Blood flow in the brain and liver of chicken in embryonal and early postembryonal periods

In chicken Leghorn, blood flow volume speed (BF, laser-Doppler flowmetry) in the brain hemispheres and in liver was measured on days 10, 14, and 19 of embryogenesis and on day 4 after hatching (in experiments on late embryos and chickens, urethane narcosis was used). It was revealed, that initial BF in investigated organs was 2-fold lower than earlier measured in skeletal muscles. In the liver, low BF remained at all periods, but it grew 5-fold greater after hatching. In the brain hemispheres, the BF during this period grows gradually reaching 4-fold size in chickens. It was shown that blood stream increase in the brain was accompanied by uniform increase in anatomic lumen of internal carotid artery; thus settlement sizes of linear speed of blood flow and wall shear stress remain in it at the same level. Lumen extension of celiac artery during the observation period lags behind increases in a blood stream of in it that leads to increase in it of the named parameters.     

A Comparative pO2 Probe and [18F]-Fluoro-Azomycinarabino-Furanoside ([18F]FAZA) PET Study Reveals Anesthesia-Induced Impairment of Oxygenation and Perfusion in Tumor and Muscle

Tumor hypoxia can be identified by [¹⁸F]FAZA positron emission tomography, or invasively using oxygen probes. The impact of anesthetics on tumor hypoxia remains controversial. The aim of this comprehensive study was to investigate the impact of isoflurane and ketamine/xylazine anesthesia on [¹⁸F]FAZA uptake and partial oxygen pressure (pO₂) in carcinoma and muscle tissue of air- and oxygen-breathing mice.

Methods

CT26 colon carcinoma-bearing mice were anesthetized with isoflurane (IF) or ketamine/xylazine (KX) while breathing air or oxygen (O₂). We performed 10 min static PET scans 1 h, 2 h and 3 h after [¹⁸F]FAZA injection and calculated the [¹⁸F]FAZA-uptake and tumor-to-muscle ratios (T/M). In another experimental group, we placed a pO₂ probe in the tumor as well as in the gastrocnemius muscle to measure the pO₂ and perfusion.

Results

Ketamine/xylazine-anesthetized mice yielded up to 3.5-fold higher T/M-ratios compared to their isoflurane-anesthetized littermates 1 h, 2 h and 3 h after [¹⁸F]FAZA injection regardless of whether the mice breathed air or oxygen (3 h, KX-air: 7.1 vs. IF-air: 1.8, p = 0.0001, KX-O₂: 4.4 vs. IF-O₂: 1.4, p < 0.0001). The enhanced T/M-ratios in ketamine/xylazine-anesthetized mice were mainly caused by an increased [¹⁸F]FAZA uptake in the carcinomas. Invasive pO₂ probe measurements yielded enhanced intra-tumoral pO₂ values in air- and oxygen-breathing ketamine/xylazine-anesthetized mice compared to isoflurane-anesthetized mice (KX-air: 1.01 mmHg, IF-air: 0.45 mmHg; KX-O₂ 9.73 mmHg, IF-O₂: 6.25 mmHg). Muscle oxygenation was significantly higher in air-breathing isoflurane-anesthetized (56.9 mmHg) than in ketamine/xylazine-anesthetized mice (33.8 mmHg, p = 0.0003).

Conclusion

[¹⁸F]FAZA tumor uptake was highest in ketamine/xylazine-anesthetized mice regardless of whether the mice breathed air or oxygen. The generally lower [¹⁸F]FAZA whole-body uptake in isoflurane-anesthetized mice could be due to the higher muscle pO₂-values in these mice compared to ketamine/xylazine-anesthetized mice. When performing preclinical in vivo hypoxia PET studies, oxygen should be avoided, and ketamine/xylazine-anesthesia might alleviate the identification of tumor hypoxia areals.     

Effects of gradual increases in o(2) concentration on nodule activity in soybean

The objectives of this study were to determine whether attached nodules of soybean (Glycine max L. Merr.) could adjust to gradual increases in rhizosphere pO₂ without nitrogenase inhibition and to determine whether the nitrogenase activity of the nodules is limited by pO₂ under ambient conditions. A computer-controlled gas blending apparatus was used to produce linear increases (ramps) in pO₂ around attached nodulated roots of soybean plants in an open gas exchange system. Nitrogenase activity (H₂ production in N₂:O₂ and Ar:O₂) and respiration (CO₂ evolution) were monitored continuously as pO₂ 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 O₂-based limitation to nitrogenase activity occurs in soybean nodules under ambient conditions.     

