Simulation of nitrogen assimilation by heterotrophic soil microbial biomass

Assimilation of N by heterotrophic soil microbial biomass is associated with decomposition of organic matter in the soil. The form of N assimilated can be either low molecular weight organic N released from the breakdown of organic matter (direct assimilation), or NH⁺₄ and NO⁻₃ from the soil inorganic N pool, into which mineralized organic N is released (mineralization immobilization turnover). The kinetics of C and N turnover in soil is quantifiable by means of computer simulation models. NCSOIL was constructed to represent the two assimilation schemes. The rate of N assimilation depends on the rate of C assimilation and microbial C/N ratio, thereby rendering it independent of the assimilation scheme. However, if any of the N forms is labeled, a different amount of labeled N assimilation will be simulated by the different schemes. Experimental data on inorganic N and ¹⁵N and on organic ¹⁵N dynamics in soils incubated with ¹⁵N added as NH⁺₄ or organic N were compared with data simulated by different model schemes. Direct assimilation could not account for the amount of ¹⁵N assimilated in any of the experimental treatments. The best fit of the model to experimental data was obtained for the mineralization immobilization turnover scheme when both NH⁺₄ and NO⁻₃ were assimilated, in proportion to their concentration in the soil.     

3D 13C/1H NMR-based assignments for side-chain resonances of Lactobacillus casei dihydrofolate reductase. Evidence for similarities between the solution and crystal structures of the enzyme

Summary ¹³C-based three-dimensional ¹H–¹H correlation experiments have been used to determine essentially complete ¹³C and ¹H resonance assignments for the amino acid side chains of uniformly ¹³C/¹⁵N labelled L. casei dihydrofolate reductase in a complex with the drug methotrexate. Excellent agreement is observed between these assignments and an earlier set of partial assignments made on the basis of correlating nuclear Overhauser effect and crystal structure data, indicating that the tertiary structure of the enzyme is similar in solution and in the crystal state.To whom correspondence should be addressed.     

Two New Triterpenoids from the Roots of Sanguisorba officinalis L.

Two new triterpenoids, octanordammar-1,11,13(17)-trien-17-ol-3,16-dione (1) and lup-12-en-15α,19β-diol-3,11-dioxo-28-oic acid (4), as well as 13 known compounds were isolated from the roots of Sanguisorba officinalis L. (Rosaceae). Their structures were determined using spectroscopic methods.     

Adenosine analogue–oligo-arginine conjugates (ARCs) serve as high-affinity inhibitors and fluorescence probes of type I cGMP-dependent protein kinase (PKGIα)

Introduction

Type I cGMP-dependent protein kinase (PKGIα) belongs to the family of cyclic nucleotide-dependent protein kinases and is one of the main effectors of cGMP. PKGIα is involved in regulation of cardiac contractility, vasorelaxation, and blood pressure; hence, the development of potent modulators of PKGIα would lead to advances in the treatment of a variety of cardiovascular diseases. Aim: Representatives of ARC-type compounds previously characterized as potent inhibitors and high-affinity fluorescent probes of PKA catalytic subunit (PKAc) were tested towards PKGIα to determine that ARCs could serve as activity regulators and sensors for the latter protein kinase both in vitro and in complex biological systems. Results: Structure–activity profiling of ARCs with PKGIα in vitro demonstrated both similarities as well as differences to corresponding profiling with PKAc, whereas ARC-903 and ARC-668 revealed low nanomolar displacement constants and inhibition IC₅₀ values with both cyclic nucleotide-dependent kinases. The ability of ARC-based fluorescent probes to penetrate cell plasma membrane was demonstrated in the smooth muscle tissue of rat cerebellum isolated arteries, and the compound with the highest affinity in vitro (ARC-903) showed also potential for in vivo applications, fully abolishing the PKG1α-induced vasodilation.     

Modulation of coupling in the Escherichia coli ATP synthase by ADP and Pi: Role of the ε subunit C-terminal domain

The ε-subunit of ATP-synthase is an endogenous inhibitor of the hydrolysis activity of the complex and its α-helical C-terminal domain (εCTD) undergoes drastic changes among at least two different conformations. Even though this domain is not essential for ATP synthesis activity, there is evidence for its involvement in the coupling mechanism of the pump. Recently, it was proposed that coupling of the ATP synthase can vary as a function of ADP and P_i concentration. In the present work, we have explored the possible role of the εCTD in this ADP- and P_i-dependent coupling, by examining an εCTD-lacking mutant of Escherichia coli. We show that the loss of P_i-dependent coupling can be observed also in the εCTD-less mutant, but the effects of P_i on both proton pumping and ATP hydrolysis were much weaker in the mutant than in the wild-type. We also show that the εCTD strongly influences the binding of ADP to a very tight binding site (half-maximal effect ≈ 1 nM); binding at this site induces higher coupling in EF_OF₁ and increases responses to P_i. It is proposed that one physiological role of the εCTD is to regulate the kinetics and affinity of ADP/P_i binding, promoting ADP/P_i-dependent coupling.     

Kidney proteome responses in the teleost fish Paralichthys olivaceus indicate a putative immune response against Streptococcus parauberis

The proteomic response to bacterial infection in a teleost fish (Paralichthys olivaceus) infected with Streptococcus parauberis was analyzed using label-free protein quantitation coupled with LC-MS(E) tandem mass spectrometry. A total of 82 proteins from whole kidney, a major lymphoid organ in this fish, were found to be differentially expressed between healthy and diseased fish analyzed 6, 24, 72 and 120 h post-infection. Among the differentially expressed proteins, those involved in mediating immune responses (e.g., heat shock proteins, cathepsins, goose-type lysozyme and complement components) were most significantly up-regulated by infection. In addition, cell division cycle 48 (CDC48) and calreticulin, which are associated with cellular recovery and glycoprotein synthesis, were up-regulated in the universal protein group, whereas the other proteins in that group were down-regulated. There was continuous activation of expression of immune-associated proteins during infection, but there was also loss of expression of proteins not involved in immune function. We expect that our findings regarding immune response at the protein level would offer new insight into the systemic response to bacterial infection of a major immune organ in teleost fish.     

