The role of carbon dioxide in the formation of end-products by Hymenolepis diminuta

The hypothesis that ambient CO₂ levels determine the end-products of energy metabolism excreted by Hymenolepis diminuta was tested by incubating the parasite in a range of CO₂ concentrations and measuring internal concentrations of adenine nucleotides and the excretion of organic acids. The strain of H. diminuta used was found to excrete mainly lactic acid and acetic acid. Succinic acid production was generally less than 5–10% of the total. At high CO₂ concentrations, the rate of excretion of lactic acid decreased while that of succinic acid increased, which conforms with the hypothesis. Acetic acid excretion did not vary significantly over the range of CO₂ concentrations used. Other results did not support the hypothesis. High CO₂ levels reduced the total amounts of acids excreted and the rate of succinic acid excretion was so small as to be ineffective in preventing the accumulation of H⁺ ions. When present in the incubation medium, succinic acid was taken up by H. diminuta. Lactic and acetic acid excretion was always sufficient to limit the accumulation of H⁺ ions. The conditions of incubation were shown not to be responsible for the low rates of succinic acid excreted. Incubation conditions and metabolic end-products were found to affect the rates of excretion of organic acids. There is thus a need, in work of this nature, to regulate and specify experimental conditions and to stipulate the strain of parasite used. The hypothesis was rejected and it was suggested that the energy metabolism of parasitic helminths is adapted to fluctuating O₂ and CO₂ tensions.     

Teladorsagia circumcincta: Survival of adults in vitro is enhanced by the presence of a mammalian cell line

Adult Teladorsagia circumcincta survival and motility in vitro was examined in a range of different cell culture media, supplements and gas mixes. Under optimum conditions, worms survived for 14 days, exhibiting high motility for 9 days and egg production for 72 h. Optimum conditions involved co-culture of worms with a HeLa cell line in a supplemented cell medium (CEM) and an atmosphere containing 10% CO₂, 5% O₂ 85% N₂, 65% humidity at 37 °C. The incubation medium consisted of Minimum Essential Medium with 10% fetal calf serum, 1% non-essential amino acids, 1% glutamax and 1% penicillin-neomycin-streptomycin cocktail mix. Compared with optimum conditions, incubation in CEM alone, cell conditioned CEM, RPMI alone, Medium 199 alone, reduced CO₂ or O₂, or when cells were replaced with Escherichia coli, both survival and motility were reduced. Optimum conditions for adult T. circumcincta maintenance for culture, anthelmintic testing or generation of excretory/secretory products are described.     

Effects of increased atmospheric CO2 on nutritional contents in poplar (Populus pseudo-simonii [Kitag.]) tissues and larval growth of gypsy moth (Lymantria dispar)

Changes in the concentrations of phytochemical compounds usually occur when plants are grown under elevated atmospheric CO₂. CO₂-induced changes in foliar chemistry tend to reduce leaf quality and may further affect insect herbivores. Increased atmospheric CO₂ also has a potential influence on decomposition because it causes variations in chemical components of plant tissues. To investigate the effects of increased atmospheric CO₂ on the nutritional contents of tree tissues and the activities of leaf-chewing forest insects, samples of Populus pseudo-simonii [Kitag.] grown in open-top chambers under ambient and elevated CO₂ (650 μmol mol^-1) conditions were collected for measuring concentrations of carbon, nitrogen, C : N ratio, soluble sugar and starch in leaves, barks, coarse roots (>2 mm in diameter) and fine roots (<2 mm in diameter). Gypsy moth (Lymantria dispar) larvae were reared on a single branch of experimental trees in a nylon bag with 1 mm 1 mm grid. The response of larval growth was observed in situ. Elevated CO₂ resulted in significant reduction in nitrogen concentration and increase in C : N ratio of all poplar tissues. In all tissues, total carbon contents were not affected by CO₂ treatments. Soluble sugar and nonstructural carbohydrate (TNC) in the poplar leaves significantly increased with CO₂ enrichment, whereas starch concentration increased only on partial sampling dates. Carbohydrate concentration in roots and barks was generally not affected by elevated CO₂, whereas soluble sugar contents in fine roots decreased in response to elevated CO₂. When second instar gypsy moth larvae consuming poplars grew under elevated CO₂ for the first 13 days, their body weight was 30.95% lower than that of larvae grown at ambient CO₂, but no significant difference was found when larvae were fed in the same treatment for the next 11 days. Elevated atmospheric CO₂ had adverse effects on the nutritional quality of Populus pseudo-simonii [Kitag.] tissues and the resultant variations in foliar chemical components had a significant but negative effect on the growth of early instar gypsy moth larvae.     

