Atmospheric stability in cell culture vessels

Summary We have investigated the atmospheric stability in polystyrene and glass cell culture vessels by measuring the dissolved O₂ and CO₂ in the media of both seeded and unseeded culture vessels incubated at 37°C. There was no diffusion of either O₂ or CO₂ through glass vessels. At low partial pressures of oxygen (P_{O 2}), oxygen diffused into the polystyrene flasks at a rate of 1 to 2 mm Hg per 24 hr, and at high P_{O 2}, oxygen diffused slowly out of polystyrene flasks. CO₂ diffused out of polystyrene flasks with a half-time of 260 hr resulting in a considerable elevation in pH. In seeded polystyrene flasks with the P_{O 2} ⩽ room air, cellular oxygen consumption was masked by the inward diffusion of oxygen. In addition, the fall in pH due to metabolic CO₂ and organic acid production during cell growth in polystyrene flasks was buffered by the diffusion of CO₂ out of the vessels. Presented, in part, by Dr. Arthur Balin in partial fulfillment of the requirements for the Ph.D. This work was supported by USPHS grants AG-00378 from the National Institute of Aging and CA-14345 from the National Cancer Institute and NR 202-005 from the Office of Naval Research. A.K.B. is a trainee of the Medical Scientist Training Program, National Institutes of Health (GM 02046).     

Availability of O2 as a Substrate in the Cytoplasm of Bacteria under Aerobic and Microaerobic Conditions

The growth rates of Pseudomonas putida KT2442 and mt-2 on benzoate, 4-hydroxybenzoate, or 4-methylbenzoate showed an exponential decrease with decreasing oxygen tensions (partial O₂ tension [pO₂] values). The oxygen tensions resulting in half-maximal growth rates were in the range of 7 to 8 mbar of O₂ (corresponding to 7 to 8 μM O₂) (1 bar = 10⁵ Pa) for aromatic compounds, compared to 1 to 2 mbar for nonaromatic compounds like glucose or succinate. The decrease in the growth rates coincided with excretion of catechol or protocatechuate, suggesting that the activity of the corresponding oxygenases became limiting. The experiments directly establish that under aerobic and microaerobic conditions (about 10 mbar of O₂), the diffusion of O₂ into the cytoplasm occurs at high rates sufficient for catabolic processes. This is in agreement with calculated O₂ diffusion rates. Below 10 mbar of O₂, oxygen became limiting for the oxygenases, probably due to their high K_m values, but the diffusion of O₂ into the cytoplasm presumably should be sufficiently rapid to maintain ambient oxygen concentrations at oxygen tensions as low as 1 mbar of O₂. The consequences of this finding for the availability of O₂ as a substrate or as a regulatory signal in the cytoplasm of bacterial cells are discussed.     

Contribution of the polychaete,Neanthes japonica (Izuka), to the oxygen uptake and carbon dioxide production of an intertidal mud-flat of the Nanakita River estuary,Japan

The benthic oxygen consumption and carbon dioxide production of undisturbed and sieved sediment cores with various values for the biomass of polychaetes collected from the intertidal mud-flat of Nanakita River estuary of Japan were measured simultaneously. The benthic oxygen consumption and carbon dioxide production increased in proportion to the biomass of a dominant polychaete species Neanthes japonica (Izuka). This increase was not explained by the respiration of the animals alone. The residual increase in benthic O₂ and CO₂ fluxes may be due to mineralization processes in the burrow wall and enhanced diffusion caused by the pumping activity of the worms. From the average biomass of polychaetes at the study site, total benthic O₂ and CO₂ fluxes were estimated to be 5.2 mmol·m⁻²·h⁻¹ and 7.3 mmol·m⁻²·h⁻¹, respectively, at 20 ° C. The worms were responsible for 79% of the total O₂ flux and 73% of the total CO₂ flux but the respiration of the worms accounted for only 53% of the total O₂ flux and 36% of the total CO₂ flux. The residual enhanced fluxes were 26% and 37% for the total O₂ and CO₂ fluxes, respectively.     

