, the depletion of NaHCO₃-extractable P (Olsen-P) extended about 1 cm into the outer compartment, but in the mycorrhizal plants a uniform P depletion zone extended up to 11.7 cm (the length of the hyphal compartment) from the root surface. In the outer compartment, the mycorrhizal hyphae length density was high (2.5–7 m cm⁻³ soil) at the various distances (0–11.7 cm) from the root surface. Uptake rate of P by mycorrhizal hyphae was in the range of 3.3–4.3×10⁻¹⁵ mol s⁻¹ cm⁻¹.     

Iron Stress and Pyoverdin Production by a Fluorescent Pseudomonad in the Rhizosphere of White Lupine (Lupinus albus L.) and Barley (Hordeum vulgare L.)

Induction of high-affinity iron transport during root colonization by Pseudomonas fluorescens Pf-5 (pvd-inaZ) was examined in lupine and barley growing in microcosms. P. fluorescens Pf-5 (pvd-inaZ) contains a plasmid carrying pvd-inaZ; thus, in this strain, ice nucleation activity is regulated by pyoverdin production. Lupine or barley plants were grown for 18 or 8 days, respectively, in soil amended with 2% calcium carbonate and inoculated with P. fluorescens Pf-5 (pvd-inaZ) at a density of 4 x 10(sup8) CFU g (dry weight) of soil(sup-1). A filter paper blotting technique was used to sample cells from the rhizosphere in different root zones, and then the cells were resuspended for enumeration and measurement of ice nucleation activity. The population density of P. fluorescens Pf-5 (pvd-inaZ) in the rhizosphere decreased by one order of magnitude in both lupine and barley over time. The ice nucleation activity ranged from -3.4 to -3.0 log ice nuclei CFU(sup-1) for lupine and -3.0 to -2.8 log ice nuclei CFU(sup-1) for barley, was similar in all root zones, and did not change over time. An in vitro experiment was conducted to determine the relationship between ice nucleation activity and pyoverdin production in P. fluorescens Pf-5 (pvd-inaZ). An ice nucleation activity of approximately -3.0 log ice nuclei CFU(sup-1) was measured in the in vitro experiment at 25 to 50 (mu)M FeCl(inf3). By using the regression between ice nucleation activity and pyoverdin production determined in vitro and assuming a P. fluorescens Pf-5 (pvd-inaZ) population density of 10(sup8) CFU g of root(sup-1), the maximum possible pyoverdin accumulation by P. fluorescens Pf-5 (pvd-inaZ) in the rhizosphere was estimated to be 0.5 and 0.8 nmol g of root(sup-1) for lupine and barley, respectively. The low ice nucleation activity measured in the rhizosphere suggests that nutritional competition for iron in the rhizosphere may not be a major factor influencing root colonization by P. fluorescens Pf-5 (pvd-inaZ).     

Mobilization of iron by phytosiderophores as affected by other micronutrients

It has been shown previously (Treeby et al., 1989) that phytosiderophores, released by roots of iron deficient grasses (Gramineae), mobilize from calcareous soils not only iron (Fe) but also zinc (Zn), manganese (Mn) and copper (Cu). Mobilization of Fe may therefore be impaired by other micronutrient cations. This has been studied in both, model experiments with Fe hydroxide and with a calcareous soil (15% CaCO₃, pH 8.6) amended with micronutrients as sulfate salts.Mobilization of Fe from Fe hydroxide by phytosiderophores (epi-3-hydroxymugineic acid) was not affected by the addition of CaCl₂, MgSO₄ and MnSO₄, slightly inhibited by ZnSO₄ and strongly inhibited by CuSO₄. In a calcareous soil amended with increasing levels of ZnSO₄, MnSO₄ and CuSO₄, mobilization of Fe by phytosiderophores remained uneffected by Zn and Mn amendments but was progressively impaired by increasing levels of Cu amendment, correlated with corresponding enhancement of Cu mobilization.High concentrations of ZnSO₄ and MnSO₄ and relatively high concentrations of CuSO₄ were required for inhibition of Fe mobilization by phytosiderophores. It is therefore concluded that in most calcareous soils phytosiderophores efficiently mobilize Fe, and that phytosiderophores play an important role in Fe acquisition by grasses grown on calcareous soils.     

Cation exchange capacity and lead sorption in ectomycorrhizal fungi

Two ectomycorrhizal fungi, Paxillus involutus 533 and Laccaria bicolor S238, differing greatly in their mycelial characteristics, were investigated with regard to their cation exchange capacity and Pb-binding capacity in vitro after growth with either NO₃ ^- or NH₄ ⁺ as N source. The CECs of 800–1200 mol g-1 dry weight for Paxillus involutus 533 and 2000–3000 mol g-1 dry weight for Laccaria bicolor S238, were high compared to plant roots. The fungal mycelium also had a high Pb sorption capacity. It was higher in Laccaria bicolor S238 than in Paxillus involutus 533 and higher after pregrowth in NO₃ ^- compared to NH₄ ⁺. Both the higher CEC and the higher Pb sorption capacity of Laccaria bicolor S238 compared to Paxillus involutus 533 might have been the result of the hydrophilic nature of the of Laccaria bicolor S238 mycelium. It would have absorbed the solutions better than the hydrophobic mycelium of Paxillus involutus 533. X-ray microanalysis of the cell walls revealed that the Pb content of the cell walls was higher in Paxillus involutus 533 than in Laccaria bicolor S238. Nevertheless, electron dense deposits in the cell walls of Laccaria bicolor S238 contained large amounts of Pb, P and S. Thus, while Pb was evenly distributed in the cell walls of Paxillus involutus 533, Pb was accumulated in electron dense deposits in Laccaria bicolor S238. The results are discussed in view of their significance for the mycorrhizal symbiosis.     

