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Artificial Intelligence (AI) is being increasingly implemented within road transport systems worldwide. Next generation of AI, Artificial General Intelligence (AGI) is imminent, and is anticipated to be more powerful than current AI. AGI systems will have a broad range of abilities and be able to perform multiple cognitive tasks akin to humans that will likely produce many expected benefits, but also potential risks. This study applied the EAST Broken Links approach to forecast the functioning of an AGI system tasked with managing a road transport system and identify potential risks. In total, 363 risks were identified that could have adverse impacts on the stated goals of safety, efficiency, environmental sustainability, and economic performance of the road system. Further, risks beyond the stated goals were identified; removal from human control, mismanaging public relations, and self-preservation. A diverse set of systemic controls will be required when designing, implementing, and operating future advanced technologies. Practitioner summary: This study demonstrated the utility of HFE methods for formally considering risks associated with the design, implementation, and operation of future technologies. This study has implications for AGI research, design, and development to ensure safe and ethical AGI implementation.

A significant number of lambs born each yr in Australia die within 72 h of birth. Periods of high wind, combined with rain and low temperatures, can lead to marked increases in the level of mortality. Under these weather conditions mortality levels may be reduced with the provision of shelter, provided it is utilized by lambs. This study used GPS collars to determine the use of shelter by ewes and lambs, to compare the movement of ewes with twin lambs across 2 types of shelter (hedgerows and shrubs), while also comparing ewes with single and twin lambs in a single shelter type (hedgerows). Additionally, the birth sites of 364 lambs and death sites of 252 lambs were recorded across the 3 shelter type and litter size combinations (Twins in shrubs, Twins in hedgerows, Singles in hedgerows) plus an unsheltered group (Singles in unsheltered). A higher (P < 0.001) than randomly expected percentage of ewes lambed in the areas closest to both shelter types; in the shrub shelter 42% of ewes lambed within 2.5 m of shrub rows compared to an expected 11% based on the proportion of the paddock this area constituted. Despite the higher than expected percentage of ewes lambing close to the shelter rows, ewes in both twin lamb shelter types avoided the areas close to the shelter before and after lambing (Hedgerows-2.5 m; Shrubs- 6.25 m) and single bearing ewes showed no preference for or against these areas. With a high proportion of twin bearing ewes lambing close to the shelter, a design that reduces the potential for ewe and offspring separation while providing good shelter will offer the greatest potential reduction in newborn twin lamb mortality arising from exposure.

Background: Intraprostatic injection of ethanol has been previously tested in clinical trials as a potential treatment of BPH, with variable outcomes. As evident from animal studies, the inconsistency was owing to various degrees of ethanol backflow along the needle tract. In acute canine experiments, we previously documented that using convection enhanced delivery (CED) eliminates backflow and improves ethanol distribution. The goal of this study was to compare the diffusion pattern between a microporous hollow fiber catheter (MiHFC) and a standard needle in human prostates from organ donors., Methods: Prostates were harvested from cadaveric organ donors immediately after removal of organs for transplant. After trimming off excess fat and weighing, prostates were injected with absolute ethanol. The total injected volume was 25% of the calculated prostate volume. One lateral lobe was injected using a single lumen 21-gauge control needle. The contralateral lobe was injected with the same volume but using a MiHFC. Immediately after injection, prostates were fixed en bloc in 10% neutral-buffered formalin, and then sectioned. Three-dimensional reconstruction was performed to determine lesion volume based on hematoxylin- and eosin-stained cross-sections., Results: Three fresh human prostates were harvested and injected. The time from harvest to intraprostatic injection was 15-35 min. The lesion created by the MiHFC was 1.14±0.52 cm(3), whereas that from the control needle was 0.28±0.10 cm(3) (P=0.038). No backflow was observed along the needle tract of the MiHFC., Conclusions: This study shows that freshly harvested human prostates can be used to evaluate new treatments using intraprostatic injection. Similar to in vivo canine experiments, the ethanol lesion sizes were significantly bigger with the use of a MiHFC when compared with a standard single lumen needle.

