Microbial transformation of labile dissolved organic matter into humic-like matter in seawater

Abstract Microbial transformation of labile, low molecular weight dissolved organic matter (DOM) into dissolved humic matter (DHM) was studied in seawater. Surface water samples were amended with [¹⁴C into ¹⁴CO₂, TO¹⁴C (total organic ¹⁴C), and PO¹⁴C (particulate organic ¹⁴C), was measured over time in confined samples. The humic and non-humic fractions of DO¹⁴C (dissolved organic ¹⁴C) were separated according to a common operational definition of DHM based on adsorption on XAD-8 macroporous resin. Both TO¹⁴C and non-humic DO¹⁴C decreased during the experiments. However, ¹⁴C-labelled DHM increased during the first week of the incubations, to a level where it comprised 15% of the TO¹⁴C remaining in the samples, or 3% of the initially added ¹⁴C. Towards the end of experiments (ca 70 days), the humic fraction of DO¹⁴C gradually approached the background level of poisoned control samples. Provided that the XAD-8 operational definition of DHM is accepted, this study indicates that humic matter may be formed in seawater within days from labile monomers such as glucose.     

Partitioning of CO2 Production Between Glucose and Lactate in Excised Sympathetic Ganglia, with Implications for Brain

Abstract: Chains of lumbar sympathetic ganglia from 15-day-old chicken embryos were incubated for 4 h at 36°C in a bicarbonate-buffered salt solution equilibrated with 5% CO₂-95% O₂. Glucose (1–10 m M ), lactate (1–10 m M ), [U-¹⁴C]glucose, [1-¹⁴C]glucose, [6-¹⁴C]glucose, and [U-¹⁴C]lactate were added as needed. ¹⁴CO₂ output was measured continuously by counting the radioactivity in gas that had passed through the incubation chamber. Lactate reduced the output of CO₂ from [U-¹⁴C]glucose, and glucose reduced that from [U-¹⁴C]lactate. When using uniformly labeled substrates in the presence of 5.5 m M glucose, the output of CO₂ from lactate exceeded that from glucose when the lactate concentration was >2 m M . The combined outputs at each concentration tested were greater than those from either substrate alone. The ¹⁴CO₂ output from [1-¹⁴C]glucose always exceeded that from [6-¹⁴C]glucose, indicating activity of the hexose monophosphate shunt. Lactate reduced both of these outputs, with the maximum difference between them during incubation remaining constant as the lactate concentration was increased, suggesting that lactate may not affect the shunt. Modeling revealed many details of lactate metabolism as a function of its concentration. Addition of a blood-brain barrier to the model suggested that lactate can be a significant metabolite for brain during hyperlactemia, especially at the high levels reached physiologically during exercise.     

Effects of In Vivo Hypoxia on Acetylcholine Synthesis by Rat Brain Synaptosomes

Abstract: Synaptosomes from normoxic and hypoxic rats were incubated aerobically in the presence and absence of veratridine. In the absence of veratridine, no significant difference was observed between the two types of preparation regarding either ATP/ADP ratio or ¹⁴CO₂ or [¹⁴C]acetylcholine synthesis from D-[U-¹⁴C]glucose. However, in the presence of veratridine, significant reductions in the output of ¹⁴CO₂ and [¹⁴C]acetylcholine by synaptosomes from hypoxic rats were apparent. It was concluded that irreversible metabolic lesions occur at the synapse as a result of hypoxia, which are apparent only when the metabolism of the preparation is accelerated to a level comparable with the maximal rate occurring in vivo. The presence of such lesions is further evidenced by the significant reductions in ATP/ADP ratio, ¹⁴CO₂ output, and [¹⁴C]acetylcholine synthesis that occur in synaptosomes from hypoxic rats made anoxic in vitro and permitted to recover. Such decreases are not seen when synaptosomes from normoxic rats are similarly treated.     

