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.     

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.     

THE RELEASE OF AMINO ACIDS FROM THE HEMISECTED SPINAL CORD DURING STIMULATION

Abstract— Isolated frog or toad hemicords were incubated for 40 min with either [¹⁴C]glycine, [³H]GABA, l -[¹⁴C]glutamate. l -[¹⁴C]aspartate, l -[¹⁴C]serine, l [¹⁴C]threonine or l -[³H]leucine, and the release of these compounds from the cord was measured under resting conditions and during electrical stimulation. Stimulation of spinal roots produced no significant change in the efflux of any of the compounds tested. Direct stimulation of the rostral cord however, produced a large increase in the efflux of [¹⁴C]glycine, [³H]GABA, l -[¹⁴C]glutamate and l -[¹⁴C]aspartate. These increased effluxes were calcium dependent, the effects of stimulation being reduced in a calcium-free, or magnesium-supplemented (10 mM) medium. Stimulation failed to produce an increase in the efflux of l -[¹⁴C]serine, l -[¹⁴C]threonine, l -[¹⁴H]leucine, [¹⁴C]mannitol or [¹⁴C]urea. These results are consistent with the suggestions that glycine, GABA, glutamate and aspartate may be synaptic transmitters in the spinal cord.     

Lumiflavin and Lumichrome Transport in the Central Nervous System

Abstract: The transport of the lipid-soluble sugarless flavins, [¹⁴C]lumiflavin and [¹⁴C]lumichrome, into and from the isolated choroid plexus and brain slices was studied in vitro. The isolated choroid plexus accumulated both [¹⁴C] flavins by a saturable, energy-requiring process that did not depend on binding or intracellular metabolism of the [¹⁴C] flavins. Both sugar-containing and sugarless flavins, as well as cyclic organic acids, significantly inhibited [¹⁴C]lumiflavin and [¹⁴C]Iumichrome uptake by the isolated choroid plexus. Within 2.5 min, 75% of the [¹⁴C]lumiflavin accumulated by the isolated choroid plexus was released into the medium. Brain slices accumulated [¹⁴C]lumiflavin by a saturable process that did not meet all the criteria for active transport. Ninety-five percent of the [¹⁴C]lumiflavin accumulated by brain slices was released into the medium within 7.5 min. In vivo , 2 h after the intraventricular injection of 6.5 nmol [¹⁴C]lumiflavin, almost all of the [¹⁴C]flavin was cleared from the CNS. Addition of 3.5 μmol FMN to the intraventricular injectate significantly decreased the clearance of [¹⁴C]lumiflavin from the CNS. These studies document that the sugarless flavins are transported by the flavin transport systems in the CNS.     

Isoprenoid biosynthesis in bacteria: Two different pathways?

Abstract The biosynthesis of isopentenylpyrophosphate, a central intermediate of isoprenoid formation, was investigated in six different bacterial organisms. Cell-free extracts of Myxococcus fulvus, Staphylococcus carnosus, Lactobacillus plantarum and Halobacterium cutirubrum converted [¹⁴C]acetyl-CoA or [¹⁴C]hydroxymethylglutaryl-CoA to [¹⁴C]mevalonic acid. Furthermore, [¹⁴C]mevalonic acid, [¹⁴C]mevalonate-5-phosphate and [¹⁴C]mevalonate-5-pyrophosphate were metabolized to [¹⁴C]isopentenylpyrophosphate in bacteria. In contrast, no intermediates of this reaction sequence could be detected using cell-free extracts of Zymomonas mobilis and Escherichia coli . These results indicate that at least two different pathways for the biosynthesis of isopentenylpyrophosphate are present in bacteria.     

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.     

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.     

Oligodendroglial Glycerophospholipid Synthesis: Incorporation of Radioactive Precursors into Ethanolamine Glycerophospholipids by Calf Oligodendroglia Prepared by a Percoll Procedure and Maintained in Suspension Culture

Abstract: Oligodendroglia prepared from minced calf cerebral white matter by trypsinization at pH 7.4, screening, and isosmotic Percoll (polyvinylpyr-rolidone-coated silica gel) density gradient centrifugation survived in culture on polylysine-coated glass, extending processes and maintaining phenotypic characteristics of oligodendroglia. In the present study, ethanolamine glycerophospholipid (EGP) metabolism of the freshly isolated cells was examined during short-term suspension culture by dual label time course and substrate concentration dependence experiments with [2-³H]glycerol and either [1,2-¹⁴C]ethanolamine or L-[U-¹⁴C]serine. Rates of incorporation of ³H from the glycerol and of ¹⁴C from the ethanolamine into EGP were constant for 14 h. In medium containing 3 mM-[1,2-¹⁴C]ethanolamine and 4.8 mM-[2-³H]glycerol, rates of incorporation of ¹⁴C and ³H into diacyl glycerophosphoethanolamine (diacyl GPE) were similar. Under the same conditions, ³H specific activities of alkylacyl GPE and alkenylacyl GPE were much lower than ¹⁴C specific activities, likely as a result of the loss of tritium during synthesis of these forms of EGP via dihydroxyacetone phosphate. L-[U-¹⁴C]serine was incorporated into serine glycerophospholipid (SGP) by base exchange rather than de novo synthesis. ¹⁴C from L-[U-¹⁴C]serine also appeared in EGP after an initial lag period of several hours. Methylation of oligodendroglial EGP to choline glycerophospholipid (CGP) was not detected.     