Cryptosporidium are ubiquitous and significant enteropathogens of all classes of vertebrates and a major cause of human morbidity and mortality worldwide. Of the 24 recognized species, the zoonotic Cryptosporidium parvum and the host-specific Cryptosporidium hominis cause the majority of cases of human cryptosporidiosis. Here, we report on structural and transcriptional variability between C. parvum and C. hominis at the MIC1 locus, which encodes a microneme localized thrombospondin-like domain containing protein previously demonstrated to be critical for host cell infection by C. parvum. We demonstrate, using reverse transcription quantitative PCR with the aid of genomic data from the EuPathDB site, that the transcribed product in C. hominis is both truncated and significantly down-regulated in the sporozoite. We hypothesize that CpMIC1 may be a genetic factor involved in facilitating the wider host range of C. parvum in comparison with the specific host range of C. hominis. Furthermore, we show that the presence of a microsatellite (ML-2) within the C. parvum MIC-1 locus enables the development of a PCR marker that can rapidly distinguish the zoonotic C. parvum from C. hominis and other significant human infectious Cryptosporidium species due to reproducible PCR slippage across the ML-2 microsatellite. Additionally, we demonstrate that this locus is tightly linked to the GP60 locus, a locus commonly used in the genetic characterization of C. parvum and C. hominis isolates. This marker should provide a robust and additional tool to aid in the rapid identification of C. parvum from other Cryptosporidium species., (© 2013 Blackwell Verlag GmbH.)

Bile salts have been identified as an important trigger for excystation of Cryptosporidium oocysts but the hierarchy or synergism of this signal in relation to other triggers involved in excystation is poorly understood. In addition to excystation, bile salts have also been reported to increase the invasiveness of sporozoites within in vitro culture, possibly by affecting the secretory pathway via modification of intracellular calcium signalling. Nevertheless, incorporation of bile or bile salts into in vitro assays is not universal, with recent reports of negative effects on parasite growth. Here we report that bile and sodium taurocholate significantly affect both excystation rate and parasite in vitro growth. We demonstrate that their effect on excystation is dose, time and pre-treatment temperature dependent, while increases in parasite replication appear to be associated with modulation of parasite intracellular calcium and increased host cell susceptibility to infection. Notably, we illustrate that bile has a significant effect on host cells and can be cytotoxic at concentrations not much higher than those currently used for in vitro assays. This work should assist with more rational design of in vitro culture systems, with significant considerations for assay format when incorporating bile or bile salts as an excystation trigger.

Electronic physician documentation is an essential element of a complete electronic medical record (EMR). At Lucile Packard Children's Hospital, a teaching hospital affiliated with Stanford University, we implemented an inpatient electronic documentation system for physicians over a 12-month period. Using an EMR-based free-text editor coupled with automated import of system data elements, we were able to achieve voluntary, widespread adoption of the electronic documentation process. When given the choice between electronic versus dictated report creation, the vast majority of users preferred the electronic method. In addition to increasing the legibility and accessibility of clinical notes, we also decreased the volume of dictated notes and scanning of handwritten notes, which provides the opportunity for cost savings to the institution.

The positive effects of fat and energy supplementation on improvements in reproduction are well documented. However, the specific effects of omega-3 polyunsaturated fatty acids (n-3) on reproductive success in ruminants have not been examined in detail. While the link between n-3 and markers associated with reproduction, in particular, prostaglandin F(2α) (PGF(2α)) and the link between PGF(2α) and reproductive outcomes are well established, evidence of a direct effect of high n-3 diets on measurable reproductive outcomes in ruminants is lacking. Therefore, the aim of the current review was to examine the effect of n-3 on a number of reproductive markers and measurable outcomes in sheep and cattle. There is strong evidence linking consumption of diets high in n-3 with reduced circulating peripheral inflammatory markers such as PGF(2α). Inflammatory eicosanoids including PGF(2α), in particular, can significantly affect reproduction outcomes such as the onset of oestrus, embryo survival and parturition. While there is also evidence linking n-3 supplementation with longer time to oestrus and parturition associated with reduced PGF(2α), the effects of n-3 on other measurable outcomes of reproductive success, such as pregnancy rate, embryo survival and intergenerational effects on the health and production of offspring are largely unknown. Similarly, the effects of diets high in n-3 or n-6 polyunsaturated fatty acids on male fertility are also unknown., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Members of the genus Cryptosporidium, which cause the gastrointestinal disease cryptosporidiosis, still represent a significant cause of water-borne disease worldwide. While intensive efforts have been invested in the development of techniques for parasite culture, in vitro growth has been hampered by a number of factors including low levels of infectivity as well as delayed life-cycle development and poor synchronicity. In this study we examined factors affecting the timing of contact between excysted sporozoites and target host cells and the subsequent impact of this upon the establishment of infection. We demonstrate that excystation rate impacts upon establishment of infection and that in our standard assay format the majority of sporozoites are not close enough to the cell monolayer when they are released from the oocyst to successfully establish infection. However, this can be easily overcome by centrifugation of oocysts onto the cell monolayer, resulting in approximately 4-fold increases in sporozoite attachment and subsequent infection. We further demonstrate that excystation procedures can be tailored to control excystation rate to match the assay end purpose and that excystation rate can influence data interpretation. Finally, the addition of both a centrifugation and washing step post-sporozoite attachment may be appropriate when considering the design of in vitro culture experiments for developmental analysis and stage-specific gene expression as this appears to increase the synchronicity of early developmental stages.

