Nitrogen fixation in Faidherbia albida,Acacia raddiana,Acacia senegal and Acacia seyal estimated using the 15N isotope dilution technique

A pot experiment was conducted in a greenhouse using the ¹⁵N isotope dilution method and two reference plants, Parkia biglobosa and Tamarindus indica to estimate nitrogen fixed in four Acacia species: A raddiana, A. senegal, A. seyal and Faidherbia albida (synonym Acacia albida). For the reference plants, the ¹⁵N enrichments in leaves, stems and roots were similar. With the fixing plants, leaves and stems had similar ¹⁵N enrichments; they were higher than the ¹⁵N enrichment of roots. The amounts of nitrogen fixed at 5 months after planting were similar using either reference plant. Estimates of the percentage of N derived from fixation (%Ndfa) for the above ground parts, in contrast to %Ndfa in roots, were similar to those for the whole plant. However, none of the individual plant parts estimated accurately total N fixed in the whole plant, and excluding the roots resulted in at least 30% underestimation of the amounts of N fixed. Between species, differences in N₂ fixation were observed, both for %Ndfa and total N fixed. For %Ndfa, the best were A. seyal (average, 63%) and A. raddiana (average, 62%), being at least twice the %Ndfa in A. senegal and F. albida. Because of its very high N content, A. seyal was clearly the best in total N fixed, fixing 1.62 g N plant^–1 compared to an average of 0.48 g N plant^–1 for the other Acacia species. Our results show the wide variability existing between Acacia species in terms of both %Ndfa and total N fixed: A. seyal was classified as having a high N₂ fixing potential (NFP) while the other Acacia species had a low NFP.     

Root water uptake of field-growing plants indicated by measurements of natural-abundance deuterium

Measurements of stable-isotope ratios of water extracted from stems and, in some studies, soils are increasingly being used to study the integrated root function of field-growing plants. This study explored if additional measurements on water extracted from roots could indicate the activity of roots in different areas of the soil profile and their influence on canopy water sources, so providing advantages over more common sampling strategies. Studies were conducted on trees and shrubs located in diverse habitats: a saline, semi-arid floodplain, a subhumid mountain-range front and a cold desert. At each site, roots, soil immediately surrounding the roots, and plant stems were sampled. Roots were taken from different depths in the soil, to approximately 2 m at one site. Overall, 80% of roots sampled had H isotope ratios different from the surrounding soil. The differences up to 37, were significant (p<0.05) at two of the sites. Thus water in most of the roots sampled did not come entirely, if at all, from the surrounding soil, illustrating movement and possible mixing of water within the root system. This condition was not simply related to the availability of water surrounding the soil, which was also measured. There were also differences in root and stem H isotope ratios (up to 17) in 67% of samples, although the difference was only significant in shallow samples from the floodplain. The general similarity in stem and root ²H values indicates that most roots sampled were involved in the main supply of water to the canopy. Patterns of root function varied between the individual sites. Trees were primarily using groundwater at the floodplain and mountain front sites, as the surface soils had mean matric potentials of-1800 kPa. At the mountain front site, the surface roots were transporting groundwater to the canopy in isolation form the surrounding soil. In contrast, surface roots at the floodplain were taking up water from the surrounding soil, although this water was not a significant source in the trees' overall water supply. This activity of surface roots would not have been evident from the ²H data without the root samples. At the cold desert the roots in moist surface soil provided the main source of water for the shrubs. There too the root data indicated different water uptake patterns than otherwise would have been assumed. The root data showed that groundwater could not have been a water source, a conclusion which had been reached in a previous study. Thus measurements of stable isotope ratios in root water may be a valuable tool in assessing water uptake patterns and root function.     

Symbiotic N2-fixation by the cover crop Pueraria phaseoloides as influenced by litter mineralization

