Auto-induction medium for the production of [U-15N]- and [U-13C, U-15N]-labeled proteins for NMR screening and structure determination

Protocols have been developed and applied for the high-throughput production of [U-15N]- or [U-13C-, U-15N]-labeled proteins using the conditional methionine auxotroph Escherichia coli B834. The large-scale growth and expression uses a chemically defined auto-induction medium containing salts and trace metals, vitamins including vitamin B12, and glucose, glycerol, and lactose. The results from nine expression trials in 2-L of the auto-induction medium (500 mL in each of four polyethylene terephthalate beverage bottles) gave an average final optical density at 600 nm of approximately 5, an average wet cell mass yield of approximately 9.5 g L(-1), and an average yield of approximately 20 mg of labeled protein in the six instances in which proteolysis of the fusion protein was observed. Correlations between the cell mass recovered, the level of protein expression, and the relative amounts of glucose, glycerol, and lactose in the auto-induction medium were noted. Mass spectral analysis showed that the purified proteins contained both 15N and 13C at levels greater than 95%. 1H-15N heteronuclear single quantum correlation spectroscopy as well as 13C; 15N-edited spectroscopy showed that the purified [U-15N]- and [U-13C, U-15N]-labeled proteins were suitable for structure analysis.     

Stable Isotope Analysis of Amphidromous Hawaiian Gobies Suggests Their Larvae Spend a Substantial Period of Time in Freshwater River Plumes

Synopsis We employed stable isotope analysis (δ¹³C, δ¹⁵N) to evaluate the sources of nutrients used by amphidromous gobiid fishes (Lentipes concolor, Sicyopterus stimpsoni, Awaous guamensis) caught migrating into and living in Hakalau Stream, Hawaii. Although considerable variation amongst the stable isotope values of stream items was noted across all 4 years of our study, the relationships between the fishes were relatively constant. Stable isotope values of recruiting gobies were consistently closer to those of both inshore plankton and freshwater adults than those of offshore plankton, suggesting that the larvae of these species derive much of their nutrition from inshore environments influenced by fresh water. Small differences between the stable values of these species further suggested that their larvae come from different inshore locations. After entering fresh water all species appear to swim rapidly upstream without feeding. Finally, once well upstream, adult L. concolor and A. guamensis appear to assume an omnivorous diet while adult S. stimpsoni rely upon autochthonous production within streams. We propose that freshwater food webs play an integral yet complex role in the lives of both larval and adult amphidromous Hawaiian fishes.     

The uptake, metabolism, transport and transfer of nitrogen in an arbuscular mycorrhizal symbiosis

Nitrogen (N) is known to be transferred from fungus to plant in the arbuscular mycorrhizal (AM) symbiosis, yet its metabolism, storage and transport are poorly understood. In vitro mycorrhizas of Glomus intra-radices and Ri T-DNA-transformed carrot roots were grown in two-compartment Petri dishes. (15)N- and/or (13)C-labeled substrates were supplied to either the fungal compartment or to separate dishes containing uncolonized roots. The levels and labeling of free amino acids (AAs) in the extra-radical mycelium (ERM) in mycorrhizal roots and in uncolonized roots were measured by gas chromatography/mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC). Arginine (Arg) was the predominant free AA in the ERM, and almost all Arg molecules became labeled within 3 wk of supplying (15)NH(4) (+) to the fungal compartment. Labeling in Arg represented > 90% of the total (15)N in the free AAs of the ERM. [Guanido-2-(15)N]Arg taken up by the ERM and transported to the intra-radical mycelium (IRM) gave rise to (15)N-labeled AAs. [U-(13)C]Arg added to the fungal compartment did not produce any (13)C labeling of other AAs in the mycorrhizal root. Arg is the major form of N synthesized and stored in the ERM and transported to the IRM. However, NH(4) (+) is the most likely form of N transferred to host cells following its generation from Arg breakdown.     

ent-Kauranoid derivatives from Sideritis moorei

Seven new ent-kauranoid derivatives ent-7alpha,18-dihydroxykaur-16-en-3-one, ent-18-acetoxy-3beta,7alpha-dihydroxykaur-15-en-17-al, ent-3beta-acetoxy-7alpha,18-dihydroxykaur-15-en-17-al, ent-18-acetoxy-3beta,7alpha,17-trihydroxykaur-15-ene, ent-3beta-acetoxy-7alpha,17,18-trihydroxykaur-15-ene, ent-18-acetoxy-3beta,7alpha,17-trihydroxy-15beta,16beta-epoxykaurane and ent-3beta-acetoxy-7alpha,17,18-trihydroxy-15beta,16beta-epoxykaurane have been isolated from Sideritis moorei. The structures of these compounds have been established by spectroscopic means and chemical correlations.     

