O2-filled swimbladder employs monocarboxylate transporters for the generation of O2 by lactate-induced root effect hemoglobin

The swimbladder volume is regulated by O(2) transfer between the luminal space and the blood In the swimbladder, lactic acid generation by anaerobic glycolysis in the gas gland epithelial cells and its recycling through the rete mirabile bundles of countercurrent capillaries are essential for local blood acidification and oxygen liberation from hemoglobin by the "Root effect." While O(2) generation is critical for fish flotation, the molecular mechanism of the secretion and recycling of lactic acid in this critical process is not clear. To clarify molecules that are involved in the blood acidification and visualize the route of lactic acid movement, we analyzed the expression of 17 members of the H(+)/monocarboxylate transporter (MCT) family in the fugu genome and found that only MCT1b and MCT4b are highly expressed in the fugu swimbladder. Electrophysiological analyses demonstrated that MCT1b is a high-affinity lactate transporter whereas MCT4b is a low-affinity/high-conductance lactate transporter. Immunohistochemistry demonstrated that (i) MCT4b expresses in gas gland cells together with the glycolytic enzyme GAPDH at high level and mediate lactic acid secretion by gas gland cells, and (ii) MCT1b expresses in arterial, but not venous, capillary endothelial cells in rete mirabile and mediates recycling of lactic acid in the rete mirabile by solute-specific transcellular transport. These results clarified the mechanism of the blood acidification in the swimbladder by spatially organized two lactic acid transporters MCT4b and MCT1b.     

The active transport of oxygen and carbon dioxide into the swim-bladder of fish

The active transport of oxygen and carbon dioxide into the swim-bladder of fish is discussed. The rete mirabile is a capillary network which is involved in the gas secretion into the bladder. The rete is regarded as a counter-current multiplier. Lactic acid which is produced in the gas gland generates in the rete single concentrating effects for oxygen and carbon dioxide; i.e., for equal partial pressures the concentrations of the gases in the afferent rete capillaries are higher than those in the efferent ones. The single concentrating effects were calculated from measurements of sea robin blood (Root, 1931). The multiplication of these effects within the rete for different rete lengths and different transport rates was numerically evaluated. The calculated O₂ and CO₂ pressures in the bladder are in good agreement with the experimental results of Scholander and van Dam (1953). The descent velocities at equilibrium between bladder pressure and hydrostatic pressure are discussed for fishes with different rete lengths.     

Histological demonstration of glucose transporters,fructose-1,6-bisphosphatase,and glycogen in gas gland cells of the swimbladder: Is a metabolic futile cycle operating?

Luminal surface of the swimbladder is covered by gas gland epithelial cells and is responsible for inflating the swimbladder by generating O(2) from Root-effect hemoglobin that releases O(2) under acidic conditions. Acidification of blood is achieved by lactic acid secreted from gas gland cells, which are poor in mitochondria but rich in the glycolytic activity. The acidic conditions are locally maintained by a countercurrent capillary system called rete mirabile. To understand the regulation of anaerobic metabolism of glucose in the gas gland cells, we analyzed the glucose transporter expressed there and the fate of ATP generated by glycolysis. The latter is important because the ATP should be immediately consumed otherwise it strongly inhibits the glycolysis rendering the cells unable to produce lactic acid anymore. Expression analyses of glucose transporter (glut) genes in the swimbladder of fugu (Takifugu rubripes) by RT-PCR and in situ hybridization demonstrated that glut1a and glut6 are expressed in gas gland cells. Immunohistochemical analyses of metabolic enzymes demonstrated that a gluconeogenesis enzyme fructose-1,6-bisphosphatase (Fbp1) and a glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (Gapdh) are highly expressed in gas gland cells. The simultaneous catalyses of glycolysis and gluconeogenesis reactions suggest the presence of a futile cycle in gas gland cells to maintain the levels of ATP low and to generate heat that helps reduce the solubility of O(2).     