THE EFFECT OF DIABETES, INSULIN AND WALLERIAN DEGENERATION ON LEUCINE METABOLISM OF ISOLATED RAT SCIATIC NERVES

Abstract– ¹⁴CO₂ production and ¹⁴C incorporation into proteins was studied in isolated rat sciatic nerves during incubation with 0.1 mM-[1-¹⁴C]leucine. Rats were made diabetic with streptozotocin. Nerves from diabetic rats incubated with glucose oxidized more [¹⁴C]leucine than controls. This difference was abolished in the presence of insulin (1 mU/ml). The effects of diabetes and insulin on leucine oxidation could not be demonstrated in the absence of glucose. Insulin stimulated the incorporation of [¹⁴C] from leucine into proteins by nerves from controls and diabetic rats.
Nerves undergoing Wallerian degeneration showed a marked increase in DNA content and stimulated incorporation of [¹⁴C]leucine into proteins. ¹⁴CO₂ production from leucine proceeded at 75% of the rate observed in intact nerves. Neither insulin nor diabetes affected leucine metabolism in degenerating nerves.
Neither the extracellular space nor the concentration of free amino acids were significantly different in nerves obtained from control and diabetic rats, except for lower glutamine content in the latter.
In vitro leucine metabolism of nerves is affected by diabetes, insulin and the integrity of the axon. The Schwann cell is suggested as a possible site of the observed changes in leucine metabolism.     

Analysis of [14C] indole-3-acetic acid metabolites from the primary roots of Zea mays seedlings using reverse-phase high-performance liquid chromatography

Methanolic extracts of Zea mays L. cv. Fronica root segments which had been incubated in [¹⁴C] indole-3-acetie acid were analysed by reverse-phase high-performance liquid chromatography. Metabolism of indole-3-acetic acid was found to be rapid and extensive with at least 11 products apparent after a 2 h incubation. A comparison of metabolites of [1-¹⁴C]– and [2-¹⁴C] IAA, calculations of ¹⁴CO₂ evolution, and data on the polarity of products indicated that decarboxylation had not occurred. An average of 34% of the radioactivity remained associated with the indole-3-acetic acid peak.     

Production of [14C]Acetylcholine and [14C]Carbon Dioxide from [U–14C]Glucose in Tissue Prisms from Aging Rat Brain

Abstract: Production of [¹⁴C]acetylcholine and ¹⁴CO₂ was examined by using tissue prisms from neocortex, hippocampus, and striatum from rats aged approximately 5 months, 13 months, and 27 months. [¹⁴C]Acetylcholine synthesis in the striatum showed highly significant decreases with age for measurements in the presence of both 5 m m - and 31 m m -K⁺, contrasting with the lack of significant change in ¹⁴CO₂ production in this region. The neocortex and hippocampus showed only small changes, especially when comparison was made between 13-month and senescent animals. Measurements of the release of [¹⁴C]acetylcholine and influence of atropine on this release confirmed the relative stability with age of the cholinergic system in the neocortex.     

L-GLUTAMIC ACID DECARBOXYLASE IN NON-NEURAL TISSUES OF THE MOUSE

Abstract— Low levels of γ-aminobutyric acid (GABA) and of glutamic acid decarboxylase (GAD) activity have been detected in mouse kidney, liver, spleen and pancreas. Quantitation of both ¹⁴CO₂ and [¹⁴C]GABA produced in radiometric assays from [U-¹⁴CJglutamic acid has shown that measurement of ¹⁴CO₂ evolution alone is not, in all cases, a valid estimate of true GAD activity. As evidenced by increased ^,14CO₂ production upon addition of NAD and CoA to assay mixtures, radiometric assay of GAD activity in crude homogenates may yield ¹⁴CO₂ via the coupled reactions of glutamic acid dehydrogenase and a-ketoglutarate dehydrogenase. The addition of 1 mM aminooxyacetic acid (AOAA) to assays of kidney homogenates inhibited [^,14C]GABA production 92 per cent while ¹⁴CO₂ production was inhibited only 53 per cent. No evidence was found to confirm the reported existence of a second form of the enzyme, GAD II. previously described by Haber el al. (H aber B., K uriyama K. & R oberts E. (1970) Biochem. Pharmac. 19, 1119-1136). Based on sensitivity-to AOAA and chloride inhibition, the GAD activity in mouse kidney is. apparently, indistinguishable from that of neural origin.     