Oxygen regulates the effective diffusion distance of nitric oxide in the aortic wall

Endothelium-derived nitric oxide (NO) is critical in maintaining vascular tone. Accumulating evidence shows that NO bioavailability is regulated by oxygen concentration. However, it is unclear to what extent the oxygen concentration regulates NO bioavailability in the vascular wall. In this study, a recently developed experimental setup was used to measure the NO diffusion flux across the aortic wall at various oxygen concentrations. It was observed that for a constant NO concentration at the endothelial surface, the measured NO diffusion flux out of the adventitial surface at [O₂] = 0 μM is around fivefold greater than at [O₂] = 150 μM, indicating that NO is consumed in the aortic wall in an oxygen-dependent manner. Analysis of experimental data shows that the rate of NO consumption in the aortic wall is first order with respect to [NO] and first order with respect to [O₂], and the rate constant k₁ was determined as (4.0 ± 0.3) × 10³ M^?1 s^?1. Computer simulations demonstrate that NO concentration distribution significantly changes with oxygen concentration and the effective NO diffusion distance at low oxygen level ([O₂] ≤ 25 μM) is significantly longer than that at high oxygen level ([O₂] = 200 μM). These results suggest that oxygen-dependent NO consumption may play an important role in dilating blood vessels during hypoxia by increasing the effective NO diffusion distance.     

The effect of pyruvate on nitrogenase activity in the blue-green alga Anabaena cyclindrica

Exogenous pyruvate added to cultures of the bluegreen alga, Anabaena cylindrica stimulated nitrogenase activity (measured by acetylene reduction) only in the dark under low pO₂ (0.05 atmospheres). Under aerobic conditions or in the light, stimulation was absent and replaced by an inhibition of activity above 5 mM added pyruvate. The curve of nitrogenase activity versus oxygen concentration had a similar maximal value of ethylene production with or without added pyruvate, but in the presence of pyruvate this maximum occurred at 0.05 atmospheres O₂, whilst in the absence of pyruvate the maximum occurred at 0.10 atmospheres O₂. Malate, citrate, α-ketoglutarate, glucose and fructose were tested also, but none gave a similar effect to pyruvate. Addition of ¹⁴C-pyruvate and autoradiography indicated that exogenous pyruvate is metabolized through the interrupted Krebs cycle. These results are explained in terms of the activity of pyruvate: ferredoxin oxidoreductase and the ATP-induced oxygen sensitivity of nitrogenase.     

Erythrocruorins (extracellular haemoglobins) from the cirratulid polychaetes cirriformia tentaculata (Montagu) and Cirratulus cirratus (Müller) with special reference to saturation dependent characteristics of the oxygen equilibria

Erythrocruorins from the intertidal cirratulid polychaetes Cirriformia tentaculata (Montagu) and Cirratulus cirratus (Müller) have a high affinity for oxygen and large Bohr shifts. At low pH, the O₂-equilibrium curves from both species are hyperbolic. In alkaline solution both erythrocruorins show marked increases in O₂-affmity with the retention of the hyperbolic form in Cirriformia. The presence of a pronounced Bohr shift in a non-cooperative erythrocruorin is unusual and its possible physiological rôle is discussed.In Cirratulus, the shape of the curve becomes sigmoidal at high pH. Moreover, the cooperativity coefficient, n, increases at high O₂-saturation. The Bohr effect thus increases in magnitude with rising pH and it is saturation dependent. The significance of this complex oxygen transport system is discussed. The findings illustrate the remarkable elasticity of functions possible in erythrocruorins from closely related species in response to different environmental or metabolic dictates.     

Influences of Infaunal Burrows on the Community Structure and Activity of Ammonia-Oxidizing Bacteria in Intertidal Sediments