The perennial legume Pueraria phaseoloides is widely used as a cover crop in rubber and oil palm plantations. However, very little knowledge exists on the effect of litter mineralization from P. phaseoloides on its symbiotic N₂-fixation. The contribution from symbiotic N₂-fixation (Ndfa) and litter N (Ndfl) to total plant N in P. phaseoloides was determined in a pot experiment using a ¹⁵N cross-labeling technique. For determination of N₂-fixation the non-fixing plant Axonopus compressus was used as a reference. The experiment was carried out in a growth chamber during 9 weeks with a sandy soil and 4 rates of ground litter (C/N=16,2.8% N). P. phaseoloides plants supplied with the highest amount of litter produced 26% more dry matter and fixed 23% more N than plants grown in soil with no litter application, but the percentage of Ndfa decreased slightly, but significantly, from 87 to 84%. The litter N uptake was directly proportional to the rate of application and constituted 10% of total plant N at the highest application rate. Additionally, a positive correlation was found between litter N uptake and the amount of fixed N₂. The total recovery of litter N in plants averaged 26% at harvest (shoot + root) and was not affected by the quantity added. A parallel incubation experiment also showed that, as an average of all litter levels, 26% of the litter N was present in the inorganic N pool. The amounts of fertilizer and soil N taken up by plants decreased with litter application, probably due to microbial immobilization and denitrification. It is concluded that, within the litter levels studied, litter mineralization will result in a higher amount of N₂-fixed by P. phaseoloides.     

A simple cell labeling technique by means of lectins linked to fluorochromes for the detection of cells on tissue sections

Summary— We designed a protocol for cell labeling with the lectin wheat germ agglutinin (WGA) linked to the fluorochrome tetramethyl-rhodamine isothiocyanate (TRITC) for effective detection of the B16F10 melanoma and Lewis lung carcinoma (LLc) cells on pulmonary histological sections from C57BL6; mice. We have also determined a suitable concentration of WGA-TRITC (10 μg/ml), which leads to a very intense and homogeneous labeling of the cells, as it avoids cell clumping due to the presence of the lectin WGA. In order to determine to what extent the method affects these tumor cells, we have studied some important aspects related to their metastatic behavior, taking into account three parameters: a) viability and rate of proliferation of the cells cultured in vitro; b) percentage of animals C57BL6 mice) bearing metastasis 15 days after intravenous inoculation with 10⁵ B16F10 or LLc cells; and c) pattern of distribution of tumor foci in lung. There were no significant differences in these three parameters between the WGA-TRITC labeled-cells compared to the cultures of non-labeled cells in either of the cell lines (B16F10, LLc). Thus, we conclude that B16F10 and LLc tumor cells can be labeled following the protocol set-up in our study, as it allows these cells to be neatly identified on tissue sections and it causes no important physiological changes in the cells, with regard to metastatic behavior. These points make this technique very suitable for the detection of B16F10 and LLc cells on histological sections in studying their behavior during the first stages of the metastatic process.     

Extracellular ATP binding proteins as potential receptors in mucociliary epithelium: Characterization using [32P]3′-O-(4-benzoyl)benzoyl ATP,a photoaffinity label

3-O-(4-benzoyl)benzoyl ATP (BzATP) was used as a photoaffinity analog of ATP to label potential ATP receptors in ciliated cells. Like ATP, without photoactivation, BzATP stimulated the ciliary beat frequency in tissue culture up to threefold. Irradiation of intact cells in the presence of [-³²P]BzATP followed by SDS-PAGE and autoradiography revealed two labeled proteins with molecular masses of 46 and 96 kDa (p46 and p96). Photolabeling of both proteins was susceptible to digestion with trypsin, implying that the labeled proteins are at least partially exposed on the extracellular surface of the plasma membrane. The dependence of ³²P incorporation in both proteins on [-³²P]BzATP concentration was similar. Labeling of p46 but not p96 required Ca²⁺ or Mg²⁺. Various nucleotides stimulated the ciliary frequency, and inhibited the photolabeling of p46 and p96. The rank order of apparent affinity for p46 is: ATP ÃDP>GTPS>ADP S, UTP, 2MeSATP, AMP-PNP >AMP-PCP>AMP>adenosine; for p96 it is: ADPADP S ATP AMP-PCP, AMP-PNP>GTPS AMP>2MeSATP, UTP, adenosine. The rank of stimulation of ciliary beat frequency is: ADPS, UTP 2MeSATP, GTPS, AMP-PNP, ATPADP>AMPPCP>adenosine>AMP. These results suggest the involvement of p46 in the stimulatory effect of extracellular ATP on the ciliary beat, as a P₂ purinoceptor. On the other hand, p96 may represent a P₂ purinoceptor or an ectonucleotidase.This work was supported by grants (to Z.P. and to V.S-B.) from the Fund for Basic Research administered by the Israel Academy of Science and Humanities.     