Effect of pO(2) on the Formation and Status of Leghemoglobin in Nodules of Cowpea and Soybean

Nodulated cowpea (Vigna unguiculata [L.] Walp. cv Vita 3: Bradyrhizobium strain CB756) and soybean (Glycine max [L.] Merr. cv White Eye: Bradyrhizobium strain CB1809) were grown with their root systems maintained in a flowing gas stream containing a range of pO₂ (1-80%, v/v) in N₂ for up to 28 days after planting. At the extremes of sub- and supra-ambient pO₂, the levels of leghemoglobin (Lb) in nodules were reduced. However, neither the proportional composition of Lb component proteins (eight in soybean, three in cowpea) nor their oxidation state was affected by pO₂. Short-term changes in pO₂ (transferring plants grown with sub- or supra-ambient pO₂ in the rhizosphere to air or vice versa) caused a significant decline in Lb content and, in cowpea but not soybean, where pO₂ was increased, a higher percentage of oxidation of Lb. Combining data on changes in Lb level of cowpea nodules grown in sub-ambient pO₂ with those for their structural adaptation to an under supply of O₂ indicated that, despite the nodules having a lower level of Lb, the amount per infected cell was increased by up to twofold and per bacteroid up to fivefold (in those from 1% O₂) compared to those grown in air. Progressive decline in pO₂ resulted in a progressive increase on this basis, indicating a close relationship between Lb content and the adaptation of nodule functioning to external O₂ level.     

Transient responses of nitrogenase to acetylene and oxygen in actinorhizal nodules and cultured frankia

Nitrogenase activity in root nodules of four species of actinorhizal plants showed varying declines in response to exposure to acetylene (10% v/v). Gymnostoma papuanum (S. Moore) L. Johnson. and Casuarina equisetifolia L. nodules showed a small decline (5-15%) with little or no recovery over 15 minutes. Myrica gale L. nodules showed a sharp decline followed by a rapid return to peak activity. Alnus incana ssp. rugosa (Du Roi) Clausen. nodules usually showed varying degrees of decline followed by a slower return to peak or near-peak activity. We call these effects acetylene-induced transients. Rapid increases in oxygen tension also caused dramatic transient decreases in nitrogenase activity in all species. The magnitude of the transient decrease was related to the size of the O₂ partial pressure (pO₂) rise, to the proximity of the starting and ending oxygen tensions to the pO₂ optimum, and to the time for which the plant was exposed to the lower pO₂. Oxygen-induced transients, induced both by step jumps in pO₂ and by O₂ pulses, were also observed in cultures of Frankia. The effects seen in nodules are purely a response by the bacterium and not a nodule effect per se. Oxygen-induced nitrogenase transients in actinorhizal nodules from the plant genera tested here do not appear to be a result of changes in nodule diffusion resistance.     

Steady and nonsteady state gas exchange characteristics of soybean nodules in relation to the oxygen diffusion barrier

An open gas exchange system was used to monitor the nonsteady state and steady state changes in nitrogenase activity (H₂ evolution in N₂:O₂ and Ar:O₂) and respiration (CO₂ evolution) in attached, excised, and sliced nodules of soybean (Glycine max L. Merr.) exposed to external pO₂ of 5 to 100%. In attached nodules, increases in external pO₂ in steps of 10 or 20% resulted in sharp declines in the rates of H₂ and CO₂ 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 pO₂. Recovery was more rapid at higher initial pO₂ and in Ar:O₂ compared to N₂:O₂. Sequential 10% increments in pO₂ to 100% O₂ resulted in rates of H₂ evolution which were 1.4 to 1.7 times the steady state rate at 20% O₂ in Ar. This was attributed to a relief at high pO₂ from the 40% decline in nitrogenase activity that was induced by Ar at a pO₂ of 20%. Changes in nodule respiration rate could not account for the nodules' ability to adjust to high external pO₂, supporting the hypothesis that soybean nodules have a variable barrier to O₂ diffusion which responds slowly (within minutes) to changes in pO₂. Nodule excision and slicing resulted in 45 and 78% declines, respectively, in total specific nitrogenase activity at 20% O₂. In contrast with the result obtained with intact nodules, subsequent 10% increases in pO₂ in Ar:O₂ did not result in transient declines in H₂ evolution rates, but in the rapid attainment of new steady state rates. Also, distinct optima in nitrogenase activity were observed at about 60% O₂. 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 O₂ diffusion and nitrogenase activity in legume nodules.     