Leishmania mexicana: Energy metabolism of amastigotes and promastigotes

The utilisation of substrates by Leishmania mexicana amastigotes and promastigotes differed significantly. The rates of uptake and catabolism of nonesterified fatty acids were up to 10-fold higher with amastigotes. Almost all the available exogenous fatty acids were consumed during amastigote transformation and by stationary phase of promastigote growth. The results suggest that fatty acids are important energy substrates for amastigotes, whereas promastigote utilisation may reflect the requirement for these substrates in anabolism. Glucose was utilised by amastigotes and promastigotes but the rate of catabolism was up to 10-fold higher in promastigotes. Uptake of glucose occurred throughout amastigote transformation and growth in vitro of promastigotes. High-subpassage promastigotes exhibited markedly lower glucose but higher amino acid utilisation than low-subpassage promastigotes. Asparagine, glutamine, glutamate, leucine, lysine, methionine, and threonine were consumed in large quantities by amastigotes and promastigotes, whereas alanine and glycine were excreted. Proline was catabolised to CO₂ by amastigotes and promastigotes but only at a low rate, and it was excreted in large amounts throughout promastigote growth. The major end products of energy metabolism were found to be CO₂ and succinate with both forms of the parasite and there was a secretion of up to 12 and 16% of the total protein synthesised by transforming amastigotes and growing promastigotes, respectively. Catabolism in amastigotes and promastigotes was found to be sensitive to cyanide and amytal, whereas 2-mercaptoacetate and 4-pentenoate primarily affected β-oxidation in the amastigote.     

Determination of volatile nematode exudates and their effects on a nematode-trapping fungus

Volatile compounds exuded from axenically grown free-living nematodes were determined with gas chromatographic and mass spectrometric techniques. Carbon dioxide evolved from 5–200 nematodes was determined with an ampoule technique, whereas total ammonia (NH₃ + NH₄ ⁺) and acetic and propionic acids were determined by direct injection of water in which nematodes had been suspended for 1–3 days. CO₂ amounted to about 80 ng nematode^–1 d^–1, total ammonia to 1–5 ng, and acetic and propionic acids to 0.5 and 1.0 pg nematode^–1 d^–1.The effects of these compounds on induction of trap formation in the nematodetrapping fungusArthrobotrys oligospora were tested. CO₂ inhibited trap formation at 5–10% CO₂ in air (v/v), whereas ammonia stimulated trap formation in a certain concentration range. No effects of acetic and propionic acids were noted for the concentrations tested. The combined effects of these volatiles in the aqueous environment are discussed on the basis of stoichiometric considerations.     

Aminooxyacetate stimulation of glycolate formation and excretion by chlamydomonas

Aminooxyacetate (1 millimolar) did not inhibit photosynthetic ¹⁴CO₂ fixation by Chlamydomonas reinhardtii Dangeard, (−) strain (N.90) but greatly stimulated the biosynthesis and excretion of glycolate. Similar results were obtained from cells grown with 5% CO₂ or low CO₂ (air). After 2 minutes with air-grown cells, [¹⁴C]glycolate increased from 0.3% of the total ¹⁴C fixed by the control to 11.7% in the presence of aminooxyacetate and after 10 minutes from 3.8% to 41.1%. Ammonium nitrate (0.2 millimolar) in the media blocked the aminooxyacetate stimulation of glycolate excretion. Chromatographic analyses of the labeled products in the cells and supernatant media indicated that aminooxyacetate also completely inhibited the labeling of alanine while some pyruvate accumulated and was excreted. A high percentage (35%) of initial ¹⁴CO₂ fixation was into C₄ acids. Initial products of ¹⁴CO₂ fixation included phosphate esters as well as malate, aspartate, and glutamate in treated or untreated cells. Lactate was also a major early product of photosynthesis, and its labeling was reduced by aminooxyacetate. Inasmuch as lactate was not excreted, glycolate excretion seemed to be specific. When photosynthesis was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, labeled organic and amino acids but not phosphate esters were lost from the cells. Aminooxyacetate did not inhibit the enzymes associated with glycolate synthesis from ribulose bisphosphate.     