TIME-DEPENDENT PARTITIONING OF GLUCOSE CARBONS METABOLIZED BY THE SUPERIOR CERVICAL GANGLION FROM CALVES

Abstract— Glucose metabolism in the superior cervical ganglion for calves has been studied by incubating slices with [1-¹⁴C]-, [6-¹⁴C]- and [U-¹⁴C]-labelled glucose at 37°C and pH 7.4. Glucose utilization and the metabolic partitioning of glucose carbon in products during different incubation periods ranging from 5 to 60 min were determined by isotopic methods.
Separation and identification of labelled compounds have been achieved by anion and cation exchange chromatography as well as by TLC and enzymatic analyses.
From the data obtained a carbon balance could be constructed showing lactate to be the major product of glucose metabolism followed by CO₂ and amino acids. Measuring the release of ¹⁴CO₂ from differently ⁴C-labelled glucose, the existence of an active pentose phosphate pathway in the ganglion could be demonstrated although this pathway seems to contribute only to a small extent to glucose metabolism. The marked decrease of the C-U: C-6 and the C-U:C-1 ratios in ¹⁴CO₂ observed in the course of incubation is discussed in terms of a time-dependent change in the rate of synthesis of amino acids which are directly connected with intermediates of the citric acid cycle.     

Influence of Temperature on Lactate Utilization by Round Spermatids from Rat Testes

Rotenone-sensitive ¹⁴CO₂ formation from [¹⁴C]lactate and oxygen consumption by round spermatids were found to be greater at elevated temperatures than at 34°C. More than 96% of the total radioactivity of the metabolized [¹⁴C]lactate was recovered in the released CO₂ and the acid soluble fraction of the cells. There was practically no incorporation of [¹⁴C]latctate into the lipid, nucleic acid, and protein fractions. Intracellular level of ATP in spermatids was enhanced in the presence of lactate (20 mM) at 34°C (scrotal temperature), whereas it was decrease at 37°C (body temperature). However, this was reversible when the cells were transferred from the elevated temperature to 34°C. It was also found that oxygen consumption and CO₂ production were increased at 34°C by 2, 4-dinitrophenol (DNP), but decreased by oligomycin. On the other hand, oligomycin and DNP had no effect on oxygen consumption and ¹⁴CO₂ formation at the elevated temperature.
These findings provide evidence that lactate utilization by spermatids is coupled with oxidative phosphorylation at scrotal temperature, but becomes uncoupled at elevated temperature, although more lactate is consumed.     

Effect of Denervation and Reinnervation on Oxidation of [6-14C]Gucose by Rat Skeletal Muscle Homogenates

Abstract: We studied the effects of denervation and reinnervation of the rat extensor digitorum longus muscle (EDL) on the oxidation of [6-¹⁴C]glucose to ¹⁴CO₂. The rate of ¹⁴CO₂ production decreased dramatically following denervation, and the decrease became significant 20 days after nerve section. Prior to day 20, changes apparently reflected the decline of muscle mass. Decreased ¹⁴CO₂ production was due to reduced capacity of the enzymatic system (apparent V_max); there was no change in apparent affinity for glucose (apparent K _m). Mixing experiments revealed that the loss of oxidative capacity following denervation is not caused by production of soluble inhibitors by degenerating muscle. Oxidative metabolism, as measured by ¹⁴CO₂ evolution, recovered during reinnervation. Surprisingly, the specific activity in reinnervated muscles displayed an "overshoot" of approximately 50%, which returned to control by day 60, possibly reflecting increased energy demand by the growing muscle. The time-course of the denervation-mediated change indicates that altered oxidative capacity is secondary to events that initiate denervation changes in muscle. Nevertheless, diminished oxidative capacity may be of considerable metabolic significance in denervated muscle.     