Influences of infaunal burrows constructed by the polychaete (Tylorrhynchus heterochaetus) on O₂ concentrations and community structures and abundances of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in intertidal sediments were analyzed by the combined use of a 16S rRNA gene-based molecular approach and microelectrodes. The microelectrode measurements performed in an experimental system developed in an aquarium showed direct evidence of O₂ transport down to a depth of 350 mm of the sediment through a burrow. The 16S rRNA gene-cloning analysis revealed that the betaproteobacterial AOB communities in the sediment surface and the burrow walls were dominated by Nitrosomonas sp. strain Nm143-like sequences, and most of the clones in Nitrospira-like NOB clone libraries of the sediment surface and the burrow walls were related to the Nitrospira marina lineage. Furthermore, we investigated vertical distributions of AOB and NOB in the infaunal burrow walls and the bulk sediments by real-time quantitative PCR (Q-PCR) assay. The AOB and Nitrospira-like NOB-specific 16S rRNA gene copy numbers in the burrow walls were comparable with those in the sediment surfaces. These numbers in the burrow wall at a depth of 50 to 55 mm from the surface were, however, higher than those in the bulk sediment at the same depth. The microelectrode measurements showed higher NH₄⁺ consumption activity at the burrow wall than those at the surrounding sediment. This result was consistent with the results of microcosm experiments showing that the consumption rates of NH₄⁺ and total inorganic nitrogen increased with increasing infaunal density in the sediment. These results clearly demonstrated that the infaunal burrows stimulated O₂ transport into the sediment in which otherwise reducing conditions prevailed, resulting in development of high NH₄⁺ consumption capacity. Consequently, the infaunal burrow became an important site for NH₄⁺ consumption in the intertidal sediment.     

Reaction of Oxyhemoglobin with Carbon Monoxide

The reaction of oxyhemoglobin and carbon monoxide was studied kinetically at pH 7.8 in a variety of suspending media. The dielectric constant of the suspending media, as well as the viscosity (and hence the Fick diffusion coefficients), was varied with the use of glycine, glycerol, and sucrose. The results showed that the reaction was unaltered by the various additions to the media, provided that the pO₂ and the concentration of carbon monoxide were held constant. Since the concentration of oxygen varies from medium to medium at constant pO₂ while the pCO varies at constant concentration of carbon monoxide, the differences in the reactions with oxygen and carbon monoxide were emphasized. The lack of variation of the rate constants with changes in dielectric constant can be interpreted as indicating that electrostatic effects are unimportant in this reaction.     

Flow velocity affects internal oxygen conditions in the seagrass Cymodocea nodosa

The internal oxygen status of seagrass tissues, which is believed to play an important role in events of seagrass die-off, is partly determined by the rates of gas exchange between leaves and water column. In this study, we examined whether water column flow velocity has an effect on gas exchange, and hence on internal oxygen partial pressures (pO₂) in the Mediterranean seagrass, Cymodocea nodosa. We measured the internal pO₂ in the horizontal rhizomes of C. nodosa in darkness at different mainstream flow velocities, combined with different levels of water column oxygen pO₂ using an experimental flume in the laboratory. Flow velocity clearly had an effect on the internal oxygen status. In stagnant, but fully aerated water the mean internal pO₂ was 6.9 kPa, corresponding to about 30% of air saturation. The internal pO₂ increased with increasing flow velocity reaching saturation of around 12.2 kPa (60% of air saturation) at flow velocities ≥7 cm s⁻¹. Flow had a relatively larger influence on internal pO₂ at lower water column oxygen concentrations. By extrapolating linear relationships between internal and water column pO₂ in this experimental setup, rhizomes would become anoxic at a water column oxygen pO₂ of 4–4.5 kPa (∼20% of air saturation) in flowing water, but already at 6.4 kPa (∼30% of air saturation) in stagnant water. Water flow may play an important role for seagrass performance and survival in areas with poor water column oxygen conditions and may, in general, be of importance for the distribution of submerged rooted plants.     

The role of strong hypoxia in tumors after treatment in the outcome of bacteriochlorin-based photodynamic therapy

