Symbioses between salamander embryos and green algae

The symbiosis between Ambystoma maculatum (spotted salamander) embryos and green algae was initially described over 120 years ago. Algae populate the egg capsules that surround individual A. maculatum embryos, giving the intracapsular fluid a characteristic green hue. Early work established this symbiosis to be a mutualism, while subsequent studies sought to identify the material benefits of this association to both symbiont and host. These studies have shown that salamander embryos benefit from increased oxygen concentrations provided by their symbiotic algae. The algae, in turn, may benefit from ammonia excreted by the embryos. All of these early studies considered the association to be an ectosymbiotic mutualism. However our recent work has shown that algae invade both embryonic salamander cells and tissues during development. The unexpected invasion of algal cells into a salamander host changes our understanding of this symbiosis. This review will summarize the earlier research on this association in the context of these recent findings. It will also emphasize gaps in our understanding of this and other amphibian embryo-algal interactions and suggest various research avenues to address these unanswered questions.     

Ammonia excretion via Rhcg1 facilitates Na⁺ uptake in larval zebrafish, Danio rerio, in acidic water

The involvement of a Na(+)/H(+) exchanger (NHE) in mediating Na(+) uptake by freshwater fish is currently debated. Although supported indirectly by empirical molecular and pharmacological data, theoretically its operation should be constrained thermodynamically, owing to unfavorable chemical gradients. Recently, there has been an increasing focus on ammonia channels (Rh proteins) as potentially contributing to Na(+) uptake across the freshwater fish gill. In this study, we tested the hypothesis that Rhcg1, a specific apical isoform of Rh protein, is critically important in facilitating Na(+) uptake in zebrafish larvae via its interaction with NHE. Treating larvae (4 days postfertilization) with 5-(N-ethyl-N-isopropyl) amiloride (EIPA), an inhibitor of NHE, caused a significant reduction in Na(+) uptake in fish reared in acidic water (pH ～ 4.0). A role for NHE in Na(+) uptake was further confirmed by translational knockdown of NHE3b, an isoform of NHE thought to be responsible for Na(+)/H(+) exchange in zebrafish larvae. Exposing the larvae reared in acidic water to 5 mM external ammonium sulfate or increasing the buffering capacity of the water with 10 mM HEPES caused concurrent reductions in ammonia excretion and Na(+) uptake. Furthermore, translational knockdown of Rhcg1 significantly reduced ammonia excretion and Na(+) uptake in larvae chronically (4 days) or acutely (24 h) exposed to acidic water. Unlike in sham-injected larvae, EIPA did not affect Na(+) uptake in fish experiencing Rhcg1 knockdown. Additionally, exposure of larvae to bafilomycin A1 (an inhibitor of H(+)-ATPase) significantly reduced Na(+) uptake in fish reared in acidic water. These observations suggest the existence of multiple mechanisms of Na(+) uptake in larval zebrafish in acidic water: one in which Na(+) uptake via NHE3b is linked to ammonia excretion via Rhcg1, and another facilitated by H(+)-ATPase.     

Interaction and inhibitory effect of ammonium cation in the oxygen evolving center of photosystem II

Photosynthetic O(2) evolution takes place at the Mn cluster in photosystem II (PSII) by oxidation of water. It has been proposed that ammonia, one of water analogues, functions as an inhibitor of O(2) evolution at alkaline pH. However, the detailed mechanism of inhibition has not been understood yet. In this study, we investigated the mechanism of ammonia inhibition by examining the NH(4)Cl-induced inhibition of O(2) evolution in a wide pH range (pH 5.0-8.0) and by detecting the interaction site using Fourier transform infrared (FTIR) spectroscopy. In addition to intact PSII membranes from spinach, PSII membranes depleted of the PsbP and PsbQ extrinsic proteins were used as samples to avoid the effect of the release of these proteins by salt treatments. In both types of samples, oxygen evolution activity decreased by approximately 40% by addition of 100 mM NH(4)Cl in the range of pH 5.0-8.0. The presence of inhibition at acidic pH without significant pH dependence strongly suggests that NH(4)(+) cation functions as a major inhibitor in the acidic pH region, where neutral NH(3) scarcely exists in the buffer. The NH(4)Cl treatment at pH 6.5 and 5.5 induced prominent changes in the COO(-) stretching regions in FTIR difference spectra upon the S(1) → S(2) transition measured at 283 K. The NH(4)Cl concentration dependence of the amplitude of the spectral changes showed a good correlation with that of the inhibition of O(2) evolution. From this observation, it is proposed that NH(4)(+) cation interacts with carboxylate groups coupled to the Mn cluster as direct ligands or proton transfer mediators, causing inhibition of the O(2) evolving reaction.     