The role of interphase nitrogen transport in the dynamic measurement of the overall volumetric mass transfer cefficient in air-sparged systems

It has been shown both theoretically and experimentally that interphase nitrogen transport may have a significant influence on the rate of interphase oxygen transport, and thereby also on the value of the volumetric mass transfer coefficient of oxygen, k_la, determined in mechanically agitated bubble fermentors using the variants of dynamic method presented in the literature. The experiments were carried out in 1M KCI solution at five stirrer frequencies and two gas inlet levels. The gas interchanges were performed either without interrupting the aeration and agitation of the charge (A) or with the aeration and agitation of the charge turned on at the same time (B). The applied variants of the interchange were N₂→ O₂→, O₂→ N₂, N₂→ air, air→ N₂, O→ O₂, and O→ air. In the two last variants the oxygen dissolved in the charge was removed by reacting with sulfite ions. The k_la values calculated by allowing for the nitrogen transport for procedure A were approximately equal to the values obtained by disregarding the nitrogen transport, whereas those for procedure B were higher (up to 40%), than the values obtained disregarding the nitrogen transport.     

A method to estimate net community metabolism from profiles of dissolved O2 and N2

A method to estimate net community metabolism (NCM) in natural waters using vertical profiles of water temperature, salinity, dissolved O₂, gas tension, and calculated dissolved N₂ is presented. The method utilizes the disparate biological activity of dissolved O₂ and N₂ to estimate metabolism at different depths in the water column. For well-mixed surface waters, N₂ saturation levels are assumed to be the result of a quasi steady state balance of net warming or cooling and air–water gas exchange. Dissolved O₂ levels are assumed to maintain a similar balance, subject to net biological activity, and NCM is then calculated based on the difference between N₂ and O₂ saturation levels and the estimated timescale required to equilibrate the layer with the atmosphere. For deeper stratified layers of water that warmed after layer formation in isolation from the atmosphere, the temperature at formation is calculated using the measured N₂ concentration and an assumed N₂ saturation level of 100% at formation. By assuming that initial N₂ and O₂ saturation levels were equal, the initial O₂ concentration is calculated based on solubility relationships. NCM of the deeper waters is then estimated based on this information and knowledge of the general seasonal heating cycle of the waters. Daily mean water temperature and dissolved gas levels are used in the calculations. The method was assessed using profile measurements collected at Long Pond, Plymouth, Massachusetts, USA, on 23 August 2002. Oxygen was supersaturated relative to N₂ by approximately 4% in the 0–6 m deep epilimnion, and undersaturated relative to N₂ by approximately 7% in the stratified water at 9 m depth. The estimated 4-day average NCM for the epilimnion was 140 ± 70 mgC m⁻²day⁻¹. For waters at 9 m depth, the temperature at formation was calculated to be 6.58 °C, and the estimated 100-day average NCM was −2.5 ± 0.6 mgC m⁻³ day⁻¹. An independent estimate of −4.6 ± 0.9 mgC m⁻³ day⁻¹ was derived from the measured O₂ decline at 9 m depth over the same period of 2003.     

Buoyancy control of four species of eleotrid fishes during aquatic surface respiration

Synopsis Four species of Australian Eleotridae from hypoxic habitats were examined in the laboratory to study buoyancy control in hypoxic water (<10 torr) when performing aquatic surface respiration (ASR; irrigating gills with upper millimeter of surface water). A conflict can arise here because O₂ can be reabsorbed from the swimbladder (reducing buoyancy) at a time when additional lift may be required to perform ASR. Three species were negatively buoyant and initially performed ASR while resting on the bottom in shallow water. After 24 h swimbladder lift increased to nearly neutral and ASR was performed while fish were pelagic. The fourth species remained pelagic at near neutral buoyancy in hypoxic water. With sudden exposure to hypoxia these physoclists reabsorbed between 5–27% (depending on species) of swimbladder volume (standard pressure) during the initial 30–90 min exposure to hypoxia. Additional experiments on one species (Hypseleotris galii) showed such loss to occur at O₂ tensions below 68 torr and when O₂ declined rapidly (2.17 torr min^-1). Secretion of gas compensated for losses under slower, natural rates of nocturnal O₂ decline. Eleotrids appear to reduce the conflict between respiration and buoyancy control in hypoxia by restricting gas reabsorbtion from the swimbladder and by rapidly secreting gases into the swimbladder.     

Effects of ethanol and 3,4,-dihydron-2,2-dimethyl-2h-1-benzopyran-6-butyric acid on the solubility of sickle hemoglobin.