Oxygen supply of the brain cortex in acute nitrite hypoxia in rodents with different ecological specialization

Microhemodynamics and oxygen tension (pO₂) in the brain cortex tissues as well as the heart rate were studied in rodents with different ecological specialization during hypoxia produced by subcutaneous injection of sodium nitrite (3 mg/100 g body mass). It was shown that the blood flow in animals with low (rats) and high (muskrats) resistance to hypoxia decreased by the 30th min of the nitrite action, with its subsequent restoration to 85% and 83% of the initial level by the 60th min. The interspecies difference consisted in an increase of the brain blood flow (by 24%) in muskrats and a decrease (by 33%) in rats 15 min after the injection. In rats, simultaneously with the blood-flow dynamics, a pO₂ increase was observed in some brain cortex microareas, while in others—a pO₂ decrease 15 min after the NaNO₂ injection: meanwhile, in muskrats, at this time period a significant pO₂ decrease was observed on the background of a blood flow increase. In both animal species, the pO₂ minimal value was reached by the 45th min, while restoration almost to the initial levels—by the 60th min of the nitrite action. Changes in the rats, synchronous and unidirectional with the heart rate frequency, of the brain blood-flow, as well as tachycardia developing throughout the whole experiment in rats allow suggesting that restoration of the oxygen regime in the brain cortex microareas is provided by activation of systemic mechanisms of regulation of circulation.     

Prediction of Selenoprotein T Structure and Its Response to Selenium Deficiency in Chicken Immune Organs

Selenoprotein T (SelT) is associated with the regulation of calcium homeostasis and neuroendocrine secretion. SelT can also change cell adhesion and is involved in redox regulation and cell fixation. However, the structure and function of chicken SelT and its response to selenium (Se) remains unclear. In the present study, 150 1-day-old chickens were randomly divided into a low Se group (L group, fed a Se-deficient diet containing 0.020 mg/kg Se) and a control group (C group, fed a diet containing sodium selenite at 0.2 mg/kg Se). The immune organs (spleen, thymus, and bursa of Fabricius) were collected at 15, 25, 35, 45, and 55 days of age. We performed a sequence analysis and predicted the structure and function of SelT. We also investigated the effects of Se deficiency on the expression of SelT, selenophosphate synthetase-1 (SPS1), and selenocysteine synthase (SecS) using RT-PCR and the oxidative stress in the chicken immune organs. The data showed that the coding sequence (CDS) and deduced amino acid sequence of SelT were highly similar to those of 17 other animals. Se deficiency induced lower (P?<?0.05) levels of SelT, SPS1, and SecS, reduced the catalase (CAT) activity, and increased the levels of hydrogen peroxide (H₂O₂) and hydroxyl radical (–OH) in immune organs. In conclusion, the CDS and deduced amino acid sequence of chicken SelT are highly homologous to those of various mammals. The redox function and response to the Se deficiency of chicken SelT may be conserved. A Se-deficient diet led to a decrease in SelT, SecS, and SPS1 and induced oxidative stress in the chicken immune organs. To our knowledge, this is the first report of predictions of chicken SelT structure and function. The present study demonstrated the relationship between the selenoprotein synthases (SPS1, SecS) and SelT expression in the chicken immune organs and further confirmed oxidative stress caused by Se deficiency. Thus, the information presented in this study is helpful to understand chicken SelT structure and function. Meanwhile, the present research also confirmed the negative effects of Se deficiency on chicken immune organs.     

Cyclic nucleotide phosphodiesterase and protein activator in fetal rabbit tissues.

