Amperometric method for determining nitrous oxide in denitrification and in nitrogenase-catalyzed nitrous oxide reduction

A conventional Clark-type O₂ probe was used to determine N₂O concentrations in suspensions. At a polarizing voltage of–0.95 V versus the reference Ag/AgCl electrode, the probe is almost half as sensitive for N₂O as for O₂, and the detection limit is less than 1 M N₂O. The probe can also be used to determine NO for which the suitable polarizing voltage is–0.7 V. The method was successfully applied for continuously recording dissimilatory formation or utilization of N₂O by intactAzospirillum brasilense Sp 7, NO production by extracts from this bacterium, and N₂O reduction catalyzed by nitrogenase in intactKlebsiella pneumoniae. It is concluded that the probe is useful for measuring N₂O or NO contents in bacterial suspensions when the O₂ level is zero or kept constant during the assays.     

Electron transport pathways to nitrous oxide in Rhodobacter species

1. Electron transport components involved in nitrous oxide reduction in several strains of Rhodobacter capsulatus and in the denitrifying strain of Rhodobacter sphaeroides (f. sp. denitrificans) have been investigated. Detailed titrations with antimycin A and myxothiazol, inhibitors of the cytochrome bc1 complex, show that part of the electron flow to nitrous oxide passes through this complex. The sensitivity to myxothiazol varies between strains and growth conditions of R. capsulatus; the higher rates of nitrous oxide reduction correlate with the higher sensitivities. Partial inhibition of the nitrous oxide reductase enzyme with azide decreased the sensitivity to myxothiazol of the strains that had the highest nitrous oxide reductase activity. 2. Inhibition of nitrous oxide reduction in cells of R. capsulatus by myxothiazol could be restored under dark conditions by addition of N,N,N',N'-tetramethyl-p-phenylene diamine. The highest activities observed after addition of this electron carrier were found in the strains that had the highest sensitivity to myxothiazol, consistent with the premise that this inhibitor is more effective at the higher flux rates to nitrous oxide. 3. Addition of nitrous oxide to cells of R. capsulatus strain N22DNAR+ under darkness caused oxidation of both b- and c-type cytochromes. The oxidation of b cytochromes was less pronounced in the presence of myxothiazol, consistent with a role for the cytochrome bc1 complex in the electron pathway to nitrous oxide. Ferricyanide, in the absence of myxothiazol, caused a similar extent of oxidation of b cytochromes, but a greater oxidation of c-type, suggesting that there was a pool of c-type cytochrome that was not oxidisable by nitrous oxide. The time course showed that both the b- and c-type cytochromes were oxidised within a few seconds of the addition of nitrous oxide. During the following seconds there was a partial re-reduction of the cytochromes such that after approximately 1 min a lower steady-state of oxidation was attained and this persisted until the nitrous oxide was exhausted. 4. A mutant, MTCBC1, of R. capsulatus that specifically lacked a functional cytochrome bc1 complex reduced nitrous oxide, albeit at 30% of the rate shown by the parent strain MT1131. A reduced minus nitrous-oxide-oxidised difference spectrum for MTCBC1 in the absence of myxothiazol was similar to the corresponding difference spectrum observed for strain N22DNAR+ in the presence of myxothiazol. It is suggested that these difference spectra identify the cytochrome components, including a b-type, involved in a pathway that is alternative to, and independent of, the cytochrome bc1 complex.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hormonal responses to a graded mental workload

Three mental arithmetic tests of graded difficulty were presented to eight young male volunteers on consecutive days. The effort compensation patterns were investigated using urinary catecholamines and salivary cortisol. A significant increase in adrenaline excretion was found in response to all three tests. Urinary adrenaline was partially graded according to the level of difficulty of the tests. The response patterns to mental workload also included a significantly lower noradrenaline/adrenaline ratio and a higher adrenaline/dopamine ratio after the tests. No significant increase was found after the tests for noradrenaline and dopamine. Changes in salivary cortisol concentration during the tests were graded with respect to test difficulty between the easiest and both of the more difficult tests. Salivary cortisol concentration changes are proposed as the basis for field observations involving mental workload. It is concluded that mental workload causes distinguishable effort compensation patterns and that under certain conditions urinary adrenaline and salivary cortisol allow one to distinguish different mental workload levels.     

Exposure of midwives to nitrous oxide in four hospitals.

The exposure of midwives to nitrous oxide in four hospitals was measured with personal samplers. In three of the four hospitals the average exposure was not significantly less than 100 parts per million (ppm). In one hospital the average exposure was 360 ppm; this was reduced by a factor of about 2.5 when a trial scavenging system was used. Differences in working practices and in the layout, size, and ventilation of the labour suites contributed to the observed differences in average exposure. Midwives and other staff working in the labour room are potentially at risk from excessive occupational exposure to nitrous oxide.     