Blood flow and pO₂ changes after vascular-targeted photodynamic therapy (V-PDT) or cellular-targeted PDT (C-PDT) using 5,10,15,20-tetrakis(2,6-difluoro-3-N-methylsulfamoylphenyl) bacteriochlorin (F₂BMet) as photosensitizer were investigated in DBA/2 mice with S91 Cloudman mouse melanoma, and correlated with long-term tumor responses. F₂BMet generates both singlet oxygen and hydroxyl radicals under near-infrared radiation, which consume oxygen. Partial oxygen pressure was lowered in PDT-treated tumors and this was ascribed both to oxygen consumption during PDT and to fluctuations in oxygen transport after PDT. Similarly, microcirculatory blood flow changed as a result of the disruption of blood vessels by the treatment. A novel noninvasive approach combining electron paramagnetic resonance oximetry and laser Doppler blood perfusion measurements allowed longitudinal monitoring of hypoxia and vascular function changes in the same animals, after PDT. C-PDT induced parallel changes in tumor pO₂ and blood flow, i.e., an initial decrease immediately after treatment, followed by a slow increase. In contrast, V-PDT led to a strong and persistent depletion of pO₂, although the microcirculatory blood flow increased. Strong hypoxia after V-PDT led to a slight increase in VEGF level 24 h after treatment. C-PDT caused a ca. 5-day delay in tumor growth, whereas V-PDT was much more efficient and led to tumor growth inhibition in 90% of animals. The tumors of 44% of mice treated with V-PDT regressed completely and did not reappear for over 1 year. In conclusion, mild and transient hypoxia after C-PDT led to intense pO₂ compensatory effects and modest tumor inhibition, but strong and persistent local hypoxia after V-PDT caused tumor growth inhibition.     

Oxygen exchange mechanisms in the human placenta: mathematical modelling and simulation

An exact knowledge of the human fetus's respiratory mechanisms is still lacking; in particular, the role of human placental anatomy in oxygen exchange has not yet been studied satisfactorily. In this paper, a mathematical model of placenta as O₂ exchanger between maternal and fetal blood was developed; it led to the solution of equations based upon diffusion laws and the haemoglobin dissociation curve. Particular care was taken to represent the regimen of laminar motion or whirling into the capillaries. Theoretical results were compared, under physiological conditions, with clinical data relating to fetal oxygenated blood pO₂ during the second half of gestation (20th–38th weeks), and a theoretical confirmation of the decreasing effectiveness of placental O₂ exchange during gestation was found. The result was able to describe oxygen exchange during a period in which clinical data are scanty (23rd–30th weeks). The effects of some pathological events on O₂ exchange were then simulated. Model parameters were changed to simulate the effects on oxygen exchange of some typical pathological variations of placental anatomical features: exchange surface thickness and capillary length. The curves obtained for different gestational ages can easily be correlated with echographic measures of placental volume and dimensions of placental capillaries. The results also show that the human placenta is more sensitive to pathologies when it is young than at term of gestation.     

The effects of feeding on the swimming performance and metabolic response of juvenile southern catfish,Silurus meridionalis,acclimated at different temperatures

To test whether the effects of feeding on swimming performance vary with acclimation temperature in juvenile southern catfish (Silurus meridionalis), we investigated the specific dynamic action (SDA) and swimming performance of fasting and feeding fish at acclimation temperatures of 15, 21, 27, and 33 °C. Feeding had no effect on the critical swimming speeding (U_crit) of fish acclimated at 15 °C (p = 0.66), whereas it elicited a 12.04, 18.70, and 20.98% decrease in U_crit for fish acclimated at 21, 27 and 33 °C, respectively (p < 0.05). Both the maximal postprandial oxygen consumption rate (VO_2peak) and the active metabolic rate (VO_2active, maximal aerobic sustainable metabolic rate of fasting fish) increased significantly with temperature (p < 0.05). The postprandial maximum oxygen consumption rates during swimming (VO_2max) were higher than the VO_2active of fasting fish at all temperature groups (p < 0.05). The VO_2max increased with increasing temperature, but the relative residual metabolic scope (VO_2max? VO_2peak) during swimming decreased with increasing in temperature. The present study showed that the impairment of postprandial swimming performance increased with increasing temperature due to the unparalleled changes in the catfish's central cardio-respiratory, peripheral digestive and locomotory capacities. The different metabolic strategies of juvenile southern catfish at different temperatures may relate to changes in oxygen demand, imbalances in ion fluxes and dissolved oxygen levels with changes in temperature.     

