Effects of Riparian Denitrification on Stream Nitrate-Evidence from Isotope Analysis and Extreme Nitrate Leaching during Rainfall-

The effects of riparian denitrification on stream nitrate were investigated by detailed soil water observations and isotope analysis at a small headwater catchment in an urban area near Tokyo, central Japan. In the base flow period, stream nitrate concentration (<100 µM) was comparable with that of riparian ground water which had less nitrate than unsaturated soil water. Nitrogen isotope analysis showed that the consumption of nitrate by denitrification took place in riparian ground water, suggesting that denitrification is an important process to control nitrate leaching to streams. During rainfall, the concentration of stream nitrate increased up to 400 µM, which was comparable with that of pre-event soil water. The fact that soil water nitrate directly leached to streams indicated that the riparian denitrification process did not work during rainfall because of the rapid discharge of water. A decrease of denitrification effects is a possible reason for high stream nitrate concentration during rainfall.     

Split luciferase complementation assay to detect regulated protein-protein interactions in rice protoplasts in a large-scale format

Background

The rice interactome, in which a network of protein-protein interactions has been elucidated in rice, is a useful resource to identify functional modules of rice signal transduction pathways. Protein-protein interactions occur in cells in two ways, constitutive and regulative. While a yeast-based high-throughput method has been widely used to identify the constitutive interactions, a method to detect the regulated interactions is rarely developed for a large-scale analysis.

Results

A split luciferase complementation assay was applied to detect the regulated interactions in rice. A transformation method of rice protoplasts in a 96-well plate was first established for a large-scale analysis. In addition, an antibody that specifically recognizes a carboxyl-terminal fragment of Renilla luciferase was newly developed. A pair of antibodies that recognize amino- and carboxyl- terminal fragments of Renilla luciferase, respectively, was then used to monitor quality and quantity of interacting recombinant-proteins accumulated in the cells. For a proof-of-concept, the method was applied to detect the gibberellin-dependent interaction between GIBBERELLIN INSENSITIVE DWARF1 and SLENDER RICE 1.

Conclusions

A method to detect regulated protein-protein interactions was developed towards establishment of the rice interactome.     

Lime-nitrogen application affects nitrification, denitrification, and N2O emission in an acidic tea soil

Lime-N (calcium cyanamide, CaCN₂) acts as both fertilizer and pesticide. Lime-N may reduce nitrous oxide (N₂O) emission from soil, although its effectiveness and the relative mechanisms are not well understood. The aim of the study was to quantify the effect of lime-N on N₂O emission from the acidic soil of tea fields. The study design consisted of two treatments: conventional fertilizer (CF) (application of conventional organo-chemical fertilizer) and lime-N (LN) (application of approximately 53 % of the applied N as lime-N and the remaining as conventional organo-chemical fertilizer). Both treatments had the same amount of N, P₂O₅, and K₂O applied to soil between plant canopies; fertilizer was incorporated into soil. We measured N₂O emissions and environmental and microbial parameters of soil between plant canopies and under the canopy of tea plants, including the concentrations of dicyandiamide and cyanamide derived from lime-N. Nitrous oxide emission from soil between plant canopies was lower in the LN treatment than in the CF treatment, and soil ammonium oxidation activity and soil denitrification rate decreased after lime-N application. We applied the acetylene inhibition technique and analyzed isotopomer ratios of N₂O; the results of both techniques suggested that denitrification was the major process of N₂O production in the soil between plant canopies, despite relatively low water-filled pore space. Cumulative N₂O emission over the 366 days of the experiment was 36.0 % lower in the LN treatment than in the CF treatment (P?<?0.05). Our results suggest that lime-N application decreases N₂O emission by inhibiting both nitrification and denitrification processes in the acidic soil.     