The inhibition of photosynthesis and photorespiration in isolated mesophyll cells of Phaseolus and Lycopersicon by reduced osmotic potentials

Mesophyll cells isolated from Phaseolus vulgaris and Lycopersicon esculentum show decreasing photosynthetic rates when suspended in media containing increasing concentrations of osmoticum. The photosynthetic activity was sensitive to small changes in osmotic potential over a range of sorbitol concentrations from 0.44 M (−1.08 MPa) to 0.77 M (−1.88 MPa). Photorespiration assayed by ¹⁴CO₂ release in CO₂-free air and by ¹⁴CO₂ release from the oxidation of [1–¹⁴C] glycolate also decreased as the osmotic potential of the incubation medium was reduced. The CO₂ compensation points of the cells increased with increasing concentration of osmoticum from approximately 60 μ I⁻¹1 at −1.08 MPa to 130 μl 1⁻¹ for cells stressed at −1.88 MPa. Changes in photosynthetic and photorespiratory activities occurred at moderate osmotic potentials in these cells suggesting that in whole leaves during a reduction in water potential, non- stomatal inhibition of CO₂ assimilation and glycolate pathway metabolism occurs simultaneously with stomatal closure.     

Confirmation of operative acetate, H2/CO2 and methanol methanogenic pathways in the solid-state refuse fermentation

By use of the radiolabelled substrates sodium [1–¹⁴C] acetate, sodium [2–¹⁴C] acetate, NaH¹⁴CO₃ and ¹⁴CH₃OH, three of the possible methanogenic pathways in fermenting refuse were confirmed. Due to the absence of a methanol pool, however, the relative contribution of each could not be determined. Circumstantial evidence for an operative trimethylamine pathway was gained but not confirmed whilst preliminary attempts to stimulate methanogenesis in refuse by supplementation with mono-and dimethylamine proved unsuccessful.     

Effects of Osmotic Pressure on the Oxidative Metabolism of Leishmania major Promastigotes

Leishmania major promastigotes were washed and resuspended in an iso-osmotic buffer. The rate of oxidation of ¹⁴C-labeled substrates was then measured as a function of osmolality. An acute decrease in osmolality (achieved by adding H₂O to the cell suspension) caused an increase in the rates of ¹⁴CO₂ production from [6-¹⁴C]glucose and, to a lesser extent, from [1, (3)-¹⁴C]glycerol. An acute increase in osmolality (achieved by adding NaCl, KCl, or mannitol) strongly inhibited the rates of ¹⁴CO₂ production from [1-: ¹⁴C]alanine, [1-¹⁴C]glutamate, and [1, (3)-¹⁴C]glycerol. The rates of ¹⁴CO₂ formation from [1-¹⁴C]laurate, [1-¹⁴C]acetate, and [2-¹⁴C]glucose (all of which form [1-¹⁴C]acetyl CoA prior to oxidation) were also inhibited, but less strongly, by increasing osmolality. These data suggest that with increasing osmolality there is an inhibition of mitochondrial oxidative capacity, which could facilitate the increase in alanine pool size that occurs in response to hyper-osmotic stress. Similarly, an increase in oxidative capacity would help prevent a rebuild up of the alanine pool after its rapid loss to the medium in response to hypo-osmotic stress.     