The effect of 3,4-dihydro-2,2-dimethyl-2H-1 benzopyran-6-butyric acid (DBA) on the solubility of deoxy-Hb S was evaluated by measuring saturation concentration, c_sat. Plots of c_sat versus DBA concentration in the presence or absence of ethanol gave two parallel lines, indicative of the additive fashion in which ethanol and DBA increase the solubility of deoxy-Hb S. At a $\text{DBA}\text{Hb}$ molar ratio of 10:1, c_sat was increased 16%. Ethanol alone increased c_sat comparably, but at a much higher molar excess (200:1). DBA had no effect on the oxygenation parameters of Hb S. Complementary solubility studies using the salting-out method showed that DBA had no effect on deoxy-Hb S, but decreased the solubility of deoxy-Hb A, oxy-Hb A and oxy-Hb S. Hence, no correlation exists between the effect of DBA on the solubility of deoxy-Hb S measured as c_sat and that measured by the salting-out technique.     

ANATOMY OF THE SWIMBLADDERS OF SEVEN FAMILIES OF TRANSVAAL FRESHWATER FISHES

SUMMARY

IN the genera Barbus and Labeo of the family Cyprinidae there is a typical twolobed, cylindrical swimbladder: a shorter anterior and a longer posterior lobe, connected by an isthmus. The pneumatic duct passes from the anteroventral end of the posterior lobe to the oesophagus. In the genus Labeo two spiral bands encircle the posterior lobe twice. No rete mirabile, nor any indication of a gas gland, was observed.

The species Hydrocynus vittutus of the family Characidae has a very similarly shaped swimbladder to that of the Cyprinidea. Inside the anterior lobe, however, there is a peculiar structure, which is evidently the gas glad, although a rete mirabile was not observed.

In the families of the Siluriformes, studied, with the exception of the Clariidae, a single lobed, heartshaped swimbladder is present. It is divided by a longitudinal and a transverse. septum into three chambers: an anterior, a right and a left posterior chamber. The pneumatic duct originates from the medial posteroventral part of the anterior chamber. In Clarias gariepinus the two-lobed, right and left lobed, swimbladder lies in a bony capsule, which is attached transversely to the posteroventral part of the skull. In all the Siluriformes, studied, no trace of a gas gland, nor of a rete mirabile was found.

The Cichlid swimbladder has no pneumatic duct, nor any other exit, hence it is physoclistic. In the Cichlids the retroperitoneal position of the swimbladder is accentuated, as the peritoneum and the outer tectum of the swimbladder have united to form a thick, tough membrane, which divides the body cavity into a distinct ventral, or visceral cavity, and a dorsal, or swimbladder cavity. The swimbladder cavity acts as an outer swimbladder. It contains an inner, smaller bladder whose internal ventro-anterior surface is covered with arborescently arranged patches of gas glands.

The attachment of the swimbladder to the tripus and also to the ossa suspensoris is discussed.     

Nitrogen Fixation and Hydrogen Metabolism in Relation to the Dissolved Oxygen Tension in Chemostat Cultures of the Wild Type and a Hydrogenase-Negative Mutant of Azorhizobium caulinodans

Both the wild type and an isogenic hydrogenase-negative mutant of Azorhizobium caulinodans growing ex planta on N₂ as the N source were studied in succinate-limited steady-state chemostat cultures under 0.2 to 3.0% dissolved O₂ tension. Production or consumption of O₂, H₂, and CO₂ was measured with an on-line-connected mass spectrometer. In the range of 0.2 to 3.0%, growth of both the wild type and the mutant was equally dependent on the dissolved O₂ tension: the growth yield decreased, and the specific O₂ consumption and CO₂ production increased. A similar dependency on the dissolved O₂ tension was found for the mutant with 2.5% H₂ in the influent gas. The H₂/N₂ ratio (moles of H₂ evolved per mole of N₂ consumed via nitrogenase) of the mutant, growing with or without 2.5% H₂, increased with increasing dissolved O₂ tensions. This increase in the H₂/N₂ ratio was small but significant. The dependencies of the ATP/N₂ ratio (moles of ATP consumed per mole of N₂ fixed) and the ATP/2e^- ratio [moles of ATP consumed per mole of electron pairs transferred from NAD(P)H to nitrogenase] on the dissolved O₂ tension were estimated. These dependencies were interpreted in terms of the physiological concepts of respiratory protection and autoprotection.     