Bradykinin effects on phospholipid metabolism and its relation to arachidonic acid turnovers in neuroblastoma × glioma hybrid cells (NG 108-15)

In neuroblastoma × glioma hybrid cells (NG 108-15) labelled with [³²P]-trisodium phosphate, [³H]-inositol and [¹⁴C]-arachidonic acid, bradykinin stimulated the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) while it had no effect on the release of [¹⁴C]-arachidonic acid (AA). The effect on PIP, was time- and dose-dependent with a maximal effect on [³H]-inositol- and [³²P]-labelled cells after 10–30 s of stimulation with 10⁻⁶ M bradykinin. However, the hydrolysis of [¹⁴C]-AA labelled PIP₂ was delayed compared to the effect on [³H]- and [¹⁴C]-PIP₂ and was not detectable until after 60 s of stimulation. Bradykinin stimulation resulted in an increased formation of [³H]-inositol phosphates (IP) and [³²P]- and [¹⁴P]- and [¹⁴C]-phosphatidic acid (PA) but the time course for PA formation did not allow the time-course for PIP₂ hydrolysis. A reduced labelling of [²³P]- and [¹⁴C]-phosphatidylcholine was also found in stimulated cells suggesting that PA may derive from other sources than PIP₂. In conclusion, our results indicate that bradykinin activates phospholipase C, but not phospholipase A₂, in NG 108-15 cells.     

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.     

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.     

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.     

METABOLISM OF GLUCOSE AND FREE AMINO ACIDS IN BRAIN, STUDIED WITH 14C-LABELLED GLUCOSE AND BUTYRATE IN RATS INTOXICATED WITH CARBON DISULPHIDE

Abstract— The effect of 15 h continuous exposure to CS₂ on the metaboliam of glucose and free amino acids in the brain of rats was studied. CS₂ caused a moderate hypoglycaemia. There were also changes in the amounts of some amino acids in the brain. Glutamate and γ-aminobutyrate were lower whereas glutamine was markedly increased. Comparative studies in vivo of the metabolism of [2-¹⁴C]glucose and [1-¹⁴C]butyrate indicated that CS₂ did not affect glycolysis or the incorporation of ¹⁴C from glucose into amino acids except into γ-aminobutyrate which was reduced. Contrary to the findings with [¹⁴C]glucose, CS₂ provoked distinct changes in the labelling of amino acids when [¹⁴C]butyrate was the precursor. The most notable change was a markedly increased incorporation of ¹⁴C into glutamine. Based on the two-compartment model of brain glutamate the experimental findings indicated that CS₂ affected metabolism associated with the 'small' pool of glutamate but had a minimal effect on metabolism associated with the 'large' glutamate pool. The possibility is suggested that the changes observed involved an increased rate of ammonia removal. The low incorporation of ¹⁴C into γ-aminobutyrate from either precursor is consistent with other evidence showing that CS₂ interferes with pyridoxal phosphate-dependent enzymes.     

Metabolism of CO2 by leaves of different photosynthetic types of Neurachne species

Following a series of continuous exposures to ¹⁴CO₂ for different lengths of time, leaves from Neurachne munroi (C₄), N. minor (C₃-C₄) and N. tenuifolia (C₃| were estimated to assimilate 100%, 9% and 2–4%, respectively, of atmospheric CO₂ by the C₄ pathway. The percentage of ¹⁴C-label appearing in malate and aspartate in leaves of N. minor progressively increased with longer exposure times indicating that a significant proportion of its C₄ acids are formed as secondary products. In ¹⁴CO₂/¹²CO₂ pulse/chase experiments, the ¹⁴C-label in leaves of N. munroi was rapidly transferred from C₄ acids to sugar monophosphates plus sugar diphosphates, and finally to sucrose. In leaves of N. minor, the ¹⁴C-label was slowly metabolized from the C-4 carboxyl of malate and asparate (apparent half-time = 250 s), and the formation of C₄ acids as secondary products was again evident. ¹⁴C-label in serine/glycine accumulated to comparable magnitudes in both N. minor and in N. tenuifolia, but there was an initial lag phase in the accumulation of label in N. minor. C₄ photosynthesis is apparently of minimal importance in reducing photorespiration in N. minor, but leaf anatomical specializations and a possible compartmentation of photorespiratory metabolism may be of considerable importance.     