Altered resource allocation during seed development in Arabidopsis caused by the abi3 mutation

The regulation of whole-plant resource allocation during seed development in Arabidopsis thaliana was investigated by examining growth rate and partitioning of ¹⁴CO₂ in wild-type plants and those carrying the abi3 mutation. Plants carrying the abi3 mutation partitioned more resources into seed development than the wild type. The extra resources were available as a result of delayed senescence of the cauline leaves in the mutant. After supply of ¹⁴CO₂ at later stages of reproductive development differences in patterns of ¹⁴C distribution between mutant and wild type were consistent with long-term changes in growth and allocation. The role of long-distance signals in the regulation of seed yield in Arabidopsis is discussed.     

Factors affecting the validity of the 13C-urea breath test for in vivo determination of Helicobacter pylori infection status in a mouse model

Background. The mouse model using a human isolate of Helicobacter pylori is being widely accepted as an economical means of studying gastric infection. A noninvasive monitoring method would be useful for repeated testing to establish the time course of infection and the efficacy of treatments. In this study, we describe factors that affect interpretation of ¹³C urea breath test results for the assessment of H. pylori infection status in this model.
Materials and Methods. Female C57Bl/6 mice that underwent gavage with H. pylori or saline were breath-tested using 50 μg of ¹³C urea at intervals up to 2 months after inoculation. The generation of ¹³CO₂ (excess δ¹³CO₂) by infected mice was compared to that of uninfected controls. The effects of diet, fasting, and coprophagy on the reliability of the ¹³C urea breath test were quantitated.
Results. Both commercial and synthetic mouse diets exhibited marked in vitro urease activity. A minimum fasting time of 13 hours prior to breath testing significantly reduced this dietary contribution to excess δ¹³CO₂ values. The coprophagic tendency of the mice caused spuriously high excess δ¹³CO₂ counts in the breath of both control and H. pylori –infected mice.
Conclusions. Although the dietary contribution to spuriously high values of excess δ¹³CO₂ in mice breath-tested for H. pylori infection was reduced by fasting, the high nonspecific urease activity generated by coprophagy severely limited the reliability of the urea breath test in the assessment of H. pylori infection status.     

Biosynthesis of aromatic amino acids by highly purified spinach chloroplasts – Compartmentation and regulation of the reactions

The ability of chloroplasts to synthesize aromatic amino acids from CO₂ was investigated using highly purified, intact spinach ( Spinacia oleracea L. cv. Viking II) chloroplasts and ¹⁴CO₂. Incorporation of ¹⁴C into aromatic amino acids was very low, however, and this was assumed to be due to lack of phosphoenolpyruvate (PEP), one of the substrates for the shikimate/arogenate pathway leading to aromatic amino acids in chloroplasts. Therefore, the glycolytic enzymes phosphoglycerate mutase (EC 2.7.5.3) and enolase (EC 4.2.1.11) were added to the ¹⁴CO₂ fixation medium in order to convert labelled 3-phosphoglycerate exported from the intact chloroplasts to 2-phosphoglycerate and PEP. In this way a part of the glycolytic pathway was reconstituted outside the chloroplasts to substitute for the cytoplasm lost on isolation. The presence of both enzymes in the medium increased incorporation of ¹⁴C into Tyr and Phe more than ten-fold and incorporation into Trp about two-fold, while total ¹³CO₂ fixation rates were not affected. Our results suggest that chloroplasts do not contain phosphoglycerate mutase or enolase, and that, in vivo, PEP is synthesized in the cytoplasm and imported to the chloroplast stroma for the biosynthesis of aromatic amino acids. The biosynthesis of all three aromatic amino acids was under feedback control. Using expected physiological concentrations (below 100 μ M ), each of the aromatic amino acids exerted a strict feedback inhibition of its own biosynthesis only.     

Growth of Mycobacterium paraffinicum on Low Concentrations of Ethane in Soils

S ummary : Growth in soils of Mycobacterium paraffinicum with 0, 5, 15, 45 and 270 p/m of ethane was assayed by adding ethane-1,2-¹⁴C and measuring the ¹⁴CO₂ produced. With < 45 p/m of ethane the lag period lasted several months. Differences in growth associated with different soils suggest that the microbial method of oil prospecting is unlikely to be commercially successful.     

