Organic and inorganic acids as the stimulus for exsheathment of infective juveniles of nematodes

Petronijevic T., Rogers W. P. and Sommerville R. I. 1986. Organic and inorganic acids as the stimulus for exsheathment of infective juveniles of nematodes. International Journal for Parasitology 16: 163–168. A variety of organic and inorganic acids stimulated exsheathment of infective juveniles of Nematospiroides dubius over the range pH 2–7. Activity, in relation to pK values, suggests that the undissociated form of the acid was the active agent. Under normal conditions of exposure, exsheathment of infective juveniles of Haemonchus contortus was not obtained below pH 6, and at higher pH values activity was low except in the presence of CO₂ at pressures >9-5 kPa in the gas phase. Brief exposure to HCl at concentrations up to 2N did not induce exsheathment but in the presence of CO₂ in the gas phase, 95.3 kPa, activity was obtained in 0-O1N HCl when the times of exposure were 3–25 min.The differences in the responses of N. dubius and H. contortus to a stimulus at low pH values may be attributed to the stability of the exsheathing enzyme of N. dubius in acid media as well as to the relative sensitivity of the two species to stimuli generally.     

Carbonic acid as the host signal for the development of parasitic stages of nematodes

Petronijevic T., Rogers W. P. and Sommerville R. I. 1985. Carbonic acid as the host signal for the development of parasitic stages of nematodes. International Journal for Parasitology15: 661–667. This paper gives results on which may be based an identification of the component of the system CO₂ + H₂O ai H₂CO₃ ai H⁺ HCO₃^? which acts as the stimulus from the animal host for some nematodes. Using infective juveniles of Nematospiroides dubius and Haemonchus contortus, the effects on exsheathment of (1) low pCO₂ values, (2) the presence of carbonic anhydrase in the stimulating medium, and (3) the inhibition of carbonic anhydrase within the juveniles have been examined. The results lead to the suggestion that it is the “readily available” undissociated H₂CO₃, or H₂CO₃ + HCO₃^? which is the critical factor in the stimulus for development. The wide range of [H⁺]s over which “readily available” H₂CO₃ is present in physiological environments suggests that this host signal may be important for infection with many species.     

Gene activity and the development of early parasitic stages of nematodes

Actinomycin-D, 8 × 10^?6m, blocked the action of the stimulus from the host which normally initiates the development of the infective stage of Haemonchus contortus to the first parasitic stage. However, the action of the stimulus in initiating exaheathment was not affected by the antibiotic.The significance of these results in relation to the activity of the gene set of the first parasitic stage and the control of exsheathment of the infective stage is discussed.     

Changes in optical path difference in the oesophageal region and the excretory cells during exsheathment in Haemonchus contortus

Davey K. G. and Sommerville R. I. 1982. Changes in optical path difference in the oesophageal region and the excretory cells during exsheathment in Haemonchus contortus. International Journal for Parasitology12: 503–507. Changes in the optical path difference (opd) between various parts of the worm and the medium in which the worms were immersed were determined by quantitative interference microscopy. The opd of the oesophagus and the excretory cells both increased upon stimulation of the worms with CO₂ at 38.5°C, suggesting a decrease in volume of those structures. The oesophagus decreased markedly in length and slightly in diameter, yielding a decrease in volume of approximately 15 to 17 pl. Desheathing the worms with NaOCl produced changes in the oesophagus but not the excretory cells. This confirms previous findings that exsheathment involves at least two parallel processes, both of which are initiated by CO₂ and only one of which is stimulated by exposure to NaOCl.     

The effect of ethoxyzolamide, an analogue of insect juvenile hormone, nor-adrenaline and iodine on changes in the optical path difference in the excretory cells and oesophagus during exsheathment in Haemonchus contortus

