Cloned cattle derived from a novel zona-free embryo reconstruction system

As the demand for cloned embryos and offspring increases, the need arises for the development of nuclear transfer procedures that are improved in both efficiency and ease of operation. Here, we describe a novel zona-free cloning method that doubles the throughput in cloned bovine embryo production over current procedures and generates viable offspring with the same efficiency. Elements of the procedure include zona-free enucleation without a holding pipette, automated fusion of 5-10 oocyte-donor cell pairs and microdrop in vitro culture. Using this system, zona-free embryos were reconstructed from five independent primary cell lines and cultured either singularly (single-IVC) or as aggregates of three (triple-IVC). Blastocysts of transferable quality were obtained at similar rates from zona-free single-IVC, triple-IVC, and control zona-intact embryos (33%, 25%, and 29%, respectively). In a direct comparison, there was no significant difference in development to live calves at term between single-IVC, triple-IVC, and zona-intact embryos derived from the same adult fibroblast line (10%, 13%, and 15%, respectively). This zona-free cloning method could be straightforward for users of conventional cloning procedures to adopt and may prove a simple, fast, and efficient alternative for nuclear cloning of other species as well

State regulation and response inhibition in children with ADHD and children with early- and continuously treated phenylketonuria: An event-related potential comparison

Background: The presentation rate of stimuli plays an important role in explaining the performance inefficiency in children with ADHD. In general, children with ADHD have been found to perform more poorly in conditions of relatively slow event rates as compared with fast and moderate event rates. The state regulation hypothesis states that these children have problems in correcting their energetic state necessary to counteract a performance decrement, which requires extra effort allocation. In this study, we investigated state regulation in children with ADHD and used children with early- and continuously treated phenylketonuria (PKU) as a clinical contrast group. Method: We measured the parietal P3 during a Go/No-Go task that incorporated a condition with a fast and a slow presentation rate. Results: We were able to show that children with ADHD, relative to controls, responded more slowly and more variably in the slow condition only, which was accompanied by a smaller P3, suggesting less effort allocation. In contrast, the children with PKU did not show a state regulation deficit. The PKU group showed prolonged stimulus evaluation processing, as indexed by P3 latency, compared to controls and children with ADHD. In addition, they made more errors of commission than the controls and the ADHD group. Conclusions: Our electrophysiological data support the state regulation hypothesis of ADHD. Only the children with PKU had more problems in inhibiting pre-potent responding than controls, which is in accord with the prefrontal dysfunction hypothesis of PKU

Nitrate transport processes in Fagus-Laccaria-mycorrhizae

The contribution of influx and efflux of NO3- on NO3- net uptake has been studied in excised mycorrhizae of 18-20 week old beech (Fagus sylvatica L.) trees. Net uptake rates of NO3- followed uniphasic Michaelis-Menten kinetics in the concentration range between 10 mu M and 1.0 mM external NO3-, with an apparent K-m of 88 +/- 7 mu M, and a V-max of 110 +/- 7 nmol g(-1) root f.wt. h(-1). The relative xylem loading of N, i.e. the portion of NO3- taken up that was loaded into the xylem vessels as NO3- plus reduced N, was constant over the concentration range tested (4.6-7.7%). NO3- influx proceeded linearly with increasing external NO3- supply. When the assumed regulators of net NO3- uptake, i.e. NH4+ or L-glutamate, were applied together with NO3-, net uptake rates of NO3- decreased. This inhibitory effect was caused by a reduction of NO3- influx rather than an enhanced efflux. The comparison of the present data with a recent study with non-mycorrhizal beech roots (Kreuzwieser et al., 1997; J. Exp. Bot. 48, 1431-1438) revealed that mycorrhization leads to reduced rates of NO3- net uptake. This effect is caused by reduced influx plus enhanced efflux of NO3- as compared with non-mycorrhizal beech roots

Efficiency of nitrate uptake in spinach: impact of external nitrate concentration and relative growth rate on nitrate influx and efflux