A METHOD FOR THE REMOVAL OF THE JELLY AND VITELLINE MEMBRANE FROM THE EMBRYOS OF XENOPUS LAEVIS

A simple procedure is described for removing the jelly and vitelline membrane of Xenopus laevis embryos. The method is based on the observation that incubation of the embryos in the mixed solution of trypsin and sodium thioglycolate at pH 8.0 causes effective dissolution of these structures. This solution is equally effective in this respect on the embryos at different developmental stages. Normal development is obtained from all of the denuded neurulae and from many of the denuded earlier embryos. Some chemical properties of the jelly and the vitelline membrane of Xenopus laevis are discussed based upon these observations.     

The effects of ambient pH on nitrogen excretion in early life stages of the American toad (Bufo americanus)

Acidification of breeding ponds has been identified as a potential threat to the survival and health of North American amphibian populations. The effects of acid exposure on ion and acid-balance are well known, but there is little information on how environmental water pH influences nitrogen balance in amphibians. The aim of this study was to determine the effects of moderately acidic water (pH 6.0) on nitrogen excretion in early life stages of the toad, Bufo americanus. Acid exposure (pH 6.0, 54 h) resulted in a 20–80% increase in ammonia-N excretion rates in embryos and early, middle and late tadpoles stages, whereas there was no significant effect on urea-N excretion. Tissue ammonia concentrations were significantly higher (+ 33%) in the embryos and 35–65% lower in the three groups of tadpoles exposed to water of pH 6.0 compared to control animals (pH 8.5). In embryos, ammonia excretion accounted for greater than 90% of total nitrogen excretion (ammonia-N + urea-N), but by the late tadpole stage this value had decreased to approximately 65%. These findings indicate that exposure of embryonic and larval B. americanus to moderately acidic water disrupts nitrogen balance by increasing nitrogen loss as ammonia, with no compensatory decrease in urea excretion.     

Effect of external pH on inorganic carbon assimilation in unicellular marine green algae

Carbonic anhydrase (CA) induction has been studied in three marine green algae under acidic (pH 4.5) or alkaline (pH 8.0) conditions. An inhibition of the induction of the external CA in acidic conditions, similar to that observed in some freshwater green algae, could be observed in only Chlorella saccharophila. In the two other species, Chlorococcum littorale and Stichococcus bacillaris, no significant difference in CA induction was found under two pH conditions. The exact function of the external CA of C. saccharophila remains unclear, since cells grown under acidic conditions (under which this enzyme is repressed) possess the same abilities to use inorganic carbon (Ci) as alkaline‐grown cells. Internal pH values were not modified by the pH of the medium used to cultivate C. saccharophila. Regardless of the growth conditions, activities related to carbon fixation, that is, photosynthetic oxygen evolution, Ci uptake and assimilation were enhanced when the measurements were performed at acidic pH. This indicates that this marine alga is able to use CO₂ more efficiently than HCO₃^?. No evidence could be found for a specific Ci uptake and assimilation system in the acid‐grown cells.     

Structure and Macromolecular Composition of the Egg and Embryo Jelly Coats of the Anuran Lepidobatrachus laevis

Eggs and cleavage-stage embryos of the frog Lepidobatrachus laevis are encased by 3 μm thick vitelline/fertilization envelope and two jelly layers, termed J₁ (innermost) and J₂ (outermost). Based on light and transmission electron microscopy, J₁ had a dense reticular appearance whereas J₂ had a laminar structure. Direct dissolution of the jelly coats was accomplished by reduction of disulfide bonds with 0.08 M 2-mercaptoethanol at pH 10. Soluble jelly preparations were uncontaminated with nucleic acid (A²⁸⁰/A²⁶⁰=1.44) and yielded an average of 150 μg protein/egg or embryo (n=5). The biochemical composition of the jelly coats in unfertilized eggs was different from that in embryos. When examined via gel permeation chromatography, soluble jelly from unfertilized eggs contained macromolecules which were markedly larger and more heterogeneous (earlier eluting and broader peaks) than jelly from embryos. Differences in the components of jelly from unfertilized eggs and embryos were also observed by electrophoresis, however, a 29,700 molecular weight glycoprotein chain was common to both jelly preparations. The electrophoretic pattern of jelly obtained from parthenogenetically activated eggs was identical to that of unfertilized eggs, therefore the fertilization-associated changes are not due to the exclusive action of cortical granule products.     