Davey K. G., Sommerville R. I. and Rogers W. P. 1982. The effect of ethoxyzolamide, an analogue of insect juvenile hormone, nor-adrenaline and iodine on changes in the optical path difference in the excretory cells and oesophagus during exsheathment in Haemonchus contortus. International Journal for Parasitology12: 509–513. Ethoxyzolamide, an inhibitor of carbonic anhydrase, markedly inhibits exsheathment of Haemonchus when the larvae are subsequently exposed to an exsheathing stimulus of CO₂ at 38.5°C. Ethoxyzolamide at 2 × 10^?5M does not prevent the increase in optical path difference in the oesophageal region which normally accompanies exsheathment, but markedly inhibits the increase in optical path difference in the excretory cells. An analogue of juvenile hormone (JHA; the methyl ester of 3,7,11 trimethyl-7,11-dichloro-2-dodecenic acid) does not affect the optical path difference in either the oesophagus or the excretory cells of ensheathed worms. When worms are artificially desheathed by exposure to NaOCl, a procedure which mimics the effect of CO₂ upon the oesophagus, but which does not affect the excretory cells, subsequent exposure to JHA at room temperature increases the optical path difference in the excretory cells. This increase is enhanced by subsequent incubation of the worms at 38.5°C at 30–60 min and further enhanced when CO₂ is present during the incubation at 38.5°C. The stimulation of the excretory cells by JHA is inhibited by ethoxyzolamide at 2 × 10^?5M. Noradrenaline at 10^?3M has no effect on ensheathed larvae, but causes an increase in optical path difference in the excretory cells of larvae desheathed with NaOCl. This increase is inhibited by ethoxyzolamide. A brief exposure to I₂ blocks the response of the excretory cells of both CO₂ and JHA, but does not significantly reduce the effect of nor-adrenaline. On the basis of these and previous results, it is proposed that both CO₂ and JHA stimulate a hypothetical CO₂ receptor which leads to the release of nor-adrenaline. The noradrenaline in turn stimulates, either directly or indirectly, the excretory cells.     

Inhibition of reticulocyte iron uptake by NH4Cl and CH3NH2

The aim of this investigation was to test the hypothesis that elevation of intracellular pH would inhibit iron uptake by reticulocytes. The experiments were performed with rabbit reticulocytes and iron bound to rabbit transferrin. Incubation of the cells with NH₄Cl, (NH₄)₂CO₃, CH₃NH₂ and (CH₃)₂NH was used in an attempt to increase intracellular pH. These substances were all found to inhibit iron uptake by reticulocytes. The mechanism of action of NH₄Cl and CH₃NH₂ was investigated in detail. Similar results were found with both reagents. They inhibited iron uptake in a concentration-dependent manner, but produced a small increase in the cellular uptake of transferrin. The onset of action was rapid and the effect was reversible. There was no decrease in the number of transferrin-binding sites per cell and their apparent affinity for transferrin increased slightly, while the efficiency of iron removal from transferrin per binding site diminished greatly. The rate of transferrin release from reticulocytes was unaffected. NH₄Cl did not affect the rate of iron release from transferrin in a cell-free system. Incubation of reticulocytes with 10 mM NH₄Cl or CH₃NH₂ was found to produce an increase in intracellular pH of 0.05—0.15 pH units. The intracellular pH determined by used of the weak acid 5,5-dimethyl-oxazolidine-2,4-dione was significantly higher than that obtained with the weak base (CH₃)₂NH. By transmission electron microscopy it was shown that reticulocytes treated with NH₄Cl or CH₃NH₂ have enlarged intracellular vesicles. The results are considered to support the hypothesis that iron release from transferrin in reticulocytes occurs as a result of protonation of the transferrin within intracellular vesicles. According to this hypothesis, weak bases such as NH₃ and CH₃NH₂ inhibit iron release by neutralizing H⁺ within the vesicles.     

NH4Cl activation of the fluorescence yield in CO2-starved Chlorella pyrenoidosa

The addition of NH₄Cl to Chlorella deprived of CO₂, raises the fluorescence yield about 2.5-fold. This effect does not appear to be related to nitrate reduction, and is specific for NH₄⁺. Concentration curves, at varying pH, indicate that the unprotonated ammonia is responsible for this effect.     