This study evaluated the ability of short-term grazing of live=green pasture to increase ovulation rate during late summer when annual pasture is generally dead and of low quality. Ovulation rates, measured by the number of corpora lutea, were compared between 4 nutritional treatments: senesced phalaris (Phalaris aquatica), phalaris plus 500g lupin grain per day, lucerne (Medicago sativa) or chicory (Chicorum intybus) pastures. The study used 100 Merino ewes per treatment, divided between 2 replicates. The experiment was repeated in 3 years; February 2006, and January 2007 and 2008. Oestrus was synchronised and the ewes grazed the pastures for 9 days prior to ovulation at times corresponding to days 8-17 of the cycle in 2006, and days 6-14 in 2007 and 2008. The proportion of ewes producing multiple ovulations was higher (P<0.05) in the lucerne and chicory (0.36, 0.38) than the phalaris (0.27), and intermediate in the lupin (0.33) treatment. Regression analysis showed that the proportion of ewes with multiple ovulations increased with the quantity of live herbage (P<0.04). Responses were achieved even at low levels of live herbage with 90% of the maximum proportion of multiples occurring at 350kg DM/ha. It is concluded that providing short-term grazing of live chicory or lucerne to ewes can increase ovulation rates relative to ewes grazing senesced phalaris and to levels similar to those achieved by lupin grain supplementation., (2010. Published by Elsevier B.V. All rights reserved.)

Cryptosporidium parvum are protozoan parasites responsible for outbreaks of gastrointestinal disease worldwide. Within the apical complex of this organism reside numerous vesicular secretory organelles and their discharge has been identified as essential for sporozoite motility, cell attachment and penetration. Traditionally, investigation of apical organelle discharge has relied on microscopic and immunochemical hybridization techniques. In this study we demonstrate for the first time how flow cytometry, in combination with vital dye staining, provides an avenue for discrimination of distinct physiological events occurring within Cryptosporidium sporozoites post-excystation. Time-course studies of freshly excysted sporozoites were carried out at 37 degrees C in cell-free medium, stained with the fluorescent dyes SYTO9/PI, DiBAC4(3), Fluo-4 AM or FM1-43 and analysed by flow cytometry. Significant decreases in sporozoite plasma membrane permeability and increased membrane depolarization were found to be accompanied by concomitant increases in intracellular calcium. Subsequent to these changes, large increases in exocytosed vesicular membrane were apparent. In addition, by measuring side and forward angle light scatter we were able to assess changes in internal granularity and size of sporozoites post-excystation. These observations were suggestive of rapid mobilization, utilization and discharge of apical organelles within sporozoites, which we relate to changes in sporozoite infectivity, ATP levels and total secreted soluble protein.

Aims: To determine the effect of solar radiation on Cryptosporidium parvum in tap and environmental waters., Methods and Results: Outdoor tank experiments and a cell culture infectivity assay were used to measure solar inactivation of C. parvum oocysts in different waters. Experiments conducted on days with different levels of solar insolation identified rapid inactivation of oocysts in tap water (up to 90% inactivation within the first hour). Increased dissolved organic carbon content in environmental waters decreased solar inactivation. The role of solar ultraviolet (UV) in inactivation was confirmed by long-pass filter experiments, where UV-B was identified as the most germicidal wavelength. Reductions in oocyst infectivity following solar radiation were not related to a loss of excystation capacity., Conclusions: Solar UV can rapidly inactivate C. parvum in environmental waters., Significance and Impact of the Study: This is the first study to assess natural sunlight inactivation of C. parvum oocysts in surface waters and drinking water using an infectivity measure and determines the wavelengths of light responsible for the inactivation. The findings presented here provide valuable information for determining the relative risks associated with Cryptosporidium oocysts in aquatic environments and identify solar radiation as a critical process affecting the oocyst survival in the environment.