Cyclic nucleotide phosphodiesterase activity (EC 3.1.4.17) was studied in fetal and newborn rabbit brain, heart, liver, kidney, and lung. Kinetic analysis of phosphodiesterase activity from homogenates of organs from the 25-day embryo suggested the presence of a high K_m and a low K_m activity for both cyclic AMP and cyclic GMP hydrolysis. The addition of 1 μm cyclic GMP to the assay stimulated the hydrolysis of cyclic AMP by whole homogenates of liver, brain, lung, and kidney, but not heart, at all of the ages studied. The addition of micromolar levels of calcium ion stimulated cyclic GMP hydrolysis by homogenates of fetal brain, heart, and kidney, with or without added protein activator. Cyclic GMP phosphodiesterase activity was not stimulated by the addition of calcium ion in homogenates of early fetal rabbit liver and lung, but stimulation was detected in the late embryo and newborn. The presence of the heat-stable protein activator was demonstrated in brain, heart, kidney, liver, and lung tissue at all of the fetal ages studied, and in the newborn rabbit. DEAE-cellulose chromatography demonstrated the presence of three separable enzymes in brain and liver at 15 days, heart at 19 days, and lung and kidney at 25 days of gestation, with no changes in the kinetic properties of the isolated enzymes during development. These experiments suggest that all of the organs studied have the mature array of phosphodiesterases early in development, but an enzyme from liver and lung becomes sensitive to regulatory control by calcium only late in gestation.     

Soybean seed growth in response to long-term exposures to differing oxygen partial pressures

Short-term studies have indicated that alterations in the oxygen partial pressure (pO₂) around developing soybean (Glycine max [L.] Merr.) seeds may alter seed growth characteristics. A 2-year field study was undertaken to determine the effects on seed development of long-term exposures of individual pods to either sub-ambient or supra-ambient pO₂. Pod chambers were used through which fixed pO₂ were continuously flowed throughout seed development. No effects on maturity date were observed from exposures to either sub-ambient or supra-ambient pO₂. On the other hand, seed weight was reduced by 0.10 pO₂ in both years of the study implicating an O₂ limitation on seed growth rate at this fairly high pO₂. In 1 of the 2 years, supra-ambient pO₂ resulted in increased seed weight.     

Adaptation of Nitrogen Fixation by Intact Soybean Nodules to Altered Rhizosphere pO(2)

The N₂-fixing legume nodule requires O₂ for ATP production; however, the O₂ sensitivity of nitrogenase dictates a requirement for a low pO₂ inside the nodule. The effects of long term exposures to various pO₂s on N₂[C₂H₂] fixation were evaluated with intact soybean (Glycine max [L.] Merr., var. Wye) plants. Continuous exposure of their rhizosphere to a pO₂ of 0.06 atmospheres initially reduced nitrogenase activity by 37 to 45% with restoration of original activity in 4 to 24 hours and with no further change in tests up to 95 hours; continuous exposure to 0.02 atmosphere of O₂ initially reduced nitrogenase activity 72%, with only partial recovery by 95 hours. Similar exposures to a pO₂ of 0.32 atmospheres had little effect on N₂[C₂H₂] fixation; a pO₂ of 0.89 atmospheres initially reduced nitrogenase activity by 98% with restoration to only 14 to 24% of that of the ambient O₂ controls by 95 hours. Re-exposure to ambient pO₂ of plants adapted to nonambient pO₂s reduced N₂[C₂H₂] fixation to similar magnitudes as the reductions which occurred upon initial exposure to variant pO₂ conditions, and a time period was required to readapt to ambient O₂. It is concluded that the N₂[C₂H₂]-fixing system of intact soybean plants is able to adapt to a wide range of external pO₂s as probably occur in soil. We postulate that this occurs through an undefined mechanism which enables the nodule to maintain an internal pO₂ optimal for nitrogenase activity.     

Oxygen regime in rat brain tissues under conditions of acute nitrite methemoglobinemia

Oxygen regime in rat brain tissues was studied under conditions of nitrite hypoxia. The local brain circulation (LCC) and pO₂ were recorded by polargraphic method in microareas of the brain cortex one hour after a subcutaneous injection of NaNO₂ (3 mg/100 g body mass). A LCC decrease by 55% was shown by the 30th min of the nitrite intoxication, with its restoration to 85% of the initial blood-flow by the 60th min. In some brain microareas, a pO₂ increase by 40.7% was observed by the 45th min of the action and its decrease by 32.2% by the 60th min, whereas in other microareas, a pO₂ decrease by 24.5% with its subsequent increase to 78.7% of the initial level, respectively. A statistically significant correlation is revealed between changes of the oxygen pressure in the process of development of nitrite hypoxia and the LCC dynamics. It is concluded that the circulatory disturbances developing at the first moments of the nitrite action lead to an increase of the degree of the pO₂ differentiated distribution in the brain: to hyperoxygenation in some microareas and to severe hypoxia in others, what can be the cause of functional brain disturbances under the effect of nitrogen oxides.     