Photoinhibition of respiratory CO2 release in the green alga Scenedesmus

¹⁴CO₂ evolution of prelabeled Scenedesmus obliquus Kütz, has been followed in the dark and in the light. In the light, no carbon dioxide is evolved. Addition of unlabeled NaHCO, leads to ¹⁴CO₂ release attaining 20 to 30% of the dark rate. Double-reciprocal plots of NaHCO₃ concentrations vs ¹⁴CO₂ release results in a straight line, indicative of competition between exogenously supplied bicarbonate and endogenously evolved carbon dioxide. With this method, it is possible to measure CO₂ evolved by respiration in the light and to show that true photoinhibition of respiration occurs in Scenedesmus . In the light. DCMU substantially increases ¹⁴CO₂ evolution; in the presence of the uncoupler carbonyl cyanide- m -chlorophenylhydrazone. ¹⁴CO₂ evolution is comparable to that in the dark. ¹⁴CO₂ release and oxygen uptake in the dark are only slightly affected by cyanide, indicative of a cyanide-resistant respiration and/or fermentation as the essential CO₂-yielding processes in the presence of cyanide. These results, compared with concurrent ATP levels, lead us to assume that energy charge is not the only factor responsible for photoinhibition of respiration.     

Lignin-modifying enzymes from selected white-rot fungi: production and role from in lignin degradation

Abstract: White-rot fungi produce extracellular lignin-modifying enzymes, the best characterized of which are laccase (EC 1.10.3.2), lignin peroxidases (EC 1.11.1.7) and manganese peroxidases (EC 1.11.1.7). Lignin biodegradation studies have been carried out mostly using the white-rot fungus Phanerochaete chrysosporium which produces multiple isoenzymes of lignin peroxidase and manganese peroxidase but does not produce laccase. Many other white-rot fungi produce laccase in addition to lignin and manganese peroxidases and in varying combinations. Based on the enzyme production patterns of an array of white-rot fungi, three categories of fungi are suggested: (i) lignin-manganese peroxidase group (e.g. P. chrysosporium and Phlebia radiata ), (ii) manganese peroxidase-laccase group (e.g. Dichomitus squalens and Rigidoporus lignosus ), and (iii) lignin peroxidase-laccase group (e.g. Phlebia ochraceofulva and Junghuhnia separabilima ). The most efficient lignin degraders, estimated by ¹⁴CO₂ evolution from ¹⁴C-[Ring]-labelled synthetic lignin (DHP), belong to the first group, whereas many of the most selective lignin-degrading fungi belong to the second, although only moderate to good [¹⁴C]DHP mineralization is obtained using fungi from this group. The lignin peroxidase-laccase fungi only poorly degrade [¹⁴C]DHP.     

SYNTHESIS AND RELEASE OF [14C]ACETYLCH0LINE IN SYNAPTOSOMES

Abstract— Synaptosomes took up [¹⁴C]choline, about half or more of which was converted to [^I4C]acetylcholine when incubated in an appropriate medium containing 1 to 5 μ M-[¹⁴C] choline and neostigmine. The amount of [¹⁴C]acetylcholine synthesized in synaptosomes increased in parallel with the increase of Na⁺ concentration in the incubation medium. The effect of Na⁺ on the uptake of [^I4C]choline into synaptosomes was dependent on the concentration of choline in the incubation medium.
About 25 per cent of [¹⁴C]acetylcholine synthesized in synaptosomes was released rapidly into the medium by increasing the K⁺ concentration in the medium from 5 m m to 35 m m . The change of Na⁺ concentration hardly affected the release of [¹⁴C]acetylcholine. The effect of K⁺ on the release of [¹⁴C]choline was rather small compared to that on [¹⁴C] acetylcholine. Ouabain promoted the release of [¹⁴C]acetylcholine.