Effect of Ammonium Chloride and Methionine Sulfoximine on the Acetylene Reduction of Detached Root Nodules of Peas (Pisum sativum)

Acetylene-reducing activity of detached pea nodules was determined by submerging the nodules in buffer solution [tris(hydroxymethyl)aminomethane-hydrochloride, pH 7.4] containing 100 mM sodium succinate and incubating under a gas phase of 90% O₂ and 10% C₂H₂. The nitrogenase activity was 4 to 8 μmol of C₂H₄ formed per g of nodule fresh weight per h and remained constant for at least 4 h. Addition of NH₄Cl to the buffer solution (at a concentration of 10 mM or more) resulted in a significant decrease of nitrogenase activity, which was more pronounced at higher concentrations of ammonium chloride. The inhibition of nitrogenase activity by NH₄Cl was reversible; when the NH₄Cl-containing buffer solution was replaced by buffer without NH₄Cl, the original activity was partly restored. Treatment of the nodules with NH₄Cl had almost no effect on the amount of nitrogenase, as measured by the acetylene-reducing activity of ethyl-enediaminetetraacetate-toluene-treated bacteroid suspensions. The effect of NH₄Cl was largely eliminated by simultaneous addition of 10 mM methionine sulfoximine to the assay solution. This suggests that the assimilation of ammonium ions by glutamine synthetase controls the functioning of nitrogenase activity in the nodules. However, no effect of glutamine, glutamate, or aspartate on the acetylene reduction by detached nodules could be detected.     

Stromal acidification mediates in vivo water stress inhibition of nonstomatal-controlled photosynthesis

Stromal acidification has been reported to mediate reduced osmotic potential (ψ_π) effects on photosynthesis in the isolated spinach chloroplast (Berkowitz, Gibbs 1983 Plant Physiol 72: 1100-1109). To determine if stromal acidification mediates osmotic dehydration inhibition of photosynthesis in vivo, the effects of a weak base (NH₄Cl), which raises stromal pH, on CO₂ fixation of vacuum-infiltrated spinach leaf slices, Chlamydomonas reinhardii cells and Aphanocapsa 6308 cells under isotonic and dehydrating conditions were investigated. Five millimolar NH₄Cl stimulated spinach leaf slice CO₂ fixation by 43% under stress (0.67 molar sorbitol) conditions, and had little effect on fixation under isotonic (0.33 molar sorbitol) conditions. Chlamydomonas cells were found to be more sensitive to reduced ψ_π than spinach leaf slices. CO₂ fixation in the cells of the green alga Chlamydomonas reinhardii was 99 and 17 micromoles per milligram chlorophyll per hour, respectively, at 0.1 molar mannitol and 0.28 molar mannitol. Five millimolar NH₄Cl stimulated CO₂ fixation of Chlamydomonas cells by 147% under stress (0.28 molar mannitol) conditions. Aphanocapsa 6308 cells (blue-green alga) were also found to be sensitive to reduced ψ_π, and inhibitions in photosynthesis were partially reversed by NH₄Cl. These data indicate that in vivo water stress inhibition of photosynthesis is facilitated by stromal acidification, and that this inhibition can be at least partially reversed in situ.     

Effect of Nitrogen Source on End Products of Naphthalene Degradation

Soil cultures, enrichment cultures, and pure culture isolates produced substantial quantities of salicylic acid from naphthalene in a mineral salts medium containing NH₄Cl as the nitrogen source. However, when KNO₃ was substituted for NH₄Cl, these same cultures failed to accumulate detectable quantities of salicylic acid but did turn the medium yellow. When an isolate identified as a Pseudomonas species was used, viable cell numbers were much greater in the medium containing KNO₃, but up to 94% of the naphthalene was utilized in both media. After 48 h of incubation in a 0.1% naphthalene-mineral salts medium, the cultures containing NH₄Cl showed irregular clumped cells, a pH of 4.7, 42 μg of salicylic acid per ml, and the production of 4.4 ml of CO₂. Under the same conditions, the cultures in the medium containing KNO₃ showed uniform cellular morphology, a pH of 7.3, no salicylic acid, the production of 29.7 ml of CO₂, and a distinct yellow coloration of the medium. The differences between nitrogen sources could not be accounted for by pH alone since results obtained using buffered media were similar. Growth with NH₄NO₃ displayed a pattern similar to that obtained when NH₄Cl was used. The yellow coloration in the medium containing KNO₃ was apparently due to more than one compound, none of which were 1,2-naphthoquinone or acidic in nature, as suggested by other investigators. Further attempts to identify the yellow compounds by high-pressure liquid chromatography, infrared analysis, and gas chromatography-mass spectrometry have been unsuccessful thus far.     