Oxygen-coupled Redox Regulation of the Skeletal Muscle Ryanodine Receptor/Ca2+ Release Channel (RyR1): SITES AND NATURE OF OXIDATIVE MODIFICATION*

In mammalian skeletal muscle, Ca²⁺ release from the sarcoplasmic reticulum (SR) through the ryanodine receptor/Ca²⁺-release channel RyR1 can be enhanced by S-oxidation or S-nitrosylation of separate Cys residues, which are allosterically linked. S-Oxidation of RyR1 is coupled to muscle oxygen tension (pO₂) through O₂-dependent production of hydrogen peroxide by SR-resident NADPH oxidase 4. In isolated SR (SR vesicles), an average of six to eight Cys thiols/RyR1 monomer are reversibly oxidized at high (21% O₂) versus low pO₂ (1% O₂), but their identity among the 100 Cys residues/RyR1 monomer is unknown. Here we use isotope-coded affinity tag labeling and mass spectrometry (yielding 93% coverage of RyR1 Cys residues) to identify 13 Cys residues subject to pO₂-coupled S-oxidation in SR vesicles. Eight additional Cys residues are oxidized at high versus low pO₂ only when NADPH levels are supplemented to enhance NADPH oxidase 4 activity. pO₂-sensitive Cys residues were largely non-overlapping with those identified previously as hyperreactive by administration of exogenous reagents (three of 21) or as S-nitrosylated. Cys residues subject to pO₂-coupled oxidation are distributed widely within the cytoplasmic domain of RyR1 in multiple functional domains implicated in RyR1 activity-regulating interactions with the L-type Ca²⁺ channel (dihydropyridine receptor) and FK506-binding protein 12 as well as in “hot spot” regions containing sites of mutation implicated in malignant hyperthermia and central core disease. pO₂-coupled disulfide formation was identified, whereas neither S-glutathionylated nor sulfenamide-modified Cys residues were observed. Thus, physiological redox regulation of RyR1 by endogenously generated hydrogen peroxide is exerted through dynamic disulfide formation involving multiple Cys residues.     

Root Growth and Oxygen Relations at Low Water Potentials. Impact of Oxygen Availability in Polyethylene Glycol Solutions

Polyethylene glycol (PEG), which is often used to impose low water potentials (ψ_w) in solution culture, decreases O₂ movement by increasing solution viscosity. We investigated whether this property causes O₂ deficiency that affects the elongation or metabolism of maize (Zea mays L.) primary roots. Seedlings grown in vigorously aerated PEG solutions at ambient solution O₂ partial pressure (pO₂) had decreased steady-state root elongation rates, increased root-tip alanine concentrations, and decreased root-tip proline concentrations relative to seedlings grown in PEG solutions of above-ambient pO₂ (alanine and proline accumulation are responses to hypoxia and low ψ_w, respectively). Measurements of root pO₂ were made using an O₂ microsensor to ensure that increased solution pO₂ did not increase root pO₂ above physiological levels. In oxygenated PEG solutions that gave maximal root elongation rates, root pO₂ was similar to or less than (depending on depth in the tissue) pO₂ of roots growing in vermiculite at the same ψ_w. Even without PEG, high solution pO₂ was necessary to raise root pO₂ to the levels found in vermiculite-grown roots. Vermiculite was used for comparison because it has large air spaces that allow free movement of O₂ to the root surface. The results show that supplemental oxygenation is required to avoid hypoxia in PEG solutions. Also, the data suggest that the O₂ demand of the root elongation zone may be greater at low relative to high ψ_w, compounding the effect of PEG on O₂ supply. Under O₂-sufficient conditions root elongation was substantially less sensitive to the low ψ_w imposed by PEG than that imposed by dry vermiculite.     

Algorithm for computing oxygen dissociation curve with pH,PCO2, and CO in sheep blood

By extending the study of Samaja and Gattinoni¹, an algorithm is described for computing the oxygen dissociation curve with variations in pH, PCO₂, and CO in homozygous HbB sheep blood. The difference in the values of O₂ pressure at 50% saturation in presence of CO computed from the present algorithm and Hill's equation does not exceed 0.5%. It is shown that O₂ affinity increases as the concentration of CO or pH increases or PCO₂ decreases. The algorithm is convenient for representing the oxygen dissociation curve with variation in pH, PCO₂ and the concentration of CO in modelling oxygen transport in sheep blood even under hypoxic conditions.     