Diazotrophic growth of Rhodopseudomonas acidophila and Rhodopseudomonas capsulata under microaerobic conditions in the dark

Diazotrophy of Rhodopseudomonas acidophila and Rhodopseudomonas capsulata was not obligatorily linked to photosynthesis. In the dark R. acidophila grew with dinitrogen as sole nitrogen source at a dissolved oxygen tension of 15 Torr (= 2.0 kPa); the doubling time was 8 h. Acetylene reduction by whole cells was more sensitive to oxygen in the light than in the dark. 16.5 mg N₂ were fixed per g lactic acid consumed. R. capsulata synthesized nitrogenase and fixed dinitrogen in the dark at a dissolved oxygen tension of less than one Torr (= 0.13 kPa). The doubling time of this bacterium was 16 h and 10.5 mg N₂ were fixed per g lactic acid consumed.Abbreviation kPa kilopascal     

Accumulation of organic acids in cultivations of Neisseria meningitidis C

Aiming at the industrial production of serogroup C meningococcal vaccine, different experimental protocols were tested to cultivate Neisseria meningitidis C and to investigate the related organic acid release. Correlations were established between specific rates of acetic acid and lactic acid accumulation and specific growth rate, during cultivations carried out on the Frantz medium in a 13 l bioreactor at 35°C, 0.5 atm, 400 rpm and air flowrate of 2 l min⁻¹. A first set of nine batch runs was carried out: (1) with control of dissolved oxygen (O₂) at 10% of its saturation point, (2) with control of pH at 6.5, and (3) without any control, respectively. Additional fed-batch or partial fed-batch cultivations were performed without dissolved O₂ control, varying glucose concentration from 1.0 to 3.0 g l⁻¹, nine of which without pH control and other two with pH control at 6.5. No significant organic acid level was detected with dissolved O₂ control, whereas acetic acid formation appeared to depend on biomass growth either in the absence of any pH and dissolved O₂ control or when the pH was kept at 6.5. Under these last conditions, lactic acid was released as well, but it did not seem to be associated to biomass growth. A survey of possible metabolic causes of this behavior suggested that N. meningitidis may employ different metabolic pathways for the carbon source uptake depending on the cultivation conditions.     

Influence of O2 availability on NO and N2O release by nitrification and denitrification in soils

The availability of O₂ is believed to be one of the main factors regulating nitrification and denitrification and the release of NO and N₂O. The availability of O₂ in soil is controlled by the O₂ partial pressure in the gas phase and by the moisture content in the soil. Therefore, we investigated the influence of O₂ partial pressures and soil moisture contents on the NO and N₂O release in a sandy and a loamy silt and differentiated between nitrification and denitrification by selective inhibition of nitrification with 10 Pa acetylene. At 60% whc (maximum water holding capacity) NO and N₂O release by denitrification increased with decreasing O₂ partial pressure and reached a maximum under anoxic conditions. Under anoxic conditions NO and N₂O were only released by denitrification. NO and N₂O release by nitrification also increased with decreasing O₂ partial pressure, but reached a maximum at 0.1–0.5% O₂ and then decreased again. Nitrification was the main source of NO and N₂O at O₂ partial pressures higher than 0.1–0.5% O₂. At lower O₂ partial pressures denitrification was the main source of NO and N₂O. With decreasing O₂ partial pressure N₂O release increased more than NO release, indicating that the N₂O release was more sensitive against O₂ than the NO release. At ambient O₂ partial pressure (20.5% O₂) NO and N₂O release by denitrification increased with increasing soil moisture content. The maximum NO and N₂O release was observed at soil moisture contents of 65–80% whc and 100% whc, respectively. NO and N₂O release by nitrification also increased with increasing soil moisture content with a maximum at 45–55% whc and 90% whc, respectively. Nitrification was the main source of NO and N₂O at soil moisture contents lower than 90% whc and 80% whc, respectively. Higher soil moisture contents favoured NO and N₂O release by denitrification. Soil texture had also an effect on the release of NO and N₂O. The coarse-textured sandy silt released more NO than N₂O compared with the fine-textured loamy silt. At high soil moisture contents (80–100% whc) the fine-textured soil showed a higher N₂O release by denitrification than the coarse-textured soil. We assume that the fine-textured soil became anoxic at a lower soil moisture content than the coarse-textured soil. In conclusion, the effects of O₂ partial pressure, soil moisture and soil texture were consistent with the theory that denitrification increasingly contributes to the release of NO and in particular N₂O when conditions for soil microorganisms become increasingly anoxic.     