Human SLC4A11 Is a Novel NH3/H+ Co-transporter

SLC4A11 has been proposed to be an electrogenic membrane transporter, permeable to Na⁺, H⁺ (OH⁻), bicarbonate, borate, and NH₄⁺. Recent studies indicate, however, that neither bicarbonate or borate is a substrate. Here, we examined potential NH₄⁺, Na⁺, and H⁺ contributions to electrogenic ion transport through SLC4A11 stably expressed in Na⁺/H⁺ exchanger-deficient PS120 fibroblasts. Inward currents observed during exposure to NH₄Cl were determined by the [NH₃]_o, not [NH₄⁺]_o, and current amplitudes varied with the [H⁺] gradient. These currents were relatively unaffected by removal of Na⁺, K⁺, or Cl⁻ from the bath but could be reduced by inclusion of NH₄Cl in the pipette solution. Bath pH changes alone did not generate significant currents through SLC4A11, except immediately following exposure to NH₄Cl. Reversal potential shifts in response to changing [NH₃]_o and pH_o suggested an NH₃/H⁺-coupled transport mode for SLC4A11. Proton flux through SLC4A11 in the absence of ammonia was relatively small, suggesting that ammonia transport is of more physiological relevance. Methylammonia produced currents similar to NH₃ but with reduced amplitude. Estimated stoichiometry of SLC4A11 transport was 1:2 (NH₃/H⁺). NH₃-dependent currents were insensitive to 10 μm ethyl-isopropyl amiloride or 100 μm 4,4′- diisothiocyanatostilbene-2,2′-disulfonic acid. We propose that SLC4A11 is an NH₃/2H⁺ co-transporter exhibiting unique characteristics.     

The developmental lipidome of Haemonchus contortus

Lipids play crucial roles in the biology of organisms, particularly relating to cellular membranes, energy storage, and intra- or inter-cellular signalling. Despite the recent expansion of the lipidomics field, very little is known about the biology of lipids in metzoan pathogens, and, to date, there has been no global lipidomic study of a parasitic nematode. Using Haemonchus contortus (barber's pole worm) as a model, we describe the first known global lipidome for a parasitic nematode via high throughput LC–MS/MS-based lipidomics. We identified a total of 554 lipid species across four lipid categories, and 18 lipid classes exhibited alterations among six developmental stages (eggs; L3 and exsheathed L3 (xL3) and L4 larval stages; female and male adults) of H. contortus. The lipid composition and abundance of H. contortus changed significantly during the transition from free-living (egg, L3 and xL3) to parasitic (L4 and adult) stages. The three main changes observed were: (i) decreased synthesis of triradylglycerols; (ii) increased glycerophospholipids (predominantly glycerophosphoethanolamines and glycerophosphocholines); and (iii) a ‘cooperative’ modulation of ether-linked lipids and saturated fatty acids. These changes suggest specific adaptations, in terms of nutrient acquisition, metabolism and development, as the nematode makes its transition to the parasitic stage inside the host animal. This lipidomic data set serves as a stimulus for studies to understand lipid biology in parasitic worms, and their roles in parasite–host interactions and disease processes.     

Changes in water content and volume accompanying exsheathment of Haemonchus contortus

Davey K. G. and Rogers W. P. 1982. Changes in water content and volume accompanying exsheathment of Haemonchus contortus, International Journal for Parasitology12: 93–96. The exchange water volume was determined for ensheathed larvae, for larvae exsheathed by exposure to CO₂ and for larvae desheathed by exposure to a solution of NaOCl. Larvae exsheathed by exposure to CO₂ lose approx. 66 pl. of water while those desheathed by exposure to NaOCl lose approx. 50 pl. Larvae first desheathed by exposure to NaOCl lose a further 20 pl of water when subsequently exposed to CO₂. Volumes calculated from linear measurements of larvae demonstrate that larvae exsheathed by exposure to CO₂ lose about 69 pl of their total volume, while those desheathed by NaOCl lose about 54 pl. It is concluded that exsheathment may involve at least two parallel processes. Both processes are initiated by CO₂, while one is mimicked by exposure to NaOCl.     