稻田与旱地土壤自养微生物同化碳在土壤中的矿化与转化特征

选择亚热带地区3种典型稻田和旱地土壤,应用碳同位素^14C-CO₂标记示踪技术结合室内模拟培养试验,研究自养微生物同化碳(“新碳”)在土壤碳库中的矿化和转化特征.结果表明: 在100 d的培养期内,“新碳”的矿化经历了先上升、10 d后缓慢下降、最后渐趋稳定的3个阶段.“新碳”的矿化比例为8.0%～26.9%,矿化速率为0.01～0.22 μg ¹⁴C·g^-1·d^-1,其中,稻田土壤为0.01～0.22 μg ¹⁴C·g^-1·d^-1,旱地土壤为0.01～0.08 μg ¹⁴C·g^-1·d^-1,而原有有机碳的矿化比例为1.6%～5.7%,矿化速率为1.3～25.66 μg C·g^-1·d^-1.土壤活性碳库\[可溶性有机碳(DOC)、微生物生物量碳(MBC)\]中,¹⁴C-DOC在培养初期(0～10 d)先上升,升高幅度达0.3 mg·kg^-1,10～30 d又迅速下降,下降幅度达0.42 mg·kg^-1,至30 d后缓慢下降.¹⁴C-MBC的波动与¹⁴C-DOC不同,在培养初期(0～10 d)先迅速下降,10～30 d又迅速上升,至40 d后缓慢下降并趋于稳定.水稻土¹⁴C-DOC/DOC的转化更新速率明显大于旱地,而旱地¹⁴C-MBC/MBC的转化更新速率大于水稻土.
     

RDX (Hexahydro-1,3,5-trinitro-1,3,5-triazine) Biodegradation in Aquifer Sediments under Manganese-Reducing Conditions

A shallow, RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine)-contaminated aquifer at Naval Submarine Base Bangor has been characterized as predominantly manganese-reducing, anoxic with local pockets of oxic conditions. The potential contribution of microbial RDX degradation to localized decreases observed in aquifer RDX concentrations was assessed in sediment microcosms amended with [U-¹⁴C] RDX. Greater than 85% mineralization of ¹⁴C-RDX to ¹⁴CO₂ was observed in aquifer sediment microcosms under native, manganese-reducing, anoxic conditions. Significant increases in the mineralization of ¹⁴C-RDX to ¹⁴CO₂ were observed in anoxic microcosms under NO₃-amended or Mn(IV)-amended conditions. No evidence of ¹⁴C-RDX biodegradation was observed under oxic conditions. These results indicate that microbial degradation of RDX may contribute to natural attenuation of RDX in manganese-reducing aquifer systems.     

The effect of dichloroacetate and hydroxypyruvate on the entry of C from [1-C]alanine into urea in rat hepatocytes

Entry of metabolic ¹⁴CO₂ into urea is shown to be decreased by dichloroacetate although the production of ¹⁴CO₂ is stimulated 2-fold. Hydroxypyruvate, a product of dichloroacetate metabolism, increases the incorporation of metabolic ¹⁴CO₂ into urea. It is proposed that these effects result from changes in the cytoplasmic-mitochondrial pH gradient.     

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.     

Denitrification in sediment determined from nitrogen isotope pairing

Abstract A new method for accurate and easy measurement of denitrification in sediments is presented. The water overlying intact sediment cores was enriched with ¹⁵NO₃⁻ which mixed with the ¹⁴NO₃⁻ of the natural sources of NO₃⁻. The formation by denitrification of single-labeled (¹⁴N¹⁵N) and double-labeled (¹⁵N¹⁵N) dinitrogen pairs was measured by mass spectrometry after a few hours incubation. Total denitrification including the formation of unlabeled (¹⁴N¹⁴N) dinitrogen could be calculated assuming random isotope pairing by denitrification of the uniformly mixed NO₃⁻ species. In contrast to previous approaches, by this method it is possible to measure denitrification of both NO₃⁻ diffusing from the overlying water and NO₃⁻ from nitrification within the sediment.     

IN VIVO FORMATION AND CATABOLISM OF [14C]HISTAMINE IN MOUSE BRAIN

Abstract— The formation of histamine in brain was studied in mice injected with l -[¹⁴C]-histidine (ring 2-¹⁴C) intravenously (i.v.) or intracerebrally; [¹⁴C]histamine appeared rapidly and exhibited a rapid rate of turnover. Drugs known to block various pathways of histamine catabolism were tested for effects on brain–[¹⁴C]histamine and [¹⁴C]-methyl-histamine in mice given (1) [¹⁴C]histamine i.v., (2) [¹⁴C]histamine intracerebrally, and (3) l -[¹⁴C]histidine i.v. Blood-borne histamine did not enter brain; brain histamine was formed locally by decarboxylation of histidine Methylhistamine did cross the blood-brain barrier. Methylation was the major route of histamine catabolism in mouse brain and some of the methylhistamine formed was destroyed by monoamine oxidase. No evidence for catabolism by the action of diamine oxidase was found.