Comparative effects of organic and inorganic nitrogen sources applied to a flooded soil on rice yield and availability of N

A pot experiment was conducted to study the effect of organic and inorganic nitrogen (N) sources on the yield and N uptake of rice from applied and native soil-N. The residual effect of these N sources on a succeeding wheat crop was also studied. Organic N was applied in the form of ¹⁵N-labelled Sesbania aculeata L., a legume, and inorganic N in the form of ¹⁵N-labelled ammonium sulphate. The two sources were applied to the soil separately or together at the time of transplanting rice. Recovery of N by rice from both the applied sources was quite low but both sources caused significant increases in biomass and N yield of rice. Maximum increase was recorded in soil treated with organic N. The residual value of the two materials as source of N for wheat was not significant; the wheat took up only a small fraction of the N initially applied. Loss of N occurred from both applied N sources, the losses being more from inorganic N. Both applied N sources caused a substantial increase in the availability of soil-N to rice and wheat; most of this increase was due to organic N and was attributed to the so-called ‘priming’ effect or ANI (added nitrogen interaction) of the applied material.     

Short-term effects of urea amended with the urease inhibitor N-(n-butyl) thiophosphoric triamide on perennial ryegrass

The current study investigated the short-term physiological implications of plant nitrogen uptake of urea amended with the urease inhibitor N-(n-butyl) thiophosphoric triamide (nBTPT) under both greenhouse and field conditions. ¹⁵N labelled urea amended with 0.0, 0.01, 0.1 and 0.5% nBTPT (w/w) was surface applied at a rate equivalent to 100 kg N ha^–1 to perennial ryegrass in a greenhouse pot experiment. Root, shoot and soil fractions were destructively harvested 0.75, 1.75, 4, 7 and 10 days after fertilizer application. Urease activity was determined in each fraction together with ¹⁵N recovery and a range of chemical analyses. The effect of nBTPT amended urea on leaf tip scorch was evaluated together with the effect of the inhibitor applied on its own on plant urease activity.nBTPT-amended urea dramatically reduced shoot urease activity for the first few days after application compared to unamended urea. The higher the nBTPT concentration the longer the time required for shoot activity to return to that in the unamended treatment. At the highest inhibitor concentration of 0.5% shoot urease activity had returned to that of unamended urea by 10 days. Root urease activity was unaffected by nBTPT in the presence of urea but was affected by nBTPT in the absence of urea.Transient leaf tip scorch was observed approximately 7–15 days after nBTPT + urea application and was greatest with high concentrations of nBTPT and high urea-N application rates. New developing leaves showed no visual sign of tip necrosis.Urea hydrolysis of unamended urea was rapid with only 1.3% urea-N remaining in the soil after 1.75 days. N uptake and metabolism by ryegrass was rapid with ¹⁵N recovery from unamended urea, in the plant (shoot + root) being 33% after 1.75 days. Most of the ¹⁵N in the soil following the urea+0.5% nBTPT application was still as urea after 1.75 days, yet ¹⁵N plant recovery at this time was 25% (root+shoot). This together with other evidence, suggests that if urea hydrolysis in soil is delayed by nBTPT then urea can be taken up by ryegrass as the intact molecule, albeit at a significantly slower initial rate of uptake than NH₄ ⁺-N. Protein and water soluble carbohydrate content of the plant were not significantly affected by amending urea with nBTPT however, there was a significant effect on the composition of amino acids in the roots and shoots, suggesting a difference in metabolism.Although nBTPT-amended urea affected plant urease activity and caused some leaf-tip scorch the effects were transient and short-lived. The previously reported benefit of nBTPT in reducing NH₃ volatilization of urea would appear to far outweigh any of the observed short-term effects, as dry-matter production of ryegrass is increased.     

The 60S ribosomal subunit is altered in the skeletal muscle of dystrophic hamsters

Polysomes from the skeletal muscle of normal and dystrophic hamsters were dissociated into ribosomal subunits by treatment with puromycin and the subunits from both strains were reassociated in all possible combinations. When their protein synthesis activity was assayed in a poly(U)-directed cell-free system at a low magnesium concentration, the reassociated ribosomes from dystrophic hamsters were less active than the ribosomes from control animals. The ribosomal defect is a property of the 60S subunit and is due to a ribosomal component rather than to abnormal binding of a non-ribosomal protein.     

Effect of host shoot clipping on carbon and nitrogen sources for arbuscular mycorrhizal fungi

The effect of clipping of the host-plant shoot on the sources of carbon and nitrogen for the arbuscular mycorrhizal (AM) fungus Gigaspora margarita was determined by measuring ¹³C in spores and hyphae in cocultures of C₃ and C₄ plants and by differential ¹⁵N labeling. C₃ and C₄ plants, which have different δ¹³C values, were grown in the same container separated by a series of hyphal compartments. The C₃ and C₄ plants were applied with ¹⁴N- and ¹⁵N-urea, respectively. After clipping of the C₃ shoots, spore δ ¹³C gradually approached that of the C₄ roots. Hyphal δ ¹³C paralleled that of spores. Spore % ¹⁵N was similar to that of mineral N in the C₄ plant compartment. Thus C in G. margarita coming from the clipped plants decreased with time. This demonstrates that C in AM fungi comes from living plants, whilst the N in spores comes mostly from the soil. Accepted: 28 November 2000