Cryptosporidium are parasitic protozoans that cause gastrointestinal disease and represent a significant risk to public health. Cryptosporidium oocysts are prevalent in surface waters as a result of human, livestock and native animal faecal contamination. The resistance of oocysts to the concentrations of chlorine and monochloramine used to disinfect potable water increases the risk of waterborne transmission via drinking water. In addition to being resistant to commonly used disinfectants, it is thought that oocysts can persist in the environment and be readily mobilized by precipitation events. This paper will review the critical processes involved in the inactivation or removal of oocysts in the terrestrial and aquatic environments and consider how these processes will respond in the context of climate change.

We report on the extraction of R = sigma(L)/sigma(T) from CCFR nu(mu)-Fe and nu(mu)-Fe differential cross sections. The CCFR differential cross sections do not show the deviations from the QCD expectations that are seen in the CDHSW data at very low and very high x. R as measured in nu(mu) scattering is in agreement with R as measured in muon and electron scattering. All data on R for Q(2)>1 GeV(2) are in agreement with a NNLO QCD calculation which uses NNLO parton distribution functions and includes target mass effects. We report on the first measurements of R in the low x and Q(2)<1 GeV(2) region (where an anomalous large rise in R for nuclear targets has been observed by the HERMES Collaboration).

A new structure function analysis of CCFR deep inelastic nu-N and nu-N scattering data is presented for previously unexplored kinematic regions down to Bjorken x = 0.0045 and Q(2) = 0.3 GeV(2). Comparisons to charged lepton scattering data from NMC and E665 experiments are made and the behavior of the structure function F(2)(nu)2 is studied in the limit Q(2)-->0.

We report on the extraction of the structure functions F2 and DeltaxF(3) = xF(nu)(3)-xF(nu;)(3) from CCFR nu(mu)-Fe and nu;(mu)-Fe differential cross sections. The extraction is performed in a physics model-independent (PMI) way. This first measurement of DeltaxF(3), which is useful in testing models of heavy charm production, is higher than current theoretical predictions. The ratio of the F2 (PMI) values measured in nu(mu) and mu scattering is in agreement (within 5%) with the predictions of next-to-leading-order parton distribution functions using massive charm production schemes, thus resolving the long-standing discrepancy between the two sets of data.

Hypnotic responsiveness may depend upon the ability to inhibit the irrelevant stimuli that evoke responses that are incompatible with current goals (or the mapping between an irrelevant/disruptive stimulus and its response) in order to actively maintain task-relevant information. In a simple correlation design, the authors investigated the relationship between cognitive inhibition (negative priming) and hypnotic responsiveness. A statistically significant correlation was obtained between the extent of negative priming (measured in time latency) and hypnotic responsiveness (r = .491). Limitations of this preliminary study and implications for future work are discussed.

Background: Limitations of the surgical correction for myopia include inaccuracy, instability, treatment of the central optical zone, and lack of reversibility. KeraVision Intacs offer an alternative that addresses these shortcomings., Methods: We present 1 year of follow-up information on 95 subjects enrolled in the United States Food and Drug Administration Phase III clinical trials., Results: At 1 year, 99% of patients (89 of 90) had 20/40 uncorrected vision or better. Ninety-two percent of eyes (83 of 90) were within 1.00 D of intended correction and 76% of eyes (68 of 90) were within 0.50 D of intended correction. Stability was achieved at 3 months, with 96% of subjects (86 of 90) having less than 1.00 D of change from their previous examination. In a substudy, 89% eyes (58 of 65) varied within +/- 0.50 D over the course of a day. Corneal curvature changed as predicted, resulting in a prolate aspheric shape within the central optical zone. Most complications or adverse events experienced were managed with additional medication or surgical intervention, resulting in a favorable outcome for subjects., Conclusions: KeraVision Intacs are effective, predictable, stable, and safe. This additive technique may also offer reversibility.