Reproduction and fetal development in rats exposed to nitrous oxide

The effects of 24 hours of nitrous oxide exposure on reproductive indices and fetal development were examined in Sprague-Dawley rats. Four different experiments employing four concentrations of nitrous oxide--0.75%, 7.5%, 25% and 75%--established that the threshold of toxicity was greater than 25%. At 75% nitrous oxide there was a significant increase in early and late resorptions, and a consistent teratogenic effect (e.g., runts, ocular malformations, limb deformities). Neither the stress of shipping dams while pregnant nor the withholding of food during nitrous oxide exposure resulted in additional adverse effects. Exposure to 25% nitrous oxide was associated with increased deoxyuridine suppression values; however, adverse reproductive effects were not seen at this nitrous oxide concentration. The results of this and other studies which have examined the reproductive and teratogenic effects of nitrous oxide do not contraindicate its use in operating rooms nor, when necessary, as an anesthetic for pregnant surgical patients.     

Gross nitrous oxide production drives net nitrous oxide fluxes across a salt marsh landscape

Sea level rise will change inundation regimes in salt marshes, altering redox dynamics that control nitrification – a potential source of the potent greenhouse gas, nitrous oxide (N₂O) – and denitrification, a major nitrogen (N) loss pathway in coastal ecosystems and both a source and sink of N₂O. Measurements of net N₂O fluxes alone yield little insight into the different effects of redox conditions on N₂O production and consumption. We used in situ measurements of gross N₂O fluxes across a salt marsh elevation gradient to determine how soil N₂O emissions in coastal ecosystems may respond to future sea level rise. Soil redox declined as marsh elevation decreased, with lower soil nitrate and higher ferrous iron in the low marsh compared to the mid and high marshes (P < 0.001 for both). In addition, soil oxygen concentrations were lower in the low and mid‐marshes relative to the high marsh (P < 0.001). Net N₂O fluxes differed significantly among marsh zones (P = 0.009), averaging 9.8 ± 5.4 μg N m^?2 h^?1, ?2.2 ± 0.9 μg N m^?2 h^?1, and 0.67 ± 0.57 μg N m^?2 h^?1 in the low, mid, and high marshes, respectively. Both net N₂O release and uptake were observed in the low and high marshes, but the mid‐marsh was consistently a net N₂O sink. Gross N₂O production was highest in the low marsh and lowest in the mid‐marsh (P = 0.02), whereas gross N₂O consumption did not differ among marsh zones. Thus, variability in gross N₂O production rates drove the differences in net N₂O flux among marsh zones. Our results suggest that future studies should focus on elucidating controls on the processes producing, rather than consuming, N₂O in salt marshes to improve our predictions of changes in net N₂O fluxes caused by future sea level rise.     

Hormonal responses to different sexually related conditions in male rats

Plasma levels of corticosterone (C) and testosterone (T) increase after sexual activity in males of several species. However, the physiological significance of these increases has not been elucidated. In the present study, hormonal response to different conditions linked to sexual activity was assessed. In the first experiment, plasma levels of C and T were assessed both in sexually experienced and naive male rats after the following conditions: (A) control group, without sexual stimulation; (B) males exposed to ovariectomized females; (C) males exposed to intact, non-receptive females; (D) males exposed to receptive females with the vagina obstructed, to avoid intromission; (E) males exposed to receptive females: but separated by a grid that prevents physical contact; (F) males exposed to receptive females during 30 min. In a second experiment, experienced male rats were allowed to repeatedly copulate until reaching the criteria for sexual exhaustion, and 24 h later, they were allowed to copulate. Once sexually related conditions ended, males were killed and their blood was obtained. C and T plasma levels were assessed by HPLC with ultraviolet (UV) detection. Results indicate that T did not increase significantly in naive male in any sexual condition, while in the experienced males, significant increases were observed with the mere presence of a receptive female and also after ejaculation. These increases were significantly larger in experienced males. On the other hand, C also increased in all sexual conditions, both in experienced and naive rats; however, the increase observed was larger in experienced males. Regarding sexual satiety, both C and T increased after copulating ad libitum to satiety. T increased almost three-fold compared to control, while C increased two-fold. No significant changes were observed in either one of the steroids 24 h after sexual exhaustion, even though males remained with a receptive female during an hour. These results show that sexual experience has an important influence on the hormonal response to sexual activity. C rises could be directly related to sexual arousal involved in the different sexual conditions, while T rises seem to have a direct relationship with both the motivation and execution aspects of masculine sexual behavior.     