Entrance and Growth of Lactic Acid Bacteria in Gas-Exchanged, Brined Cucumbers

Entrance of lactic acid bacteria into the interior of brined cucumbers was found to be greatly influenced by gas composition of the cucumbers before brining. Exchange of the internal gas of fresh cucumbers with O₂ resulted in absorption of bacteria into the subsequently brined fruit within a few hours. Bacteria were absorbed into nonexchanged cucumbers to a lesser extent. Little bacterial absorption occurred in N₂-exchanged cucumbers. Stomata of the cucumber skin appeared to be a likely port for bacterial entry. When Pediococcus cerevisiae or Lactobacillus plantarum cells were added to the brine of O₂-exchanged cucumbers, the respective cell types colonized in large numbers within intercellular spaces and vascular elements of mesocarp tissue during fermentation of the cucumbers. Implications of these observations, particularly with regard to bloater formation in brined cucumbers, are discussed.     

Delivery of N2 to bacteroids in simulated soybean nodule cells: consideration of gradients of concentration of dissolved N2 in cell walls,cytoplasm, and symbiosomes

Summary The previously published simulation of physiological functions occurring in infected cells of soybean nodules has been extended to include consideration of the diffusion of N₂ from the outside of a nodule to the nitrogen-fixing bacteroids, in relation to published values for the apparentK _m(N₂) for nitrogen fixation in the soybean nodule system. Nitrogen fixation is driven by bacteroid respiration, so increases in the average relative oxygenation (Y) of cytoplasmic leghaemoglobin lead to increased bacteroid respiration, increased nitrogen fixation, and greater differences in concentration of dissolved N₂ between the cell surface and the innermost bacteroids (d[N₂]). Over the range ofY considered, values for d[N₂] were from 5.2- to 6.2-fold greater than the corresponding values for d[O₂], because of facilitation of O₂ flux by cytoplasmic leghaemoglobin. Gradients of [N₂] within symbiosomes are small relative to cytoplasmic values and at the symbiosome surface [N₂] was greater than 0.4 mol/m³ at the greatest rates of nitrogen fixation calculated. Therefore, it is unlikely that values for [N₂] anywhere in the infected cell are low enough to affect rates of nitrogen fixation significantly, unless low external atmospheric N₂ pressures are used experimentally.Abbreviations Lb leghaemoglobin - LbO₂ oxyleghaemoglobin - [O₂], [N₂ concentrations of free, dissolved oxygen and nitrogen - Y fractional oxygenation of leghaemoglobin     

A Mathematical Model of the Controlled Plant of the Réspiratory System

Ability to predict the dynamic response of oxygen, carbon dioxide tensions, and pH in blood and tissues to abrupt changes in ventilation is important in the mathematical modeling of the respiratory system. In this study, the controlled plant (the amount and distribution of O₂ and CO₂) of the respiratory system is modeled. Although the body tissues are divided into a finite number of “compartments” (three tissue groups), in contrast to earlier models, the blood and tissue gas tensions within each compartment are considered to be continuously distributed in time and in one spatial coordinate. The mass conservation equations for oxygen and carbon dioxide involved in the blood-tissue gas exchange are described by a set of partial differential equations which take into account convection of O₂ and CO₂ caused by the flow of blood as well as diffusion due to local tension gradients. Nonlinear algebraic equations for the dissociation curves, which take into account the Haldane and Bohr effects in blood, are used to obtain the relationships between concentrations and partial pressures. Time-variable delays caused by the arterial and venous transport of the respiratory gases are also included. The model so constructed successfully reproduced actual O₂ and CO₂ tensions in arterial blood, and in muscle venous and mixed venous blood when ventilation was abruptly changed.     