Two studies were completed to test whether responding to hypnotic suggestions requires intentional effort. Hypnotic suggestions for amnesia were used as an analog of thought suppression, and Wegner's model of ironic processing was applied to hypnotic responding. In the first study, participants were required to maintain suggested amnesia while performing a cancellation task with and without a cognitive load. The second study required suppression of thoughts of a favorite car, once with "blank-mind" instructions and then with a suggestion for amnesia. The results of these studies indicate that dissociated control theory provides the best explanation for hypnotic responding in one subset of highly hypnotizable participants, whereas more intentional responding provides the best explanation for others.

Electrophoretic conditions including electrode and gel buffers, acrylamide concentration, use of stacking gels, voltage, current, and run time were investigated in order to produce isozyme bands of high resolution which would facilitate densitometric quantification of enzyme activity following polyacrylamide gel electrophoresis (PAGE). Electrode buffers which provided optimal conditions for gels stained for the isozymes of malate dehydrogenase (MDH), 6-phosphogluconate dehydrogenase (6-PGD), phosphoglucose isomerase (PGI), and shikimate dehydrogenase (SkDH) were 0.02 M Tris-glycine, pH 8.5, 0.1 M sodium borate, pH 6.0, 0.1 M sodium borate, pH 8.7, and 0.07 M sodium borate, pH 7.0, respectively. A 0.5 M Tris-HCl, pH 7.5, gel buffer was optimal for gels stained for the isozymes of 6-PGD, PGI and SkDH. A 0.5 M Tris-HCl, pH 8.5, gel buffer was best for gels stained for MDH. Stacking gels were found to be detrimental to enzyme activity and showed no improvement in resolution for any of the enzymes. Acrylamide concentration for gels stained for MDH were 8.7%, gels stained for 6-PGD and PGI were 7.5%, while gels stained for SkDH had an acrylamide concentration of 5.0%. Higher concentrations above these levels caused a reduction and in some cases loss of band activity, while below this concentration there was a decrease in band resolution.(ABSTRACT TRUNCATED AT 250 WORDS)

Extracting proteins from vegetative tissues while maintaining good enzyme activity and electrophoretic resolution presents numerous problems due to the presence of phenols, quinones, proteases and other components released during cell disruption. To overcome this problem in Citrus leaves, an extraction buffer was developed which contained EDTA, potassium chloride, magnesium chloride hexahydrate, PVP-40, 2-mercaptoethanol and bovine serum albumin in a Tris-HCl buffer, pH 7.5. This extraction buffer was used in association with liquid nitrogen for sample preparation. Buffers used in previous studies for Citrus isozyme extraction for PAGE were found to provide unsatisfactory resolution and activity for the three enzyme systems investigated (malate dehydrogenase, 6-phosphogluconate dehydrogenase and shikimate dehydrogenase). This extraction buffer maintains high enzyme activity and provides good resolution in PAGE gels suitable for densitometric analysis.

The short-lived radiotracer 13N was used to study feedback regulation of nitrate influx through the inducible high-affinity transport system of barley (Hordeum vulgare L. cv Steptoe) roots. Both wild-type plants and the mutant line Az12:Az70 (genotype nar1a;nar7w), which is deficient in the NADH-specific and NAD(P)H-bispecific nitrate reductases (R.L. Warner, R.C. Huffaker [1989] Plant Physiol 91: 947-953) showed strong feedback inhibition of nitrate influx within approximately 5 d of exposure to 100 fmu]M nitrate. The result with the mutant, in which the flux of nitrogen into reduced products is greatly reduced, indicated that nitrate itself was capable of exercising feedback regulation upon its own influx. This conclusion was supported by the observation that feedback in wild-type plants occurred in both the presence and absence of L-methionine sulfoximine, an inhibitor of ammonium assimilation. Nitrite and ammonium were also found to be capable of exerting feedback inhibition upon nitrate influx, although it was not determined whether these ions themselves or subsequent metabolites were responsible for the effect. It is suggested that feed-back regulation of nitrate influx is potentially mediated through several nitrogen pools, including that of nitrate itself.