Hormonal responses to physical training in high-level peripubertal male gymnasts

The effects of performing intensive training during growth remains controversial, with claims of negative effects upon growth and maturation purportedly due at least in part to a combination of hormonal disturbances and inappropriate nutrition. We examined the training-related responses of total testosterone (T), insulin-like growth factor-1 (IGF-1), cortisol (C) and diet in 16 peripubertal (pubertal stage <2) male gymnasts [mean (SD) age 10.5 (0.9) years, training 17.2 (5.6) h x week(-1)] and 17 controls [mean (SD) age 9.6 (1.2) years] over a 10-month period. Fasted, resting morning blood samples (0730-0900 hours) were taken from all children on Monday, Wednesday and Friday during a single week towards the end of each of three phases of gymnastics training: routine development (RD), precompetition (PC) and strength conditioning (SC). Serum concentrations of T, C and IGF-1 did not differ between the groups at any time. The ratio between IGF-1 and cortisol was significantly reduced in gymnasts relative to controls during RD and SC training (P<0.05), although no differences were detected for the T:C ratio. Diet did not correlate with any of the hormonal measurements, and no intergroup differences were found for the rate of growth in height. In summary, these results suggest that either the gymnastics training performed by these subjects was not intense enough to alter adrenal function, or that the gymnasts were well adapted to the training. In contrast, the reduction in the anabolic to catabolic balance represented by the IGF-1:C ratio is suggestive of a catabolic state, perhaps resulting from overstrain, insufficient recovery and/or inadequate caloric intake relative to energy output. While physical training during growth may induce a catabolic state, further research is needed to determine the biological significance of this finding, particularly with regard to growth and maturation.     

The copper site in nitrous oxide reductase

Summary The properties of the novel copper enzyme nitrous oxide reductase from denitrifyingPseudomonas stutzeri are described. Multifrequency electron paramagnetic resonance spectroscopy is used to characterize the various forms of the enzyme. The features observed at 2.4, 3.4, 4.5, 9.31 and 35 GHz are explained by a mixed-valence \s[Cu(1.5)\3. Cu(1.5)\s]S=\12 species with the unpaired electron delocalized between the two Cu nuclei. This site is also present in the catalytically inactive derivative of nitrous oxide reductase which was obtained from a transposon Tn5-induced mutant with defective chromophore biosynthesis. The resemblance of the low-frequency electron paramagnetic resonance spectra to the spectra for the so-called Cu_A of cytochromec oxidase can be taken as a first indication that the Cu_A may have a structural and electronic arrangement similar to the electron-paramagnetic-resonance-detectable copper in nitrous oxide reductase. Results from oxidation/reduction experiments, and from a quantitative determination of sulfhydryl and disulfide residues in the various forms of nitrous oxide reductase, suggest the involvement of the redox-couple cysteine/cystine in the structural organization of the active site of nitrous oxide reductase.     

Toxicity of bone marrow in dentists exposed to nitrous oxide.

The morphology of the bone marrow of 21 dentists who habitually used nitrous oxide in their surgeries was investigated. Exposure to nitrous oxide was measured with an atmospheric sampling device, and each dentist was invited to fill in a questionnaire giving details of medical history, diet, and intake of alcohol. During the trial a full neurological and haematological investigation was carried out and a bone marrow aspirate was examined both morphologically and by the deoxyuridine suppression test. Mean exposures to nitrous oxide ranged from 159 to 4600 parts per million. In all subjects serum vitamin B12 and folate concentrations were within normal limits. Abnormal results of deoxyuridine suppression tests were obtained in three of the 20 dentists tested; two of these three had abnormal white cells in their peripheral blood films. This study provides direct evidence that occupational exposure to nitrous oxide may cause depression of vitamin B12 activity resulting in measurable changes in bone marrow secondary to impaired synthesis of deoxyribonucleic acid.     

Hormonal control of inflammatory responses

Almost any stage of inflammatory and immunological responses is affected by hormone actions. This provides the basis for the suggestion that hormones act as modulators of the host reaction against trauma and infection. Specific hormone receptors are detected in the reactive structures in inflamed areas and binding of hormone molecules to such receptors results in the generation of signals that influence cell functions relevant for the development of inflammatory responses. Diversity of hormonal functions accounts for recognized pro- and anti-inflammatory effects exerted by these substances. Most hormone systems are capable of influencing inflammatory events. Insulin and glucocorticoids, however, exert direct regulatory effects at concentrations usually found in plasma. Insulin is endowed with facilitatory actions on vascular reactivity to inflammatory mediators and inflammatory cell functions. Increased concentrations of circulating glucocorticoids at the early stages of inflammation results in downregulation of inflammatory responses. Oestrogens markedly reduce the response to injury in a variety of experimental models. Glucagon and thyroid hormones exert indirect anti-inflammatory effects mediated by the activity of the adrenal cortex. Accordingly, inflammation is not only merely a local response, but a hormone-controlled process.