Adaptation of Nodulated Soybean (Glycine max L. Merr.) to Growth in Rhizospheres Containing Nonambient pO(2)

Nodulated soybean (Glycine max L. Merr. cv White Eye inoculated with Bradyrhizobium japonicum strain CB 1809) plants were cultured in the absence of combined N from 8 to 28 days with their root systems maintained continuously in 1, 2.5, 5, 10, 20, 40, 60, or 80% O₂ (volume/volume) in N₂. Plant dry matter yield was unaffected by partial pressure of oxygen (pO₂) and N₂ fixation showed a broad plateau of maximum activity from 2.5 to 40 or 60% O₂. Slight inhibition of nitrogenase activity occurred at 1% O₂ and as much as 50% inhibition occurred at 80% O₂. Low pO₂ (less than 10%) decreased nodule mass on plants, but this was compensated for by those nodules having higher specific nitrogenase activities. Synthesis and export of ureides in xylem was maintained at a high level (70-95% of total soluble N in exudate) over the range of pO₂ used. Measurements of nitrogenase (EC 1.7.99.2) activity by acetylene reduction indicated that adaptation of nodules to low pO₂ was largely due to changes in ventilation characteristics and involved increased permeability to gases in those grown in subambient pO₂ and decreased permeability in those from plants cultured with their roots in pO₂ greater than ambient. A range of structural alterations in nodules resulting from low pO₂ were identified. These included increased frequency of lenticels, decreased nodule size, increased volume of cortex relative to the infected central tissue of the nodule, as well as changes in the size and frequency of extracellular voids in all tissues. In nodules grown in air, the inner cortex differentiated a layer of four or five cells which formed a band, 40 to 50 micrometers thick, lacking extracellular voids. This was reduced in nodules grown in low pO₂ comprising one or two cell layers and being 10 to 20 micrometers thick in those from 1% O₂. Long-term adaptation to different external pO₂ involved changes which modify diffusive resistance and are additional to adjustments in the variable diffusion barrier.     

Protective Roles of Selenium on Nitric Oxide-Mediated Apoptosis of Immune Organs Induced by Cadmium in Chickens

Little is known about the influence of subchronic cadmium exposure on apoptosis in the immune organs of birds and the protective effects on apoptosis by selenium against cadmium. The aim of this study was to investigate the effect of subchronic cadmium exposure on nitric oxide and apoptosis in the immune organs of chicken and the protective roles of selenium against cadmium-induced apoptosis. Two hundred ten 30-day-old chickens were randomly assigned to three groups and were fed a basal diet, cadmium?+?selenium (as 150 mg of CdCl₂ per kg of diet?+?10 mg of Na₂SeO₃ per kg of diet ) or cadmium (as 150 mg of CdCl₂ per kg of diet) in basic diets for 15, 30, 45, and 60 days. Then, the production of nitric oxide, messenger RNA (mRNA level), and the activity of inducible nitric oxide synthase, ultrastructural changes, TUNEL assay, and flow cytometric analysis of apoptosis and Bcl-2 and p53 mRNA levels in the immune organs were examined. The results showed that cadmium exposure caused ultrastructural damage and increased production of nitric oxide, mRNA level, and activity of inducible nitric oxide synthase, the degree, and the number of apoptotic cells in a time-dependent manner. Cadmium exposure decreased Bcl-2 mRNA level and increased p53 mRNA level in a time-dependent manner. Selenium supplementation during dietary cadmium reduced the production of nitric oxide, the mRNA level, and activity of inducible nitric oxide synthase, ultrastructural damage, and apoptosis in the immune organs of chicken. It indicated that cadmium induced nitric oxide-mediated apoptosis of immune organs, and selenium played protective effects against cadmium-induced apoptosis in the immune organs of chickens.     