Metabolism of absorbed aspartate,asparagine, and arginine by rat small intestine in vivo

l-[U-¹⁴C]aspartate, l-[U-¹⁴C]asparagine, and l-[U-¹⁴C]arginine were administered luminally into isolated segments of rat jejunum in situ, and the radioactive products appearing in venous blood from the segment were identified and quantified, in a continuation of similar studies with l-glutamate and l-glutamine (Windmueller H.G. and Spaeth, A. E. (1975) Arch. Biochem. Biophys. 171, 662–672). Aspartate, administered alone (6 mm) or with 18 other amino acids plus glucose, was absorbed more rapidly than glutamate, but, as with glutamate, less than 1% was recovered intact in intestinal venous blood. More than 50% of aspartate carbon was recovered in CO₂, 24% in organic acids, mostly lactate, 12% in other amino acids (alanine, glutamate, proline, ornithine, and citrulline), and 10% in glucose, apparently the first demonstration of gluconeogenesis by intestine in vivo. In contrast to aspartate and glutamine, nearly all asparagine was absorbed intact, less than 1% being catabolized. About 4% of the absorbed dose was incorporated into the acid-insoluble fraction of intestine, as was the case with all the amino acids studied. In conventional or germ-free rats, only 60% of arginine was absorbed intact, while 33% was hydrolyzed to ornithine and urea. The urea and 38% of the ornithine were released into the blood; the remaining ornithine was metabolized further by intestine to citrulline, proline, glutamate, organic acids, and CO₂. Catabolism of several amino acids from the lumen plus glutamine from arterial blood may provide an important energy source in small intestine.     

Effect in the cow of intraruminal infusions of volatile fatty acids and of lactic acid on the secretion of the component fatty acids of the milk fat and on the composition of blood

1. The effects in the cow of intraruminal infusions of acetic acid, propionic acid or butyric acid on the secretion of the component fatty acids of the milk fat, and of these acids and of lactic acid on the composition of the blood plasma of the jugular vein, have been studied. 2. The infusion of acetic acid or butyric acid increased the yield of the C₄–C₁₆ acids of milk fat but decreased the yield of C₁₈ acids. The infusion of propionic acid decreased the yields of all major component acids except palmitic acid and possibly lauric acid. 3. The changes in the concentrations in blood plasma of glucose and of ketone bodies were consistent with the glucogenic effect of propionic acid and the ketogenic effects of butyric acid and acetic acid. The effects of lactic acid were not consistent from cow to cow. Only with the infusion of acetic acid was a significant increase in the concentration of total volatile fatty acids in blood plasma found. Infusions of butyric acid and of propionic acid tended to depress the concentration of citric acid in the blood plasma and infusion of acetic acid increased it. No consistent effects of the infused acids on the concentration in blood plasma of esterified cholesterol, free cholesterol, triglyceride or phospholipid were observed. 4. The possibility is discussed that the effects of the infused acids on milk-fat secretion are caused through an alteration of the concentrations of precursors of milk fat in mammary arterial blood.     

Histochemical lipid studies on Schistosoma mansoni adults maintained in situ and in vitro

Haseeb M. A., Eveland L. K. and Fried B. 1984. Histochemical lipid studies on Schistosoma mansoni adults maintained in situ and in vitro. International Journal for Parasitology14: 83–88. Schistosoma mansoni male and female adults were incubated at 37°C for 0.5 and 1.0 h in Earle's balanced salt solution containing 0.1% glucose and 0.5% lactalbumin hydrolysate, then examined by histochemistry and scanning electron microscopy. Histochemical analysis of cryostat sections stained with Oil Red O showed that males contain neutral lipid mainly in the parenchyma and tubercles, while females contain neutral lipid in the vitellaria. Neutral lipids are released from the tubercles of both paired and unpaired males maintained in vitro. There is evidence of in situ lipid transfer from males to blood vessel walls. Neutral lipid was not seen in females from unisexual infections. Sudan Black B staining fo total lipids is positive in tubercles, parenchyma, and vitellaria. Nile Blue Sulphate stains acidic lipids in male caecal walls. Scanning electron microscopy reveals no tegumental damage.     