Effect of elevated CO2 concentration and nitrate: ammonium ratios on gas exchange and growth of cassava (Manihot esculenta Crantz)

Aims

This study evaluated how different nitrogen forms affect growth and photosynthetic responses of cassava to CO₂ concentration.

Methods

Cassava was grown in 14-L pots in a greenhouse at 390 or 750 ppm of CO₂. Three nitrogen treatments were applied: (a) 12?mM NO₃ ^?, (b) 6?mM NO₃ ^??+?6?mM NH₄ ⁺, and (c) 12?mM NH₄ ⁺.

Results

Thirty-six days after treatments began, plants grown under elevated CO₂ and fertilized only with NO₃ ^? (750_NO₃ ^?) had photosynthetic rates similar to plants grown under 390_NO₃ ^?, indicating significant photosynthetic acclimation to CO₂. In contrast, photosynthetic rates at elevated CO₂ increased as NH₄ ⁺ increased in the nutrient solution, such that photosynthetic acclimation was reduced for plants fertilized with only NH₄ ⁺. However, this positive effect of NH₄ ⁺ on photosynthesis was not observed in more advanced growth stages, and the toxic effects of NH₄ ⁺ severely reduced total dry mass for these plants measured at the end of the experiment.

Conclusions

Our results indicate that cassava will respond with increased biomass accumulation in response to raising atmospheric CO₂ levels, and that N form can have an important impact on the photosynthetic response. However, the positive effect of NH₄ ⁺ fertilization on cassava photosynthetic CO₂ response eventually led to a toxicity problem that reduced biomass production. The challenge is to determine how to manage NH₄ ⁺ fertilization so that the photosynthetic benefit observed in the initial phase may persist throughout the crop cycle.     

Analysis of the Microbial Community in an Acidic Hollow-Fiber Membrane Biofilm Reactor (Hf-MBfR) Used for the Biological Conversion of Carbon Dioxide to Methane

Hydrogenotrophic methanogens can use gaseous substrates, such as H₂ and CO₂, in CH₄ production. H₂ gas is used to reduce CO₂. We have successfully operated a hollow-fiber membrane biofilm reactor (Hf-MBfR) for stable and continuous CH₄ production from CO₂ and H₂. CO₂ and H₂ were diffused into the culture medium through the membrane without bubble formation in the Hf-MBfR, which was operated at pH 4.5–5.5 over 70 days. Focusing on the presence of hydrogenotrophic methanogens, we analyzed the structure of the microbial community in the reactor. Denaturing gradient gel electrophoresis (DGGE) was conducted with bacterial and archaeal 16S rDNA primers. Real-time qPCR was used to track changes in the community composition of methanogens over the course of operation. Finally, the microbial community and its diversity at the time of maximum CH₄ production were analyzed by pyrosequencing methods. Genus Methanobacterium, related to hydrogenotrophic methanogens, dominated the microbial community, but acetate consumption by bacteria, such as unclassified Clostridium sp., restricted the development of acetoclastic methanogens in the acidic CH₄ production process. The results show that acidic operation of a CH₄ production reactor without any pH adjustment inhibited acetogenic growth and enriched the hydrogenotrophic methanogens, decreasing the growth of acetoclastic methanogens.     

Hydrogen evolution as a consumption mode of reducing equivalents in green algal fermentation

Dark anaerobic fermentation in the green algae Chlamydomonas MGA 161, Chlamydomonas reinhardtii, Chlorella pyrenoidosa, and Chlorococcum minutum was studied. Our isolate, Chlamydomonas MGA 161, was unusual in having high H₂ but almost no formate. The fermentation pattern in Chlamydomonas MGA 161 was altered by changes in the NaCl or NH₄Cl concentration. Glycerol formation increased at low (0.1%) and high (7%) NaCl concentrations; starch degradation, and formation of ethanol, H₂, and CO₂ increased with the addition of NH₄Cl to above 5 millimolar in N-deficient cells. C. reinhardtii and C. pyrenoidosa exhibited a very similar anaerobic metabolism, forming formate, acetate and ethanol in a ratio of about 2:2:1. C. minutum was also unusual in forming acetate, glycerol, and CO₂ as its main products, with H₂, formate, and ethanol being formed in negligible amounts. In the presence of CO, ethanol formation increased twofold in Chlamydomonas MGA 161 and C. reinhardtii, but the fermentation pattern in C. minutum did not change. An experiment with hypophosphite addition showed that dark H₂ evolution of the Escherichia coli type could be ruled out in Chlamydomonas MGA 161 and C. reinhardtii. Among the green algae investigated, three fermentation types were identified by the distribution pattern of the end products, which reflected the consumption mode of reducing equivalents in the cells.     