Noninvasive Monitoring of Small Intestinal Oxygen in a Rat Model of Chronic Mesenteric Ischemia

We noninvasively monitored the partial pressure of oxygen (pO₂) in rat’s small intestine using a model of chronic mesenteric ischemia by electron paramagnetic resonance oximetry over a 7-day period. The particulate probe lithium octa-n-butoxynaphthalocyanine (LiNc-BuO) was embedded into the oxygen permeable material polydimethyl siloxane by cast-molding and polymerization (Oxy-Chip). A one-time surgical procedure was performed to place the Oxy-Chip on the outer wall of the small intestine (SI). The superior mesenteric artery (SMA) was banded to ~30 % of blood flow for experimental rats. Noninvasive measurement of pO₂ was performed at the baseline for control rats or immediate post-banding and on days 1, 3, and 7. The SI pO₂ for control rats remained stable over the 7-day period. The pO₂ on day-7 was 54.5 ± 0.9 mmHg (mean ± SE). SMA-banded rats were significantly different from controls with a noted reduction in pO₂ post banding with a progressive decline to a final pO₂ of 20.9 ± 4.5 mmHg (mean ± SE; p = 0.02). All SMA-banded rats developed adhesions around the Oxy-Chip, yet remained asymptomatic. The hypoxia marker Hypoxyprobe? was used to validate the low tissue pO₂. Brown cytoplasmic staining was consistent with hypoxia. Mild brown staining was noted predominantly on the villus tips in control animals. SMA-banded rats had an extended region of hypoxic involvement in the villus with a higher intensity of cytoplasmic staining. Deep brown stainings of the enteric nervous system neurons and connective tissue both within layers and in the mesentery were noted. SMA-banded rats with lower pO₂ values had a higher intensity of staining. Thus, monitoring SI pO₂ using the probe Oxy-Chip provides a valid measure of tissue oxygenation. Tracking pO₂ in conditions that produce chronic mesenteric ischemia will contribute to our understanding of intestinal tissue oxygenation and how changes impact symptom evolution and the trajectory of chronic disease.     

Frankia vesicles provide inducible and absolute oxygen protection for nitrogenase

When Frankia HFPCcI3 was grown in culture at oxygen O₂ levels ranging from 2 to 70 kilopascals O₂, under nitrogen fixing conditions, nitrogenase activity adapted to ambient pO₂ and showed a marked optimum close to growth pO₂. Vesicles were thin walled at low pO₂ and very thick walled at high pO₂. Freeze fracture transmission electron microscopy confirmed that Frankia produces vesicles with outer walls thickened by multiple lipid-like monolayers, in proportion to ambient pO₂.     

Structural and Functional Divergence of Growth Hormone-Releasing Hormone Receptors in Early Sarcopterygians: Lungfish and Xenopus

The evolutionary trajectories of growth hormone-releasing hormone (GHRH) receptor remain enigmatic since the discovery of physiologically functional GHRH-GHRH receptor (GHRHR) in non-mammalian vertebrates in 2007. Interestingly, subsequent studies have described the identification of a GHRHR₂ in chicken in addition to the GHRHR and the closely related paralogous receptor, PACAP-related peptide (PRP) receptor (PRPR). In this article, we provide information, for the first time, on the GHRHR in sarcopterygian fish and amphibians by the cloning and characterization of GHRHRs from lungfish (P. dolloi ) and X. laevis. Sequence alignment and phylogenetic analyses demonstrated structural resemblance of lungfish GHRHR to their mammalian orthologs, while the X. laevis GHRHR showed the highest homology to GHRHR₂ in zebrafish and chicken. Functionally, lungfish GHRHR displayed high affinity towards GHRH in triggering intracellular cAMP and calcium accumulation, while X. laevis GHRHR₂ was able to react with both endogenous GHRH and PRP. Tissue distribution analyses showed that both lungfish GHRHR and X. laevis GHRHR₂ had the highest expression in brain, and interestingly, X. laevis _GHRHR2 also had high abundance in the reproductive organs. These findings, together with previous reports, suggest that early in the Sarcopterygii lineage, GHRHR and PRPR have already established diverged and specific affinities towards their cognate ligands. GHRHR₂, which has only been found in xenopus, zebrafish and chicken hitherto, accommodates both GHRH and PRP.     