Continuous measurement of 14C-labeled carbon dioxide and lactic acid by cultured cells grown in Leighton tubes.

A procedure for continuous measurement of ¹⁴CO₂ production by cultured cells grown in Leighton tubes has been described. The apparatus developed also permits aliquots of the incubation medium to be taken during the experiments for analysis of labeled metabolites released into the solution. A simple method for determination of [¹⁴C]lactic acid in such aliquots has been described. The reproducibility and usefulness of the apparatus has been demonstrated by incubating fibroblasts with glucose labeled in the C-1 or C-6 position, and examining the effects of selected drugs on CO₂ and lactic acid production.     

Carbon Flux Analysis by 13C Nuclear Magnetic Resonance To Determine the Effect of CO2 on Anaerobic Succinate Production by Corynebacterium glutamicum

Wild-type Corynebacterium glutamicum produces a mixture of lactic, succinic, and acetic acids from glucose under oxygen deprivation. We investigated the effect of CO₂ on the production of organic acids in a two-stage process: cells were grown aerobically in glucose, and subsequently, organic acid production by nongrowing cells was studied under anaerobic conditions. The presence of CO₂ caused up to a 3-fold increase in the succinate yield (1 mol per mol of glucose) and about 2-fold increase in acetate, both at the expense of l-lactate production; moreover, dihydroxyacetone formation was abolished. The redistribution of carbon fluxes in response to CO₂ was estimated by using ¹³C-labeled glucose and ¹³C nuclear magnetic resonance (NMR) analysis of the labeling patterns in end products. The flux analysis showed that 97% of succinate was produced via the reductive part of the tricarboxylic acid cycle, with the low activity of the oxidative branch being sufficient to provide the reducing equivalents needed for the redox balance. The flux via the pentose phosphate pathway was low (∼5%) regardless of the presence or absence of CO₂. Moreover, there was significant channeling of carbon to storage compounds (glycogen and trehalose) and concomitant catabolism of these reserves. The intracellular and extracellular pools of lactate and succinate were measured by in vivo NMR, and the stoichiometry (H⁺:organic acid) of the respective exporters was calculated. This study shows that it is feasible to take advantage of natural cellular regulation mechanisms to obtain high yields of succinate with C. glutamicum without genetic manipulation.     

Primary metabolites of Phaseolus vulgaris nodules

More ethanol soluble material (carbohydrate and amino nitrogen) was found in both host cell and bacteroid components of Phaseolus vulgaris nodules from plants grown at 28 W/m² than from plants grown at 7 W/m². The range of compounds identified was similar at the two irradiances. On feeding ¹⁴CO₂ to the plant tops at either irradiance the labelling patterns of carbohydrates and organic acids in the nodule host cells and bacteroids suggested that any or all of the following substances could be donated by the host to the bacteroids for general metabolism: sucrose, fructose, glucose, an unidentified carbohydrate, malic acid and an organic acid co-chromatographing with 6-phosphogluconate. Distribution and labelling patterns of nodule amino compounds were consistent with the hypothesis that ammonia is the primary product of nitrogen fixation within bacteroids, and that this ammonia is transported to host cells for assimilation, initially into glutamine and glutamate.     