Optimizing Mouse Surgery with Online Rectal Temperature Monitoring and Preoperative Heat Supply. Effects on Post-Ischemic Acute Kidney Injury

Body temperature affects outcomes of tissue injury. We hypothesized that online body core temperature recording and selective interventions help to standardize peri-interventional temperature control and the reliability of outcomes in experimental renal ischemia reperfusion injury (IRI). We recorded core temperature in up to seven mice in parallel using a Thermes USB recorder and ret-3-iso rectal probes with three different protocols. Setup A: Heating pad during ischemia time; Setup B: Heating pad from incision to wound closure; Setup C: A ventilated heating chamber before surgery and during ischemia time with surgeries performed on a heating pad. Temperature profile recording displayed significant declines upon installing anesthesia. The profile of the baseline experimental setup A revealed that <1% of the temperature readings were within the target range of 36.5 to 38.5°C. Setup B and C increased the target range readings to 34.6 ± 28.0% and 99.3 ± 1.5%, respectively. Setup C significantly increased S3 tubular necrosis, neutrophil influx, and mRNA expression of kidney injury markers. In addition, using setup C different ischemia times generated a linear correlation with acute tubular necrosis parameters at a low variability, which further correlated with the degree of kidney atrophy 5 weeks after surgery. Changing temperature control setup A to C was equivalent to 10 minutes more ischemia time. We conclude that body temperature drops quickly in mice upon initiating anesthesia. Immediate heat supply, e.g. in a ventilated heating chamber, and online core temperature monitoring can help to standardize and optimize experimental outcomes.     

Contribution of an arbuscular mycorrhizal fungus to the uptake of cadmium and nickel in bean and maize plants

Two experiments were carried out in pots with three compartments, a central one for root and hyphal growth and two outer ones which were accessible only for hyphae of the arbuscular mycorrhizal fungus, Glomus mosseae ([Nicol. and Gerd.] Gerdemann and Trappe). In the first experiment, mycorrhizal and nonmycorrhizal bean (Phaseolus vulgaris L.) plants were grown in two soils with high geogenic cadmium (Cd) or nickel (Ni) contents. In the second experiment, mycorrhizal and nonmycorrhizal maize (Zea mays L.) or bean plants were grown in a non-contaminated soil in the central compartment, and either the Cd- or Ni-rich soil in the outer compartments. In additional pots, mycorrhizal plants were grown without hyphal access to the outer compartments. Root and shoot dry weight was not influenced by mycorrhizal inoculation, but plant uptake of metals was significantly different between mycorrhizal and nonmycorrhizal plants. In the first experiment, the contribution of mycorrhizal fungi to plant uptake accounted for up to 37% of the total Cd uptake by bean plants, for up to 33% of the total copper (Cu) uptake and up to 44% of the total zinc (Zn) uptake. In contrast, Ni uptake in shoots and roots was not increased by mycorrhizal inoculation. In the second experiment, up to 24% of the total Cd uptake and also up to 24% of the total Cu uptake by bean could be attributed to mycorrhizal colonisation and delivery by hyphae from the outer compartments. In maize, the mycorrhizal colonisation and delivery by hyphae accounted for up to 41% of the total Cd uptake and 19% of the total Cu uptake. Again, mycorrhizal colonisation did not contribute to Ni uptake by bean or maize. The results demonstrate that the arbuscular mycorrhizal fungus contributed substantially not only to Cu and Zn uptake, but also to uptake of Cd (but not Ni) by plants from soils rich in these metal cations. Deceased 21 September 1996 Deceased 21 September 1996     

Effect of nitrogen forms on growth and chemical changes in the rhizosphere of strawberry plants

The experiment was set up to examine the influence of different nitrogen forms: (NH₄)₂SO₄, Ca(NO₃)₂ or NH₄NO₃ on growth response, root induced pH changes in the rhizosphere, root-borne acid phosphatase activity in strawberry plants cv. Senga Sengana. The plants grown on sandy mineral soil were fertilized with 3 forms of nitrogen, in concentrations of 46 mg N·kg⁻¹ soil. The plants were grown in rhizoboxes with removable plexiglass lids. To ensure the root growth along the plexiglass lids, the rhizoboxes were placed at an angle of about 50° with the lid on the lower side. In case of ammonium supply, the nitrification inhibitor DIDIN was added (10 mg·kg⁻¹ of moist soil) to prevent conversion of ammonium into nitrate. The growth response (roots and shoots) of strawberry plants were determined after 11 weeks of treatment with different N forms. The best development of the root system and shoots (root and shoot dry weight and root length) was obtained, when ammonium nitrate was supplied. It is suggested therefore, that NH₄NO₃ stimulates vegetative growth of strawberry plants cv. Senga Sengana. However, there were no statistical differences in a leaf and flower number of the plants grown under different forms of N-fertilization. Determination of rhizosphere pH, and acid phosphatase activity were executed using non-destructive techniques, which enabled weekly measurement of chemical changes in the rhizosphere. The results revealed that the form of nitrogen supplied had a predominant effect on chemical changes in the rhizosphere of strawberry plants. The highest pH values (average pH 6.8) were measured in the rhizosphere of individual plants supplied with Ca(NO₃)₂. Whereas the lowest pH values (average pH 5.8) were detected in the presence of (NH₄)₂SO₄. The curve of rhizosphere pH measured along individual roots of the plants treated with Ca(NO₃)₂ represents the highest pH values whereas the curve of rhizosphere pH under (NH₄)₂SO₄ treatment had the lowest pH values. The highest activity of acid phosphatase were observed in the rhizosphere of strawberry plants grown in the presence of (NH₄)₂SO₄, at pH 5.8.