Response of the Endophytic Diazotroph Gluconacetobacter diazotrophicus on Solid Media to Changes in Atmospheric Partial O2 Pressure

Gluconacetobacter diazotrophicus is an N₂-fixing endophyte isolated from sugarcane. G. diazotrophicus was grown on solid medium at atmospheric partial O₂ pressures (pO₂) of 10, 20, and 30 kPa for 5 to 6 days. Using a flowthrough gas exchange system, nitrogenase activity and respiration rate were then measured at a range of atmospheric pO₂ (5 to 60 kPa). Nitrogenase activity was measured by H₂ evolution in N₂-O₂ and in Ar-O₂, and respiration rate was measured by CO₂ evolution in N₂-O₂. To validate the use of H₂ production as an assay for nitrogenase activity, a non-N₂-fixing (Nif⁻) mutant of G. diazotrophicus was tested and found to have a low rate of uptake hydrogenase (Hup⁺) activity (0.016± 0.009 μmol of H₂ 10¹⁰ cells⁻¹ h⁻¹) when incubated in an atmosphere enriched in H₂. However, Hup⁺ activity was not detectable under the normal assay conditions used in our experiments. G. diazotrophicus fixed nitrogen at all atmospheric pO₂ tested. However, when the assay atmospheric pO₂ was below the level at which the colonies had been grown, nitrogenase activity was decreased. Optimal atmospheric pO₂ for nitrogenase activity was 0 to 20 kPa above the pO₂ at which the bacteria had been grown. As atmospheric pO₂ was increased in 10-kPa steps to the highest levels (40 to 60 kPa), nitrogenase activity decreased in a stepwise manner. Despite the decrease in nitrogenase activity as atmospheric pO₂ was increased, respiration rate increased marginally. A large single-step increase in atmospheric pO₂ from 20 to 60 kPa caused a rapid 84% decrease in nitrogenase activity. However, upon returning to 20 kPa of O₂, 80% of nitrogenase activity was recovered within 10 min, indicating a “switch-off/switch-on” O₂ protection mechanism of nitrogenase activity. Our study demonstrates that colonies of G. diazotrophicus can fix N₂ at a wide range of atmospheric pO₂ and can adapt to maintain nitrogenase activity in response to both long-term and short-term changes in atmospheric pO₂.     

Mechanistic studies of epoxide hydrolase utilizing a continuous spectophotometric assay

A precise continuous photometric assay has been devised and utilized for mechanistic studies of chicken and rat liver microsomal epoxide hydrolase (EH). The assay is based on monitoring the hydration of p-nitrostyrene oxide (PNSO) at 310 nm. Rat liver EH hydrates S-(+)- and R-(?)-PNSO differentially, the K_m and V values for the former being ca. four times those for the latter; in contrast, enantiomeric differences are negligible with chicken liver EH. With rat EH V increases slightly from pH 7 to 8 and then falls rapidly from pH 8 to 9.5; K_m remains constant from pH 7 to 8 and then increases steadily from pH 8 to 9.5. In 86 mol% D₂O the solvent isotope effect on V ($\text{H}{}_2\text{O}\text{D}{}_2\text{O}$) is 1.103 ± 0.015. Both rat and chicken EH show a 3% inverse isotope effect for the hydration of [7-²H]PNSO and a 4% normal isotope effect for the hydration of [8-²H₂]PNSO. These observations are discussed in terms of the possible participation of acid as well as base catalysis in the enzymatic mechanism.     

Effect of pO(2) on Growth and Nodule Functioning of Symbiotic Cowpea (Vigna unguiculata L. Walp.)

Nodulated cowpea (Vigna unguiculata L. Walp. cv Vita 3:Bradyrhizobium CB 756) plants were cultured with their whole root system or crown root nodulation zone maintained for periods from 5 to 69 days after planting in atmospheres containing a range of pO₂ (1-80%, v/v) while the rest of the plant grew in normal air. Growth (dry matter yield) and N₂ fixation were largely unaffected by pO₂ from 10 to 40%. Decrease in fixation at pO₂ below 5% was due to lower nodulation and nodule mass and, at pO₂ above 60%, to a fall in specific N₂-fixing activity of nodules. Root:shoot ratios were significantly lower at pO₂ below 2.5%. The effect of pO₂ on nitrogenase activity (acetylene reduction), both of whole nodulated root systems and crown root nodulation zones, varied with plant age but was generally lower at supra- and subambient extremes of O₂. H₂ evolution showed a sharp optimum at 20% O₂ but was at most 4% of total nitrogenase activity. The ratio of CO₂ evolved to substrate (C₂H₂+H⁺) reduced by crown root nodulation zones was constant (6 moles CO₂ per mole substrate reduced) from 2.5 to 60% O₂ but at levels below 2.5 and above 80% O₂ reached values between 20 and 30 moles CO₂ per mole substrate reduced. Effects of long-term growth with nonambient pO₂ on adaptation and efficiency of functioning of nodules are discussed.