Nitrification in a biofilm at low pH values: role of in situ microenvironments and acid tolerance

The sensitivity of nitrifying bacteria to acidic conditions is a well-known phenomenon and generally attributed to the lack and/or toxicity of substrates (NH3 and HNO2) with decreasing pHs. In contrast, we observed strong nitrification at a pH around 4 in biofilms grown on chalk particles and investigated the following hypotheses: the presence of less acidic microenvironments and/or the existence of acid-tolerant nitrifiers. Microelectrode measurements (in situ and under various experimental conditions) showed no evidence of a neutral microenvironment, either within the highly active biofilm colonizing the chalk surface or within a control biofilm grown on a nonbuffering (i.e., sintered glass) surface under acidic pH. A 16S rRNA approach (clone libraries and fluorescence in situ hybridizations) did not reveal uncommon nitrifying (potentially acid-tolerant) strains. Instead, we found a strongly acidic microenvironment, evidence for a clear adaptation to the low pH in situ, and the presence of nitrifying populations related to subgroups with low Km s for ammonia (Nitrosopira spp., Nitrosomonas oligotropha, and Nitrospira spp.). Acid-consuming (chalk dissolution) and acid-producing (ammonia oxidation) processes are equilibrated on a low-pH steady state that is controlled by mass transfer limitation through the biofilm. Strong affinity to ammonia and possibly the expression of additional functions, e.g., ammonium transporters, are adaptations that allow nitrifiers to cope with acidic conditions in biofilms and other habitats.     

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.     

Effect of copper on liver key enzymes of anaerobic glucose metabolism from freshwater tropical fish Prochilodus lineatus

We investigated the effect of copper on liver key enzymes of the anaerobic glucose metabolism (hexokinase, HK; phosphofructokinase, PFK; pyruvate kinase, PK; lactate dehydrogenase, LDH) as well as of the pentose pathway (glycose-6-phosphate dehydrogenase, G6PDH) from the fish Prochilodus lineatus. The fish were acclimated at either 20 degrees C or 30 degrees C at pH 7.0, transferred to water at pH 4.5 or 8.0, and exposed to 96 h-CL(50) copper concentrations. Copper accumulation in liver was higher in fish acclimated at 20 degrees C and maintained in water pH 8.0. Three-way analysis of variance revealed a significant effect of temperature on all enzymes, a significant effect of pH on all enzymes except for PK, and a significant effect of copper on only PFK, and LDH in pH 4.5 at 20 degrees C and, at 30 degrees C, on PFK and PK at pH 4.5 and 8.0, HK at pH 4.5 and G6PDH at pH 8.0. There were significant interactions between treatments for many enzymes. These changes suggest that the activity of enzymes in question is modified by a change in ambient water. At least at 30 degrees C, the overall reduction in the glycolytic enzyme activities of copper-exposed fish seems to reduce energy availability via glucose metabolism, thereby contributing to enhance copper toxic effects.     

Nitrification in a Biofilm at Low pH Values: Role of In Situ Microenvironments and Acid Tolerance

The sensitivity of nitrifying bacteria to acidic conditions is a well-known phenomenon and generally attributed to the lack and/or toxicity of substrates (NH₃ and HNO₂) with decreasing pHs. In contrast, we observed strong nitrification at a pH around 4 in biofilms grown on chalk particles and investigated the following hypotheses: the presence of less acidic microenvironments and/or the existence of acid-tolerant nitrifiers. Microelectrode measurements (in situ and under various experimental conditions) showed no evidence of a neutral microenvironment, either within the highly active biofilm colonizing the chalk surface or within a control biofilm grown on a nonbuffering (i.e., sintered glass) surface under acidic pH. A 16S rRNA approach (clone libraries and fluorescence in situ hybridizations) did not reveal uncommon nitrifying (potentially acid-tolerant) strains. Instead, we found a strongly acidic microenvironment, evidence for a clear adaptation to the low pH in situ, and the presence of nitrifying populations related to subgroups with low K_ms for ammonia (Nitrosopira spp., Nitrosomonas oligotropha, and Nitrospira spp.). Acid-consuming (chalk dissolution) and acid-producing (ammonia oxidation) processes are equilibrated on a low-pH steady state that is controlled by mass transfer limitation through the biofilm. Strong affinity to ammonia and possibly the expression of additional functions, e.g., ammonium transporters, are adaptations that allow nitrifiers to cope with acidic conditions in biofilms and other habitats.     