Effects of NO(2) (-), ClO(3) (-), and ClO(2) (-) on the induction of nitrate transport and nitrate reductase activity (NRA) as well as their effects on NO(3) (-) influx into roots of intact barley (Hordeum vulgare cv Klondike) seedlings were investigated. A 24-h pretreatment with 0.1 mol m(-3) NO(2) (-) fully induced NO(3) (-) transport but failed to induce NRA. Similar pretreatments with ClO(3) (-) and ClO(2) (-) induced neither NO(3) (-) transport nor NRA. Net ClO(3) (-) uptake was induced by NO(3) (-) but not by ClO(3) (-) itself, indicating that NO(3) (-) and ClO(3) (-) transport occur via the NO(3) (-) carrier. At the uptake step, NO(2) (-) and ClO(2) (-) strongly inhibited NO(3) (-) influx; the former exhibited classical competitive kinetics, whereas the latter exhibited complex mixed-type kinetics. ClO(3) (-) proved to be a weak inhibitor of NO(3) (-) influx (K(i) = 16 mol m(-3)) in a noncompetitive manner. The implications of these findings are discussed in the context of the suitability of these NO(3) (-) analogs as screening agents for the isolation of mutants defective in NO(3) (-) transport.

The cytoplasmic NO(3) (-) concentration ([NO(3) (-)](c)) was estimated for roots of barley (Hordeum vulgare L. cv Klondike) using a technique based on measurement of in vivo nitrate reductase activity. At zero external NO(3) (-) concentration ([NO(3) (-)](o)), [NO(3) (-)](c) was estimated to be 0.66 mm for plants previously grown in 100 mum NO(3) (-). It increased linearly with [NO(3) (-)](o) between 2 and 20 mm, up to 3.9 mm at 20 mm [NO(3) (-)](o). The values obtained are much lower than previous estimates from compartmental analysis of barley roots. These observations support the suggestion (MY Siddiqi, ADM Glass, TJ Ruth [1991] J Exp Bot 42: 1455-1463) that the nitrate reductase-based technique and compartmental analysis determine [NO(3) (-)](c) for two separate pools; an active, nitrate reductase-containing pool (possibly located in the epidermal cells) and a larger, slowly metabolized storage pool (possibly in the cortical cells), respectively. Given the values obtained for [NO(3) (-)](c) and cell membrane potentials of -200 to -300 mV (ADM Glass, JE Schaff, LV Kochian [1992] Plant Physiol 99: 456-463), it is very unlikely that passive influx of NO(3) (-) is possible via the high-concentration, low-affinity transport system for NO(3) (-). This conclusion is consistent with the suggestion by Glass et al. that this system is thermodynamically active and capable of transporting NO(3) (-) against its electrochemical potential gradient.

When intact nodulated roots of soybean (Glycine max L. Merr. nodulated with Bradyrhizobium japonicum strain USDA 16) were exposed to an atmosphere lacking N(2) gas (Ar:O(2) 80:20), total nitrogenase activity (measured as H(2) evolution) and respiration (CO(2) evolution) declined with time of exposure. In Ar-inhibited nodules, when the O(2) concentration in the rhizosphere was increased in a linear ;ramp' of 2.7% per minute, 93% of the original H(2) evolution and 99% of the CO(2) evolution could be recovered. The internal nodule O(2) concentration (estimated from leghemoglobin oxygenation) declined to 56% of its initial value after 60 minutes of Ar:O(2) exposure and could be partially recovered by the linear increases in O(2) concentration. Nodule gas permeability, as estimated from the lag in ethylene production following exposure of nodules to acetylene, decreased to 26% of its initial value during the Ar-induced decline. Collectively, the results provide direct evidence that the Ar-induced decline results from decreased nodule gas permeability and indicate that the decline in permeability, rather than being immediate, occurs gradually over the period of Ar:O(2) exposure.