Catabolic Activities of Neisseria meningitidis: Utilization of Succinate

Two strains of Saccharomycopsis guttulata, JB-1 and JB-3, isolated from stomach contents of domestic rabbits, were grown under different gas phases, and their growth rates were compared. Strain JB-1 grew exponentially at a maximal growth rate under a continuous gas phase of 15% CO₂, 2% O₂ in nitrogen. High cell yields with low cell granulation were obtained. The growth rates were almost the same between oxygen concentrations of 0.25 and 20% at 15% CO₂. Poor growth and early cell granulation occurred in the absence of oxygen at 15% CO₂. Growth increased at 2% O₂ in direct proportion to the carbon dioxide concentration up to 10 to 15% CO₂. A very high carbon dioxide content (e.g. 98%) was somewhat inhibitory. Cell granulation always occurred during the maximal stationary phase in media at pH 4, but was relatively slight at pH 5.6 or higher. Strain JB-3 responded to various gas phases in a similar manner except that it grew slowly in the absence of oxygen at 15% CO₂ (pH 4). The effect of an optimal gas phase on the growth of strain JB-1 was examined in relation to other environmental conditions. In the presence of 15% CO₂, 2% O₂, this strain grew exponentially in yeast autolysate-Proteose Peptone-glucose medium at 37 C at pH 2, 4, and 5.6 at approximately the same rate; the growth rate was somewhat lower at pH 6.2. Under similar conditions, strain JB-1 grew at 30 C and pH 4 at one-sixth its maximal growth rate. Cell granulation was greatly reduced at this temperature. With adequate CO₂ strain JB-1 also grew at a reduced rate in a yeast autolysate medium previously reported not to support growth. Results indicate that continuous gassing with an optimal gas phase increases the growth rate to the extent that the growth rate surpasses the death rate by a significant margin; as a result, granulated cells can be avoided almost entirely in the log phase.     

Spatio-temporal relief from hypoxia and production of reactive oxygen species during bud burst in grapevine (Vitis vinifera)

Background and Aims Plants regulate cellular oxygen partial pressures (pO₂), together with reduction/oxidation (redox) state in order to manage rapid developmental transitions such as bud burst after a period of quiescence. However, our understanding of pO₂ regulation in complex meristematic organs such as buds is incomplete and, in particular, lacks spatial resolution.Methods The gradients in pO₂ from the outer scales to the primary meristem complex were measured in grapevine (Vitis vinifera) buds, together with respiratory CO₂ production rates and the accumulation of superoxide and hydrogen peroxide, from ecodormancy through the first 72 h preceding bud burst, triggered by the transition from low to ambient temperatures.Key Results Steep internal pO₂ gradients were measured in dormant buds with values as low as 2·5 kPa found in the core of the bud prior to bud burst. Respiratory CO₂ production rates increased soon after the transition from low to ambient temperatures and the bud tissues gradually became oxygenated in a patterned process. Within 3 h of the transition to ambient temperatures, superoxide accumulation was observed in the cambial meristem, co-localizing with lignified cellulose associated with pro-vascular tissues. Thereafter, superoxide accumulated in other areas subtending the apical meristem complex, in the absence of significant hydrogen peroxide accumulation, except in the cambial meristem. By 72 h, the internal pO₂ gradient showed a biphasic profile, where the minimum pO₂ was external to the core of the bud complex.Conclusions Spatial and temporal control of the tissue oxygen environment occurs within quiescent buds, and the transition from quiescence to bud burst is accompanied by a regulated relaxation of the hypoxic state and accumulation of reactive oxygen species within the developing cambium and vascular tissues of the heterotrophic grapevine buds.     

Microscale oxygen distribution in various invertebrate burrow walls

Profiles of dissolved oxygen were measured in pore waters of unburrowed sediment and the burrow walls of seven invertebrate dwellings. Burrows studied include those of Corophium volutator, Heteromastus filiformis, Arenicola marina, Saccoglossus bromophenolosus, Clymenella sp., Hemigrapsus oregonensis and Cirriformia luxuriosa all from mudflats in Willapa Bay, Washington. These animals comprise a range of burrow architectures ranging from simple, unlined burrows to more complex, mucous lined burrows. Oxygen penetrated unburrowed sediment between depths of 0.4–2.6 mm, whereas oxygen penetrated the burrow walls from 0.3 mm to 2.3 mm. Three groups of burrows are recognized based on the oxygen diffusive properties relative to the unburrowed sediment including those that: (1) slightly impeded oxygen penetration, (2) clearly inhibited oxygen penetration, and (3) enhanced oxygen penetration. Differences in the diffusive properties of the burrow wall are related to the burrow microstructure and presumably the microbial communities living within the burrow microenvironment. The results of this study suggest that burrow shape and burrow‐wall architecture may play an important role in controlling the diffusion of oxygen, and possibly of other dissolved gases (i.e. CO₂, H₂S). The results further demonstrate that simplified assumptions (i.e. that bioturbation uniformly enhances oxygen diffusion into suboxic and anoxic sediments), while requisite for numerical modelling, are not necessarily representative of field data.     