Ammonia/potassium exchange in methanogenic bacteria

Methanospirillum hungatei exposed to ammonia in a K+-free buffer lost up to 98% of the cytoplasmic K+ through an ammonia/K+ exchange reaction. The exchange was immediate, and occurred in cells poisoned by air or by other metabolic inhibitors. Additions of NH4OH or various NH+4 salts (or methylamine) were most effective in causing K+ depletion in media of alkaline pH, suggesting that NH3 was the chemical species crossing the membrane. In alkaline media, the exchange reaction resulted in a dissipation of the transmembrane pH gradient (inside acidic), but had only small effects on the membrane potential until concentrations of ammonia were used above those required to abolish the K+ gradient. Through the use of NH4Cl to vary the cytoplasmic pH at a constant acidic external pH, and NH4OH to abolish the transmembrane pH gradient at various alkaline external pH values, we conclude that methanogenesis is sensitive to both the pH of the cytoplasm and the medium. Methanogenesis in Msp. hungatei and Methanosarcina barkeri was inhibited dramatically at external pH values more acidic than 6.5 or more alkaline than 7.5. Dramatic K+ depletion in response to ammonia additions at pH 8.0 occurred with Ms. barkeri, another strain of Msp. hungatei, Escherichia coli, and Bacillus polymyxa. In several other methanogens, ammonia/potassium exchange was hardly detected.     

The effects of nest temperature,nest substrate,and clutch size on the oxygenation of embryos and larvae of the Australian moss frog,Bryobatrachus nimbus

The jelly around amphibian eggs presents a formidable barrier to oxygen diffusion. Therefore, egg capsules must be thin enough, and the dimensions of globular egg masses small enough, to avoid oxygen limitation leading to developmental retardation or death. The eggs of the Australian moss frog, Bryobatrachus nimbus, have the thickest jelly capsule known for any anuran amphibian. Laboratory measurements of respirometric variables predict that single prehatching embryos should be normoxic between 5 degrees and 20 degrees C, with Po(2 in) maintained above critical levels (10.2-17.0 kPa). However, numerical models of embryos amid larger egg masses (13-20 eggs) predict hypoxia at temperatures above 5 degrees C. Contrary to model predictions, however, B. nimbus embryos rarely experience hypoxia in natural nests, because embryos occur in one or two layers and the moss substrate permits aeration of the lower surface while photosynthesis probably supplies oxygen directly. After hatching, larvae move to oxygen-rich regions of the jelly mass and disperse more widely within the mass as temperatures increase. Although nest characteristics relieve diffusive constraints, small clutch sizes, low rates of embryonic and larval respiration, and the cool climate occupied by B. nimbus are the main characteristics that prevent hypoxia.     

Toxicity of ammonia to algae in sewage oxidation ponds.

Ammonia, at concentrations over 2.0 mM and at pH values over 8.0, inhibits photosynthesis and growth of Scenedesmus obliquus, a dominant species in high-rate sewage oxidation ponds. Photosynthesis of Chlorella pyrenoidosa, Anacystis nidulans, and Plectonema boryanum is also susceptible to ammonia inhibition. Dark respiration and cell morphology were unaffected by any combination of pH and ammonia concentrations tested, thus limiting the apparent effect to inhibition of the normal function of the chloroplasts. Methylamine had the same effect as ammonia, and its penetration into the cells was found to be pH dependent. Therefore, the dependence of toxicity of amines to algae on pH apparently results from the inability to penetrate the cell membrane in the ionized form. When operated at 120-h detention time of raw wastewater, the high-rate oxidation pond maintained a steady state with respect to algal growth and oxygen concentration, and the concentration of ammonia did not exceed 1.0 mM. Shifting the pond to 48-h detention time caused an increase in ammonia concentration in the pond water to 2.5 mM, and the pond gradually turned anaerobic. Photosynthesis, which usually elevates the pH of the pond water to 9.0 to 10.0, could not proceed beyond pH 7.9 because of the high concentration of ammonia, and the algal population was washed out and reduced to a concentration that could maintain a doubling time of 48 h without photosynthesis bringing the pH to inhibitory levels. Under these conditions, the pH of the bond becomes a factor that limits the operational efficiency of the oxidation pond.     

Regulation of intracellular pH values in higher plant cells. Carbon-13 and phosphorus-31 nuclear magnetic resonance studies.