An open gas exchange system was used to monitor the nonsteady state and steady state changes in nitrogenase activity (H(2) evolution in N(2):O(2) and Ar:O(2)) and respiration (CO(2) evolution) in attached, excised, and sliced nodules of soybean (Glycine max L. Merr.) exposed to external pO(2) of 5 to 100%. In attached nodules, increases in external pO(2) in steps of 10 or 20% resulted in sharp declines in the rates of H(2) and CO(2) evolution. Recovery of these rates to values equal to or greater than their initial rates occurred within 10 to 60 minutes of exposure to the higher pO(2). Recovery was more rapid at higher initial pO(2) and in Ar:O(2) compared to N(2):O(2). Sequential 10% increments in pO(2) to 100% O(2) resulted in rates of H(2) evolution which were 1.4 to 1.7 times the steady state rate at 20% O(2) in Ar. This was attributed to a relief at high pO(2) from the 40% decline in nitrogenase activity that was induced by Ar at a pO(2) of 20%. Changes in nodule respiration rate could not account for the nodules' ability to adjust to high external pO(2), supporting the hypothesis that soybean nodules have a variable barrier to O(2) diffusion which responds slowly (within minutes) to changes in pO(2). Nodule excision and slicing resulted in 45 and 78% declines, respectively, in total specific nitrogenase activity at 20% O(2). In contrast with the result obtained with intact nodules, subsequent 10% increases in pO(2) in Ar:O(2) did not result in transient declines in H(2) evolution rates, but in the rapid attainment of new steady state rates. Also, distinct optima in nitrogenase activity were observed at about 60% O(2). These results were consistent with an increase in the diffusive resistance of the nodule cortex following nodule excision or nodule slicing. This work also shows the importance of using intact plants and continuous measurements of gas exchange in studies of O(2) diffusion and nitrogenase activity in legume nodules.

The objectives of this study were to determine whether attached nodules of soybean (Glycine max L. Merr.) could adjust to gradual increases in rhizosphere pO(2) without nitrogenase inhibition and to determine whether the nitrogenase activity of the nodules is limited by pO(2) under ambient conditions. A computer-controlled gas blending apparatus was used to produce linear increases (ramps) in pO(2) around attached nodulated roots of soybean plants in an open gas exchange system. Nitrogenase activity (H(2) production in N(2):O(2) and Ar:O(2)) and respiration (CO(2) evolution) were monitored continuously as pO(2) was ramped from 20 to 30 kilopascals over periods of 0, 5, 10, 15, and 30 minutes. The 0, 5, and 10 minute ramps caused inhibitions of nitrogenase and respiration rates followed by recoveries of these rates to their initial values within 30 minutes. Distinct oscillations in nitrogenase activity and respiration were observed during the recovery period, and the possible basis for these oscillations is discussed. The 15 and 30 minute ramps did not inhibit nitrogenase activity, suggesting that such inhibition is not a factor in the regulation of nodule diffusion resistance. During the 30 minute ramp, a stimulation of nitrogenase activity was observed, indicating that an O(2)-based limitation to nitrogenase activity occurs in soybean nodules under ambient conditions.

A fiber optic spectrophotometric system was used to monitor the in vivo oxygenation of leghemoglobin in intact, attached soybean root nodules (Glycine max L. Merr. x USDA 16 Bradyrhizobium japonicum) which were flattened during development by growth in narrow, glass-walled cuvettes. When equilibrated at an external pO(2) of 20 kilopascals, leghemoglobin was 36.6 +/- 5.4% oxygenated, a value estimated to represent an infected cell O(2) concentration of 21.5 nanomolar. Increasing the external pO(2) from 20 to 25 kilopascals caused a rapid increase in leghemoglobin oxygenation, followed by a recovery to the initial level, all within 7.5 minutes. At 25 kilopascals O(2), the rates of H(2) and CO(2) evolution were similar to those at 20 kilopascals. Since respiration had not increased, the results support the proposal that nodules adapt to increased external pO(2) by regulating their resistance to O(2) diffusion.

The magnitude and role of dark CO(2) fixation were examined in nodules of intact soybean plants (Harosoy 63 x Rhizobium japonicum strain USDA 16). The estimated rate of nodule dark CO(2) fixation, based on a 2 minute pulse-feed with (14)CO(2) under saturating conditions, was 102 micromoles per gram dry weight per hour. This was equivalent to 14% of net nodule respiration. Only 18% of this CO(2) fixation was estimated to be required for organic and amino acid synthesis for growth and export processes. The major portion (75-92%) of fixed label was released as CO(2) within 60 minutes. The labeling pattern during pulse-chase experiments was consistent with CO(2) fixation by phosphoenolpyruvate carboxylase. During the chase, the greatest loss of label occurred in organic acids. Exposure of nodulated roots to Ar:O(2) (80:20) did not affect dark CO(2) fixation, while exposure to O(2):CO(2) (95:5) resulted in 54% inhibition. From these results, it was concluded that at least 66% of dark CO(2) fixation in soybean may be involved with the production of organic acids, which when oxidized would be capable of providing at least 48% of the requirement for ATP equivalents to support nitrogenase activity.