Effects of elevated CO2 on the foraging behavior of cotton bollworm, Helicoverpa armigera

Effects of elevated CO2 on the foraging behavior of cotton bollworm Helicoverpa arrnigera Hübner reared on milky grains of spring wheat grown in ambient, 550μL/L and 750μL/L CO2 concentration atmospheres in open-top chambers （OTC） were studied. The results indicated that： （i） elevated CO2 significantly affected both the type and amount of food eaten by H.arrnigera reared on milky grains of ambient CO2-grown wheat were significant higher than those for bollworm larvae reared on wheat grains grown in 550 and 750μL/L CO2 atmospheres; （ii） when bollworm larvae were reared on mixed milky grains from different CO2-grown wheat （food-choice condition）, larval duration increased significantly-pupal weight, adult longevity, and fecundity decreased significantly, comparing with those reared on milky grains of ambient CO2-grown wheat, 550μL/L CO2-grown wheat and 750μL/L CO2-grown wheat respectively; （iii） significant decreases in the contents of fructose and gross protein （GP） and significant increases in the contents of glucose, amylose, total saccharides （TSC）, TSC： GP ratio, free amino acids and soluble protein in the wheat grains with CO2 rising; （iv） and selected-foraging amount/food-choice index of cotton bollworm H.armigera were significantly positive correlated with the contents of fructose and GP of wheat grains, but they had significantly negative relationships with the contents of glucose, amylose, TSC and TSC： GP ratio of wheat grains.     

3-mercaptopicolinic acid and energy metabolism in the liver fluke, Fasciola hepatica

Mercaptopicolinic acid inhibited ¹⁴CO₂ uptake and phosphoenolpyruvate carboxykinase activity in intact fluke. Studies with enzyme preparations showed that the inhibition was mixed-competitive with phosphoenolpyruvate and non-competitive with GTP. Inhibition was not reversed by Mn²⁺. Pyruvate kinase was not inhibited by mercaptopicolinic acid, although under certain circumstances, mercaptopicolinic acid interfered with the pyruvate kinase assay system. Intact flukes incubated with mercaptopicolinic acid showed depressed adenylate energy charge, increased lactic acid production and reduced flow of carbon from phosphoenolpyruvate to the mitochondrial substrate, malate. Additions of glutamate, alanine or aspartate did not reverse these effects even though, in each case, the amino acid was metabolised and considerably more acid end products were formed than in the absence of mercaptopicolinic acid. The changes in the concentrations of metabolites and end products are consistent with the view that, in flukes whose energy metabolism is impaired by mercaptopicolinic acid, pyruvate enters the mitochondrion and is converted to acetic and propionic acids.     

Organic acid production from CO2/H2 and CO/H2 by mixed-culture anaerobes

The gases CO, CO₂, and H₂ were used as substrates in anaerobic fermentations producing organic acids. Various mixed bacterial sources were used, including sewage sludge digester effluent, rabbit feces, and soil. Nonsterile microorganism selection was carried out using CO₂/H₂ and CO/H₂ as the primary carbon and energy sources. Cultures were grown in specially designed, high-pressure (to 70 psig) flasks. Methanogenic bacteria were eliminated from the cultures. Liquid products of the fermentations were acetic through caproic acids, with the even-numbered acids predominating. Carbon balances showed conclusively that acetic acid was formed from carbon contained in the CO or CO₂ feed gas. Measurements made included rates of acid product formation, cell density, and degree of gas utilization. Limited characterization of the microorganisms was also performed. Production of organic acids by mixed culture inocula from CO₂/H₂ or CO/H₂ had not been reported previously. Application of this work is to the production of organic chemicals from synthesis gas (SNG), produced by the gasification of fossil fuels (peat, lignite, and various ranks of coals), biomass (agricultural and forest residues, and various biomass crops grown expressly for energy recovery), and municipal solid waste.     

Brachymeria lasus: Culture in vitro of a chalcid insect parasite

The composition and formulation of an artificial medium and handling techniques are described for rearing the chalcid insect endoparasite, Brachymeria lasus (Walker) from the egg to adult stage under in vitro conditions. The medium was devoid of host materials, prepared aseptically, and contained 15% ($\text{w}\text{v}$) albumin, 1% amino acids, 4% glucose, and approximately 2.5% lipids, 0.3% inorganic salts, 0.01% B vitamins, and 0.2% lipogenic growth factors. Eggs were obtained by host dissection and developing larvae were reared in tissue culture plates. Adults were fecund.     