Regulation of nitrate influx and efflux in spinach (Spinacia oleracea L., cv. Subito), was studied in short-term label experiments with N-13- and N-15-nitrate. Nitrate fluxes were examined in relation to the N demand for growth, defined as relative growth rate (RGR) times plant N concentration. Plants were grown at different nitrate concentrations (0.8 and 4 mM), with mineral composition of growth and uptake solutions identical. Nitrate influx, efflux and net nitrate uptake rate (NNUR) were independent of the external nitrate concentration, despite differences in internal nitrate concentration. At both N regimes, NNUR was adequate to meet the N demand for growth. RGR-related signals predominantly determined the nitrate fluxes. At high RGR (0.25 g g(-1) day(-1)), nitrate influx was 20 to 40% lower and nitrate efflux was 50 to 70% lower than at lower RGR (0.17 g g(-1) day(-1)); efflux:influx ratio (E:I) declined from 0.5 at low RGR to 0.2 at higher RGR. Thus, the efficiency of NNUR substantially increased with increasing RGR. Differences in nitrate translocation between morning and afternoon coincided with differences in nitrate efflux, which is in accordance with the suggested regulation of nitrate efflux by the root cytoplasmic nitrate concentration

Interactions of NH4+ and L-glutamate with NO3- transport processes of non-mycorrhizal Fagus sylvatica roots

The processes of NO3- uptake and transport and the effects of NH4+ or L-glutamate on these processes were investigated with excised non-mycorrhizal beech (Fagus sylvatica L,) roots, NO3- net uptake followed uniphasic Michaelis-Menten kinetics in a concentration range of 10 mu M to 1 mM with an apparent K-m of 9.2 mu M and a V-max of 366 nmol g(-1) FW h(-1), NH4+, when present in excess to NO3- or 10 mM L-glutamate inhibited the net uptake of NO3-. Apparently, part of NO3- taken up was loaded into the xylem, Relative xylem loading of NO3- ranged from 3.2 +/- 1,6 to 6.4 +/- 5,1% of NO3- net uptake, It was not affected by treatment with NH4+ or L-glutamate. N-15/N-13 double labelling experiments showed that NO3- efflux from roots increased with increasing influx of NO3- and, therefore, declined if influx was reduced by NH4+ or L-glutamate exposure, From these results it is concluded that NO3- net uptake by non-mycorrhizal beech roots is reduced by NH4+ or L-glutamate at the level of influx and not at the level of efflux

Nitrate and ammonium influxes in soybean (Glycine max) roots:Direct comparison of N-13 and N-15 tracing

We compared influxes and internal transport in soybean plants (Glycine max cv. Kingsoy) of labelled N from external solutions where either ammonium or nitrate was labelled with the stable isotope N-15 and the radioactive isotope N-13. The objective was to see whether mass spectrometric determinations of tissue N-15 content were sufficiently sensitive to measure influxes accurately over short time periods. Our findings were as follows. (1) There was a close quantitative correspondence between estimates of N influx of individual plants using N-15 or N-13 measurements with either NO3- or NH4+ at 4 or 2 mol m(-3), respectively in the external solution. (2) Transport to the shoot of N from NO3 absorbed over a 5-15 min period could be monitored when the external NO3- concentration ranged from 0.05 to 4 mol m(-3). NH4+ as the N source labelled shoot tissue more slowly, and estimates of the transport between root and shoot could be made only with N-13. (3) Influx of NO3- into root tissue could be measured by N-15 enrichment after 5-10 min at concentrations approaching the probable KM of the high-affinity transport system. (4) There was some indication of isotope discrimination, especially with respect to the movement of labelled N to the shoot, when NO3- is the N source. For many purposes, N-15 tracing can be used satisfactorily to estimate influxes of both NO3- and NH4+ in soybean roots. Use of the short-lived radionuclide N-13 remains the method of choice for more refined measurements of internal distribution and assimilation