Halogenated metabolites from Japanese Laurencia spp

Further investigation of Laurencia species from Japanese waters, which were collected at three locations, yielded brominated metabolites, a labdane- type diterpene and a C15 acetogenin possessing a terminal bromoallene group. Their structures were deduced from analysis of spectroscopic data.     

Inter-row crop competition for band-injected ammonium nitrate

Inter-species competition for applied nutrients may be managed by weed control in cultivated communities. However, intra-crop competition may still occur. Thus, regarding a fertilizer band as a finite nutrient source, individual rows of a cereal crop may compete for the applied nitrogen. The aim was to explore the partition of applied N between crop rows affected by the displacement of the fertilizer band relative to the rows. In a micro-plot experiment, a ¹⁵N-ammonium–¹⁵N-nitrate solution was injected in four positions parallel to two rows of spring wheat (Triticum aestivum ssp. vulgare). The course of ¹⁵N crop uptake in each row was estimated by a sigmoid growth function using eight samplings during the elongation phase. The estimated parameters were used for calculating the asymptotic value (a), the time until a/2 was reached (T_0.5a), and the uptake rate at this time (MaxRate), and related to the distance between the crop row and fertilizer band. The a-value decreased by 5.1%-point recovery cm^–1, T_0.5a increased by 0.5 day cm^–1 and the MaxRate decreased by 0.3%-point recovery day^–1 cm^–1. Placement of the fertilizer band centred between two crop rows caused an even access to the applied nitrogen, but a parallel displacement from the centre by a few centimetres significantly affected the parameters. The proliferation of the roots in the soil and thus the time until the roots reached the fertilizer zone is assumed to cause the inter-row competition for the band-injected N source. The access to the fertilizer band was clearly reflected in the dry matter production causing an uneven crop stand. The consequences of inter-row crop competition for a band-injected N source with respect to grain yield and grain quality were not explored, but attention on these parameters are required, particularly in the production of high quality wheat grains.     

New unusual pregnane glycosides with antiproliferative activity from Solenostemma argel

Seven new 15-keto pregnane glycosides, namely Stemmosides E--K, were isolated from Solenostemma argel. Stemmosides E--J are characterized by the occurrence of an uncommon 14 beta proton configuration while stemmosides E and F possess in addition a rare enolic function in C-16. On the other hand, stemmosides G-J display an unusual C-17 alpha side chain. Their structures were established by ESI-MS and NMR experiments. Moreover, the effect of these compounds on the VEGF-induced in Kaposi's sarcoma cell proliferation was tested. Results indicated that all the compounds reduced the cell proliferation in a dose dependent manner.     

GPR80/99, proposed to be the P2Y<Subscript>15</Subscript> receptor activated by adenosine and AMP,is not a P2Y receptor

The orphan receptor GPR80 (also called GPR99) was recently reported to be the P2Y₁₅ receptor activated by AMP and adenosine and coupled to increases in cyclic AMP accumulation and intracellular Ca²⁺ mobilization (Inbe et al. J Biol Chem 2004; 279: 19790–9). However, the cell line (HEK293) used to carry out those studies endogenously expresses A_2A and A_2B adenosine receptors as well as multiple P2Y receptors, which complicates the analysis of a potential P2Y receptor. To determine unambiguously whether GPR80 is a P2Y receptor subtype, HA-tagged GPR80 was either stably expressed in CHO cells or transiently expressed in COS-7 and HEK293 cells, and cell surface expression was verified by radioimmunoassay (RIA). COS-7 cells overexpressing GPR80 showed a consistent twofold increase in basal inositol phosphate accumulation. However, neither adenosine nor AMP was capable of promoting accumulation of either cyclic AMP or inositol phosphates in any of the three GPR80-expressing cells. A recent paper (He et al. Nature 2004; 429: 188–93) reported that GPR80 is a Gq-coupled receptor activated by the citric acid cycle intermediate, -ketoglutarate. Consistent with this report, -ketoglutarate promoted inositol phosphate accumulation in CHO and HEK293 cells expressing GPR80, and pretreatment of GPR80-expressing COS-7 cells with glutamate dehydrogenase, which converts -ketoglutarate to glutamate, decreased basal levels of inositol phosphates. Taken together, these data demonstrate that GPR80 is not activated by adenosine, AMP or other nucleotides, but instead is activated by -ketoglutarate. Therefore, GPR80 is not a new member of the P2Y receptor family.     