Establishment of embryo transfer in the musk shrew (Suncus murinus)

The present study established techniques to induce pseudopregnancy, in vitro oocyte cultures from pronuclear to 2- to 4-cell stages, and embryo transfer in musk shrews, a reflex ovulator. Offspring were subsequently obtained by transferring in vivo-developed or in vitro-cultured embryos. Female musk shrews received human chronic gonadotropin (hCG), with or without mating stimuli, from vasectomized males to produce pseudopregnant recipients. Embryos at the 2- to 4-cell stage were collected 44–48 h after mating. Another set of embryos was collected 26–27 h after mating and then cultured for 20 h from the pronuclear to 2- to 4-cell stages. Subsequently, embryos were transferred into the oviducts of pseudopregnant recipients 24 or 48 h after the induction of pseudopregnancy. Offsprings were successfully obtained from recipients that received hCG 24 h before embryo transfer, regardless of mating stimuli. These techniques may be valuable for producing transgenic musk shrews.     

Central estrogen action sites involved in prepubertal restraint of pulsatile luteinizing hormone release in female rats

The present study aimed to determine estrogen feedback action sites to mediate prepubertal restraint of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) release in female rats. Wistar-Imamichi strain rats were ovariectomized (OVX) and received a local estradiol-17β (estradiol) or cholesterol microimplant in several brain areas, such as the medial preoptic area (mPOA), paraventricular nucleus, ventromedial nucleus and arcuate nucleus (ARC), at 20 or 35 days of age. Six days after receiving the estradiol microimplant, animals were bled to detect LH pulses at 26 or 41 days of age, representing the pre- or postpubertal period, respectively. Estradiol microimplants in the mPOA or ARC, but not in other brain regions, suppressed LH pulses in prepubertal OVX rats. Apparent LH pulses were found in the postpubertal period in all animals bearing estradiol or cholesterol implants. It is unlikely that pubertal changes in responsiveness to estrogen are due to a change in estrogen receptor (ER) expression, because the number of ERα-immunoreactive cells and mRNA levels of Esr1, Esr2 and Gpr30 in the mPOA and ARC were comparable between the pre- and postpubertal periods. In addition, kisspeptin or GnRH injection overrode estradiol-dependent prepubertal LH suppression, suggesting that estrogen inhibits the kisspeptin-GnRH cascade during the prepubertal period. Thus, estrogen-responsive neurons located in the mPOA and ARC may play key roles in estrogen-dependent prepubertal restraint of GnRH/LH secretion in female rats.     

Effects of initial handling and training on autonomic nervous function in young Thoroughbreds

OBJECTIVE: To determine the effects of initial handling and training on autonomic nervous functions in young Thoroughbreds. ANIMALS: 63 healthy Thoroughbreds. PROCEDURE: All horses were trained to be handled and initially ridden in September of the yearling year and then trained until the following April by conventional training regimens. To obtain the heart rate (HR), electrocardiograms were recorded in the stable before initial handling and training and following 7 months of training; variations in HR were then evaluated from the power spectrum in terms of the low frequency (LF; 0.01 to 0.07 Hz) power and high frequency (HF; 0.07 to 0.6 Hz) power as indices of autonomic nervous activity. To evaluate the fitness, the V200 (velocity at HR of 200 beat/min), which is reflective of the aerobic capacity of the horse, was measured. RESULTS: Mean (+/- SE) resting HR decreased significantly from 41.5 +/- 0.8 to 38.7 +/- 0.4 beat/min following 7 months of training. The LF power of horses increased significantly from 1,037 +/- 128 milliseconds2 in September of the yearling year to 2,944 +/- 223 milliseconds2 in the following April. Similarly, the HF power increased significantly from 326 +/- 30 milliseconds2 to 576 +/- 39 milliseconds2 at the corresponding time points. The V200 increased significantly following training. CONCLUSIONS AND CLINICAL RELEVANCE: Increases in LF and HF powers indicate that parasympathetic nervous activity increases in horses by 7 months of training. The decrease in resting HR may be dependent on the training-induced increase of parasympathetic nervous activity in Thoroughbreds.