The regulation of the cytoplasmic and vacuolar pH values (pHc and pHv) in sycamore (Acer pseudoplatanus L.) cells was analyzed using 31P and 13C nuclear magnetic resonance spectroscopy. Suspension-cultured cells were compressed in the NMR tube and perfused with the help of an original arrangement enabling a tight control of the pH (external pH, pHe) of the carefully oxygenated circulating nutrient medium. Intracellular pH values were measured from the chemical shifts of: CH2-linked carboxyl groups of citric acid below pH 5.7; orthophosphate between pH 5.7 and 8.0; 13C-enriched bicarbonate over pH 8.0. pHc and pHv were independent of pHe over the range 4.5-7.5. In contrast intracellular pH values decreased rapidly below pHe 4.5 and increased progressively at pHe over 7.5. There was an acceleration in the rate of O2 consumption accompanied with a decrease in cytoplasmic ATP concentration as pHe decreased. When the rate of O2 consumption was approaching the uncoupled O2 uptake rate, a loss of pHc control was observed. It is concluded that as pHe decreased, the plasma membrane ATPase consumed more and more ATP to reject the invading H+ ions in order to maintain pHc at a constant value. Below pHe 4.5 the efficiency of the H+ pump to react to back leakage of H+ ions became insufficient, leading to an acidification of pHc and to an alkalinization of pHe. On the other hand, over pHe 7.5 a passive influx of OH- ions was observed, and pHc increased proportionally to the increase of pHe. Simultaneously appreciable amounts of organic acids (malate and citrate) were synthesized by cells during the course of the alkalinization of the cytoplasmic compartment. The synthesis of organic acids which partially counteract the alkalinization of the cytoplasmic compartment may result from a marked activation of the cytoplasmic phosphoenolpyruvate carboxylase induced by an increase in cytoplasmic bicarbonate concentration. The fluctuations of pHv followed a similar course to that of pHc. It is concluded that the vacuole, which represents a potentially large H+ ions reservoir, can counteract H+ (or OH-) ion invasion observed at acidic (or alkaline) pHe contributing to the homeostasis of pHc.     

Toxicity of ammonia to algae in sewage oxidation ponds.

Ammonia, at concentrations over 2.0 mM and at pH values over 8.0, inhibits photosynthesis and growth of Scenedesmus obliquus, a dominant species in high-rate sewage oxidation ponds. Photosynthesis of Chlorella pyrenoidosa, Anacystis nidulans, and Plectonema boryanum is also susceptible to ammonia inhibition. Dark respiration and cell morphology were unaffected by any combination of pH and ammonia concentrations tested, thus limiting the apparent effect to inhibition of the normal function of the chloroplasts. Methylamine had the same effect as ammonia, and its penetration into the cells was found to be pH dependent. Therefore, the dependence of toxicity of amines to algae on pH apparently results from the inability to penetrate the cell membrane in the ionized form. When operated at 120-h detention time of raw wastewater, the high-rate oxidation pond maintained a steady state with respect to algal growth and oxygen concentration, and the concentration of ammonia did not exceed 1.0 mM. Shifting the pond to 48-h detention time caused an increase in ammonia concentration in the pond water to 2.5 mM, and the pond gradually turned anaerobic. Photosynthesis, which usually elevates the pH of the pond water to 9.0 to 10.0, could not proceed beyond pH 7.9 because of the high concentration of ammonia, and the algal population was washed out and reduced to a concentration that could maintain a doubling time of 48 h without photosynthesis bringing the pH to inhibitory levels. Under these conditions, the pH of the bond becomes a factor that limits the operational efficiency of the oxidation pond.     

Photosynthetic inorganic carbon utilization and growth of Porphyra linearis (Rhodophyta)