The effects of temperature and salinity on ventilation behavior of two species of ghost shrimp (Thalassinidea) from the northern Gulf of Mexico: a laboratory study

Thalassinidean shrimp are among the most important bioturbators in coastal ecosystems. The species Lepidophthalmus louisianensis and Callichirus islagrande are found in dense aggregations (up to 400 burrows m⁻²) along sandy and muddy shores of the northern Gulf of Mexico. These shrimp actively ventilate their burrows to provide oxygen and eliminate wastes. In doing so, they expel nutrient-rich burrow water to the overlying water column, potentially altering nutrient cycling and benthic primary productivity. To develop a mechanistic understanding of the role of burrowing shrimp in nutrient processes, we must first examine how changes in environmental conditions alter the frequency, strength, and duration of ventilation. Field measurements of burrow temperature and salinity suggest that the burrow serves as a buffer from the highly variable conditions found in these estuarine, intertidal habitats. Temperatures at sediment depths >30 cm were generally warmer in winter and cooler in summer than at the sediment surface. Burrow salinities, measured at low tide, were consistently higher than adjacent open water. We used these measurements to parameterize laboratory studies of burrow ventilation in artificial burrows made of plastic tubing and in more natural sediment mesocosms, and studies of oxygen consumption in small glass containers. Rates of oxygen consumption and burrow ventilation by L. louisianensis were lower than those of C. islagrande, perhaps reflecting a lower overall activity rate in the former species which resides in less permeable sediments. Generally, increased temperature had a significant positive effect on oxygen consumption for both species. Salinity had no effect on oxygen consumption by L. louisianensis, reflecting the ability of this species to exist in a wide range of salinities. In contrast, oxygen consumption rates of C. islagrande, which is less tolerant of low salinity, were significantly higher at 35‰ than at 20‰. Ventilation rates were highly variable, and shrimp in artificial burrows tended to have consistently higher ventilation rates than those in sediment mesocosms. There is a trend toward more frequent ventilation at 30 °C for both species. Salinity had no effect on ventilation for either species. Our results suggest that thalassinideans exhibit highly variable and species-specific ventilation patterns that are more likely to be affected by temperature than salinity. Increased ventilation at higher temperatures seems to coincide with increased oxygen consumption at these temperatures, although a similar finding was not made for salinity treatments.     

Use of models in understanding the mechanism of action of haemoglobin

Models with three, four and eight salt-bridges have been used to study the mechanism of action of haemoglobin. Both side chains forming a salt-bridge, i.e. the proton acceptor and the proton donor, are postulated to change pK on ligation of oxygen. The eight salt-bridge model is able to predict, as a unified theory, both the degree of oxygenation and the Bohr effect at any PH and p_O2 value; this has not been done by any other published model. The predicted pK values for the Borh groups corresponde well with those measured experiemntally. This model predicts the pK values of those side chains responsible for the acid Bohr effect, suggesting that these correspond to the proton acceptors of the salt-bridges. The model also fulfils the condition of linearity between the fractional degree of oxygenation and fractional number of protons released. It is postulated that there is a gradual change in structure on going from deoxy to oxyhaemoglobin, due to the rupture of salt-bridges. The path folowed during this process will be both pH and p_O2 dependent. A formula describing the number of intact or broken salt-bridges as a function of pH and p_O2 was developed. This formula shows that the fractional number of broken salt-bridges reaches a minimum value of 0.2 at around pH 6.3 in the absence of oxygen. However, if oxygen is added, this fractional number approaches 1.0 soon after the partial pressure of oxygen goes above 40 mm Hg.