Solubilization and reconstitution of the oat root vacuolar h/ca exchanger

Calcium is sequestered into vacuoles of oat (Avena sativa L.) root cells via a H⁺/Ca²⁺ antiporter, and vesicles derived from the vacuolar membrane (tonoplast) catalyze an uptake of calcium which is dependent on protons (pH gradient [ΔpH] dependent). The first step toward purification and identification of the H⁺/Ca²⁺ antiporter is to solubilize and reconstitute the transport activity in liposomes. The vacuolar H⁺/Ca²⁺ antiporter was solubilized with octylglucoside in the presence of soybean phospholipids and glycerol. After centrifugation, the soluble proteins were reconstituted into liposomes by detergent dilution. A ΔpH (acid inside) was generated in the proteoliposomes with an NH₄Cl gradient (NH₄⁺_in » NH₄⁺_out) as determined by methylamine uptake. Fundamental properties of ΔpH dependent calcium uptake such as the K_m for calcium (~15 micromolar) and the sensitivity to inhibitors such as N,N′-dicyclohexylcarbodiimide, ruthenium red, and lanthanum, were similar to those found in membrane vesicles, indicating that the H⁺/Ca²⁺ antiporter has been reconstituted in active form.     

Mechanism of Excitation of Aplysia Neurons by Carbon Dioxide

The abdominal ganglion of Aplysia californica was perfused with artificial seawater equilibrated at different P_COCO2's and pH's for 5 min or less. 5% CO₂ dropped perfusate pH from 8.0 to 6.5 and produced depolarization and increased discharge rate in visceromotor neurons. Half the giant cells studied had a similar response, whereas the other half were hyperpolarized. Pacemaker neurons showed little, if any, response to such changes in pH or CO₂. Membrane conductance of responsive cells was always increased. The effect of CO₂ occurred even when synaptic transmission was blocked by low calcium and high magnesium, and therefore must have been a direct result of CO₂ or the concomitant fall in pH. When extracellular pH was lowered to 6.5 using HCl or H₂SO₄ and no CO₂, the same effects were observed. Also, local application of HCl or H₂SO₄ to the external surface of the cell soma elicited depolarization and spike discharge. When extracellular pH was held constant by continual titration, 5–50% CO₂ had no effect. Intracellular pH was probably decreased at least one pH unit under these circumstances. Thus CO₂ per se, decreased intracellular pH, and increased bicarbonate ion were without effect. It is concluded that CO₂ acts solely through a decrease in extracellular pH.     

Correlation between photosynthesis and the transthylakoid proton gradient

In isolated intact chloroplasts, maximal rates of photosynthetic O₂ evolution (in saturating HCO^?₃) are associated with a critical transthylakoid proton gradient as a result of the stoichiometric consumption of 2 mol NADPH and 3 mol ATP/mol CO₂ fixed. Studies with the fluorescent probe 9-aminoacridine reveal that in the illuminated steady state the critical ΔpH is 3.9.CO₂-dependent O₂ evolution is inhibited by increases of 0.1–0.2 in ΔpH that occur when catalase is omitted from the medium, NO^?₂ is included as an electron acceptor, or when chloroplasts are illuminated under low partial pressures of O₂. Low concentrations of antimycin (0.33 μM) or NH₄Cl (0.33 mM) decrease ΔpH and relieve this inhibition of electron flow. The energy transfer inhibitor quercetin lowers the high ATP/ADP ratio associated with these conditions, but does not lower ΔpH or relieve the inhibition.A decrease of ΔpH below 3.9 by weaker illumination, millimolar levels of NH₄Cl or micromolar levels of antimycin, results in lower rates of photosynthesis owing to limitation by the phosphorylation rate.These findings show that in absence of rate limitation by the carbon cycle, the extent of thylakoid energization is related to the ratio of ATP to NADPH production and in turn, the rate of CO₂ assimilation.