Photosynthetic (oxygen evolution) and growth (biomass increase) responses to ambient pH and inorganic carbon (Ci) supply were determined for Porphyralinearis grown in 0.5 L glass cylinders in the laboratory, or in 40 L fibreglass outdoor tanks with running seawater. While net photosynthetic rates were uniform at pH 6.0–8.0, dropping only at pH 8.7, growth rates were significantly affected by pH levels other than that of seawater (c. pH 8.3). In glass cylinders, weekly growth rates averaged 76% at external pH 8.0, 13% at pH 8.7 and 26% at pH 7.0. Photosynthetic O₂ evolution on a daily basis(i.e. total O₂ evolved during day time less total O₂ consumed during night time) was similar to the growth responses at all experimental pH levels, apparently due to high dark respiration rates measured at acidic pH. Weekly growth rates averaged 53% in algae grown in fibreglass tanks aerated with regular air (360 mg L_-1 CO₂) and 28% in algae grown in tanks aerated with CO₂-enriched air (750 mg L^-1 CO₂). The pH of the seawater medium in which P. linear is was grown increased slightly during the day and only rarely reached 9.0. The pH at the boundary layer of algae submerged in seawater increased in response to light reaching, about pH 8.9 within minutes, or remained unchanged for algae submerged in a CO₂-free artificial sea water medium. Photosynthesis of P. linearissaturated at Ci concentrations of seawater (K_0.5560 μM at pH 8.2) and showed low photosynthetic affinity for CO₂(K_0.5 61 μM) at pH 6.0. It is therefore concluded that P. linearisuses primarily CO₂ with HCO₃ ^- being an alternative source of Ci for photosynthesis. Its fast growth could be related to the enzyme carbonic anhydrase whose activity was detected intra- and extracellularly. This revised version was published online in August 2006 with corrections to the Cover Date.     

Studies on the purification of wastewater from the nitrogen fertilizer industry by intensive algal cultures. I. Growth of Chlorella vulgaris in wastes.

The possibility of growth of intensive cultures of Chlorella vulgaris on industrial wastewater from nitrogen fertizer plant containing ammonia, urea and nitrate was investigated. Good growth of algae was obtained when the waste was enriched with phosphorus and inoculum contained a high number of cells. The optimal pH for the culture was 7.0--8.0. The main factor limiting growth of algae on wastes on the concentration of ammonia nitrogen. Chlorella vulgaris grows quite well in wastes containing 600 mg NH4-N/l but is inhibited at concentration about 100 mg NH4-N/l.     

Form, function, and embryonic migration in large gelatinous egg masses of arenicolid worms

Abstract. Attachment and gas exchange are design problems for the spheroidal egg masses that annelids and molluscs tether in sediments. At an intertidal site in Bodega Harbor, a high proportion of masses of arenicolid worms became detached, but they were not stranded higher than attached masses, and the embryos developed to advanced stages. The large masses, weighing 100–200 g, challenge predictions on limiting size for oxygen supply by diffusion. The estimated concentrations of embryos and thickness of the embryo layer exceed the limits predicted by a simple model for oxygen supply by diffusion from the surrounding water, but several features may enhance oxygen supply to embryos. (1) There is an internal cavity. Distal tears in the gel layer admit pulses of water into the center, thus adding an inner surface for gas exchange. (2) The motile embryos aggregate near the outer and sometimes inner surfaces of the gel layer. Embryos migrate in the gel toward regions of greater oxygen concentration. (3) Pennate diatoms colonize the masses, coating the surfaces and penetrating into the gelatinous matrix. Photosynthesis can exceed respiration when a mass is in the light. The pH in gel changes diurnally from a low of ∼7 to as much as 9.5, indicating an excess of respiration over photosynthesis at night and at least sometimes an excess of photosynthesis over respiration by day.     

Restraint of spreading-dependent activation of polymorphonuclear leukocyte NADPH oxidase in an acidified environment

Elucidation of the mechanisms by which environmental pH affects or regulates the functions of polymorphonuclear leukocytes (PMNs) is important because severe acidification of the microenvironment often prevails at sites of inflammation where they act in host defense. In the present study, we investigated the effect of an acidic environment on spreading-dependent activation of O2- -producing NADPH oxidase in PMNs. We found that PMNs underwent spreading spontaneously over type I collagen and plastic surfaces at both neutral and acidic pH, although spreading over fibrinogen surfaces, for which cellular stimulation with H2O2 is required, was inhibited by acidic pH. At acidic pH, however, PMNs were unable to undergo spreading-dependent production of O2-. Pharmacological experiments showed that p38 mitogen-activated protein kinase (MAPK) was involved in the signaling pathways mediating the spreading-dependent activation of NADPH oxidase, and that its spreading-dependent phosphorylation of Thr-180 and Tyr-182, a hallmark of activation, was impaired at acidic pH. Furthermore, the inhibition by acidic pH of O2- production as well as p38 MAPK phosphorylation subsequent to spreading induction was reversible; environmental neutralization and acidification after induction of spreading at acidic and neutral pH, respectively, up- and down-regulated the two phenomena. Acidic pH did not affect the O2- production activity of NADPH oxidase pre-activated by phorbol 12-myristate 13-acetate (PMA). These results suggest that, in PMNs, the p38 MAPK-mediated signaling pathway functions as a pH-sensing regulator of spreading-dependent NADPH oxidase activation.