Survival of Salmonella enteritidis and Listeria monocytogenes on salad vegetables

The influence of initial head-spaces of air – 4.9% CO₂/2.1% O₂/93% N₂ and 5% CO₂/5.2% O₂/89.8% N₂ – on Salmonella enteritidis and Listeria monocytogenes, and on microbial association with shredded carrots and lettuce was studied at 4 °C. Both these pathogens survived but did not grow in any vegetable regardless of the packaging system used. Total viable count, lactic acid bacteria and pseudomonads were also monitored. Lactic acid bacteria were the predominant organisms in all samples. The pH dropped significantly during the storage of vegetables.     

Modulation of epithelial cell proliferation in culture by dissolved oxygen

Modulation of epithelial cell proliferation by the dissolved oxygen concentration (P_O2) of the growth medium was assessed with primary human foreskin epithelium and a continuous monkey kidney epithelial cell line (LLC-MK₂). Direct measurement of the growth medium P_O2 provides the first quantitative evaluation of epithelial cell proliferation as a function of P_O2 provides the first quantitative evaluation of epithelial cell proliferation as a function of P_O2. Sustained proliferation of LLC-MK₂ cells occurs in serum-free medium equilibrated with a gas phase containing 18% or 30% O₂ v/v. Mid-logarithmic phase cultures rapidly consume dissolved oxygen; this results in a 60–70 mm Hg decline in P_O2 and leads to a stable growth medium P_O2 between 70 and 100 mm Hg, well above anoxic values. In contrast, if culture medium is equilibrated with a gas phase containing 0% or 1% O₂ v/v to yield a growth medium P_O2 ～ 20–40 mm Hg, proliferation of LLC-MK₂ and primary foreskin epithelial cells is retarded, and LLC-MK₂ cells use little dissolved oxygen. Gentle, continuous rocking to prevent diffusion gradient formation enhances proliferation slightly at the higher P_O2, but neither periodic fluid renewals nor continued rocking stimulates cells retarded by a lowered oxygen concentration to resume proliferation. The data collectively demonstrate that epithelial cell proliferation requires a P_O2 > 40 mm Hg, and threshold requirements are probably closer to 70 mm Hg. Glycolysis continues at a P_O2 insufficient for proliferation, but more lactic acid accumulates in actively proliferating cultures than in cultures equilibrated with 0% oxygen. We conclude that epithelial cells in vitro both consume more oxygen and require a higher P_O2 for continued proliferation, and that the oxygen requirement for epithelial cell proliferation exceeds that of a comparable population of fibroblasts for which low oxygen may enhance survival and proliferation.     

Effects of various partial pressures of oxygen and carbon dioxide on different stages of somatic embryogenesis in Picea abies

Effects of various partial pressures of oxygen (5, 20 and 45 kPa) and carbon dioxide (0.03 and 6 kPa) on initiation, proliferation and maturation of somatic embryos in Picea abies were studied. The pO₂ had a significant effect on the initiation of embryogenic tissue from mature zygotic embryos. However, the effect of pO₂ was dependent on the strength of the basal medium. Low pO₂ stimulated the formation of embryogenic tissue when the zygotic embryos were incubated on full strength medium, but was inhibitory when half-strength medium was used. Proliferation of embryogenic tissue was stimulated by higher partial pressures of both CO₂ and O₂. The effect of the gas phase on maturation of somatic embryos varied between different cell lines. However, there was a general tendency for 5 kPa O₂ and 6 kPa CO₂ to stimulate maturation.Abbreviations ABA abscisic acid - BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - ET embryogenic tissue     

Hydrogen production in anaerobic and microaerobic Thermotoga neapolitana

We have tested the hypothesis (Van Ooteghem et al. Appl Biochem Biotechnol 2002 98–100: 177–189) that microaerobic metabolism may increase the yield of H₂ from the thermophilic bacterium Thermotoga neapolitana. In anaerobic conditions, T. neapolitana converted glucose into acetic acid and lactic acid and yielded 2.4 ± 0.3 mol H₂ mol⁻¹ glucose. The bacterium tolerated low O₂ partial pressures but the H₂ yield was not improved under microaerobic conditions. Our results indicate that T. neapolitana only produces H₂ by anaerobic metabolism, and that the yield of H₂ can be maximised by minimising the production of lactic acid.