Efficient enzymatic synthesis of 13 C,15N-labeled DNA for NMR studies

The power of heteronuclear NMR spectroscopy to study macromoleculesand their complexes has been amply demonstrated over the last decade. Theobstacle to routinely applying these techniques to the study of DNA has beenthe synthesis of ¹³C,¹⁵N-labeled DNA. Here wepresent a simple and efficient method to generate isotope-labeled DNA forNMR studies that is as easy as that for isotope labeling of RNA. The methodwas used to synthesize a uniformly¹³ C,¹⁵N-labeled 32-nucleotide DNA that binds tohuman basic fibroblast growth factor with high affinity and specificity.Isotope-edited experiments were applied to the¹³ C,¹⁵N-labeled DNA bound to unlabeled protein,and the ¹³ C,¹⁵N-labeled DNA was also examined incomplex with ¹⁵N-labeled protein. The NMR experiments showthat the DNA adopts a well-defined stable structure when bound to theprotein, and illustrate the potential of¹³ C,¹⁵N-labeled DNA for structural studies ofDNA–protein complexes.     

Monitoring calcium-induced conformational changes in recoverin by electrospray mass spectrometry.

Recoverin is a calcium-binding protein that regulates the vertebrate photoresponse by inhibiting rhodopsin kinase in response to high calcium concentrations. It is heterogeneously N-acylated by myristoyl and related fatty acyl residues that are thought to act as "calcium-myristoyl switches," whereby, in the presence of Ca2+, the N-terminal acyl group is extended away from recoverin and, in the absence of calcium, it is more closely associated with the protein. Here we use electrospray ionization mass spectrometry (ESI/MS) to examine hydrogen isotopic exchange rates for specific regions of both acylated and nonacylated recoverin in the presence and absence of calcium. The deuterium exchange rates of three regions in the hydrophobic myristoyl binding pocket of acylated recoverin decreased in the absence of calcium. This effect is most likely due to the closer association of the acyl group with the protein under these conditions. In contrast, rates of deuterium incorporation increased in the absence of calcium for other regions, including the two functional calcium-binding sites. In addition to supporting the calcium-myristoyl switch hypothesis, a comparison of the behavior of acylated and unacylated recoverin revealed that the N-acyl group (N-lauroyl or N-myristoyl) exerts a significant stabilizing influence on the dynamics of recoverin. We demonstrate that the new technique of monitoring hydrogen isotopic exchange by ESI/MS can be used to obtain useful information concerning protein structures in solution using smaller amounts of protein and under more physiologically relevant conditions than is typically possible with NMR or X-ray crystallography.     

Distribution and disappearance of the radiolabeled carbon derived from L-arginine and taurine in the mouse

L-arginine and taurine are still in the center of physiological and pharmacological research. Although the fate of nitrogen of both compounds and of the ³⁵S-taurine is well-documented, the fate of the carbon skeleton has not been elucidated yet. We studied the organ distribution of ¹⁴C arginine and ¹⁴C taurine over time in the mouse using whole body autoradiography with densitometric image analysis. We describe different organ distribution patterns. Kidney, heart, lung, the Harderian gland, the central nervous system, intestine and testis showed a comparable pattern of arginine disappearance in contrast to rapid disappearance in the salivary gland and the accumulation pattern in bone and spleen. Data on ¹⁴C taurine of liver, kidneys, lung, testis and Harderian gland resembled the arginine pattern; Accumulation of taurine carbon was found in salivary gland, bone, intestine, heart and brain. Our studies challenge and demand further related studies to obtaining more information on the fate of the carbon skeleton of these amino acids.     

High-level 2H/13C/15N labeling of proteins for NMR studies

Summary The protein human carbonic anhydrase II (HCA II) has been isotopically labeled with ²H, ¹³C and ¹⁵N for high-resolution NMR assignment studies and pulse sequence development. To increase the sensitivity of several key ¹H/¹³C/¹⁵N triple-resonance correlation experiments, ²H has been incorporated into HCA II in order to decrease the rates of ¹³C and ¹H_N T₂ relaxation. NMR quantities of protein with essentially complete aliphatic ²H incorporation have been obtained by growth of E. coli in defined media containing D₂O, [1,2-¹³C₂, 99%] sodium acetate, and [¹⁵N, 99%] ammonium chloride. Complete aliphatic deuterium enrichment is optimal for ¹³C and ¹⁵N backbone NMR assignment studies, since the ¹³C and ¹H_N T₂ relaxation times and, therefore, sensitivity are maximized. In addition, complete aliphatic deuteration increases both resolution and sensitivity by eliminating the differential ²H isotopic shift observed for partially deuterated CH_nD_m moieties.