1,2 Propanediol utilization by Lactobacillus reuteri DSM 20016, role in bioconversion of glycerol to 1,3 propanediol, 3-hydroxypropionaldehyde and 3-hydroxypropionic acid

The objective of the presented work is to demonstrate the metabolism of 1,2 propandiol by Lactobacillus reuteri and to elucidate the metabolites produced during the process. This Metabolic pathway is crucial for biotechnological applications using L. reuteri in bioconversion of glycerol to industrially important plate-form chemicals. L. reuteri grown on minimal media containing 1,2 propanediol was able to utilize the compound as a sole carbon and energy source. The growth of the bacteria was linear with time; however the specific growth rate was significantly low compared to bacteria grown on the same media in the presence of glucose.The fermentation of 1,2 propanediol by L. reuteri in presence and absence of glucose was followed for 72 h and the metabolites produced during the process were detected using HPLC. 1,2 Propanediol was completely converted to propionaldhyde in a time dependent fashion, this process had a higher rate in presence of glucose. Consequently the produced propionaldhyde was converted to propionic acid and propanol in a skewed equimolar manner. In presence of glucose: acetic acid, lactic acid, succinic acid and ethanol were detected while in absence of glucose only minute amounts of acetic acid and lactic acid were detected which indicates presence of different metabolic pathways for glucose and 1,2 propanediol metabolism. Resting cells of L. reuteri induced in presence of 1,2 propanediol have shown significant capabilities to convert aqueous glycerol to 1,3 propanediol, 3-hydroxypropionaldhyde and a compound proposed to be 3-hydroxypropionic acid as detected by gas chromatographic technique.     

14CO2 dark fixation in the halophytic species mesembryanthemum crystallinum

The time course of ¹⁴CO₂ dark fixation was studied in leaves of the facultatively halophytic plant species Mesembryanthemum crystallinum cultivated with and without 400 mM NaCl in the nutrient medium. It is generally known from the literature that plants grown under saline conditions incorporate ¹⁴C predominately into amino acids. By contrast in leaves of M. crystallinum grown on NaCl and exposed to ¹⁴CO₂ in the dark, relatively more radioactivity is incorporated in the organic acids (especially malate) than in amino acids. The data obtained are discussed in relation to the NaCl induced Crassulacean acid metabolism in M. crystallinum reported earlier.     

Observations on the in vitro culture of Cotylurus erraticus (Trematoda: Strigeidae)

Mtitchell J. S., Halton D. W. and Smyth J. D. 1978. Observations on the in vitro culture of Cotylurus erraticus (Trematoda: Strigeidae). International Journal for Parasitology8: 389–397. Cotylurus erraticus metacercariae obtained from around the heart of rainbow trout were excysted and grown in vitro and in vivo to egg-producing adults. For in vitro development, tissue culture media M199 or NCTC 135 was used, together with varying amounts of chicken serum. Worms grown in media containing the highest concentration of serum (80% per volume) showed the fastest rate of development, measured by the time taken for the first eggs to appear in the uterus. The testes, ovaries and vitellaria of these worms were comparable in structure and histochemistry with those of worms reared in gulls. Eggs were produced by worms in all media containing chicken serum, but the eggs had abnormal shells and failed to embryonate.     

Effect of Modified Atmosphere Composition on the Metabolism of Glucose by Brochothrix thermosphacta

The influence of atmosphere composition on the metabolism of Brochothrix thermosphacta was studied by analyzing the consumption of glucose and the production of ethanol, acetic and lactic acids, acetaldehyde, and diacetyl-acetoin under atmospheres containing different combinations of carbon dioxide and oxygen. When glucose was metabolized under oxygen-free atmospheres, lactic acid was one of the main end products, while under atmospheres rich in oxygen mainly acetoin-diacetyl was produced. The proportions of the total consumed glucose used for the production of acetoin (aerobic metabolism) and lactic acid (anaerobic metabolism) were used to decide whether aerobic or anaerobic metabolism predominated at a given atmosphere composition. The boundary conditions between dominantly anaerobic and aerobic metabolisms were determined by logistic regression. The metabolism of glucose by B. thermosphacta was influenced not only by the oxygen content of the atmosphere but also by the carbon dioxide content. At high CO₂ percentages, glucose metabolism remained anaerobic under greater oxygen contents.