The Fate and Retention of Organic and Inorganic <Superscript>15</Superscript>N-Nitrogen in an Old-Growth Forest Soil in Western Oregon

Forests in the American Pacific Northwest receive very little nitrogen (N) through atmospheric deposition; therefore, they can provide insights into how the N cycle functioned in other regions before heavy atmospheric deposition of inorganic N began. Our objectives were to determine (a) if the fate of organic N differed from the fate of inorganic N, (b) the effect that polyphenols have on the fate of organic N, and (c) the effect of season of addition on the fate of N inputs. We traced N added to in situ soil cores as ammonium, organic N, tannin-complexed organic N, and the N₂-fixing lichen Lobaria oregana. Total ¹⁵N recovery was between 74% and 109% for all N additions. Total ¹⁵N recovery did not vary significantly from the first sampling date to the last date. The litter/organic horizon, as a bulk pool, was the largest N retention pool for all forms of N addition. Within the litter/organic horizon, the chloroform-extractable microbial biomass initially accounted for nearly all of the added N from the ammonium additions. On a different time scale, microbial biomass also played a noteworthy role in the retention of N from organic N, tannin-complexed organic N, and Lobaria. Complexing organic matter with tannin appeared to slow N cycling, but it did not significantly change the ultimate distribution of added organic N. Season of N addition had little effect on the retention of added N; however, where differences did occur, spring additions had lower recoveries than autumn additions.     

Stable isotope composition of organic compounds transported in the phloem of European beech--evaluation of different methods of phloem sap collection and assessment of gradients in carbon isotope composition during leaf-to-stem transport

The analysis of stable isotope composition (delta13C, delta15N, delta18O) of phloem-transported organic matter is a useful tool for assessing short-term carbon and water balance of trees. A major constraint of the general application of this method to trees at natural field sites is that the collection of phloem sap with the "phloem bleeding" technique is restricted to particular species and plant parts. To overcome this restriction, we compared the contents (amino compounds and sugars) and isotope signatures (delta13C, delta15N, delta18O) of phloem sap directly obtained from incisions in the bark (bleeding technique) with phloem exudates where bark pieces were incubated in aqueous solutions (phloem exudation technique with and without chelating agents [EDTA, polyphosphate] in the initial sampling solution, which prevent blocking of sieve tubes). A comparable spectrum of amino compounds and sugars was detected using the different techniques. O, C, or N compounds in the initial sampling solution originating from the chelating agents always decreased precision of determination of the respective isotopic signatures, as indicated by higher standard deviation, and/or led to a significant difference of mean delta as compared to the phloem bleeding technique. Hence, depending on the element from which the ratio of heavy to light isotope is determined, compounds lacking C, N, and/or O should be used as chelating agents in the exudation solution. In applying the different techniques, delta13C of organic compounds transported in the phloem of the twig (exudation technique with polyphosphate as chelating agent) were compared with those in the phloem of the main stem (phloem bleeding technique) in order to assess possible differences in carbon isotope composition of phloem carbohydrates along the tree axis. In July, organic compounds in the stem phloem were significantly enriched in 13C by > 1.3 per thousand as compared to the twig phloem, whereas this effect was not observed in September. Correlation analysis between delta13C and stomatal conductance (Gs) revealed the gradient from the twigs to the stem observed in July may be attributed to temporal differences rather than to spatial differences in carbon isotope composition of sugars. As various authors have produced conflicting results regarding the enrichment/depletion of 13C in organic compounds in the leaf-to-stem transition, the different techniques presented in this paper can be used to provide further insight into fractionation processes associated with transport of C compounds from leaves to branches and down the main stem.