Vanillic acid, a metabolite of 4-hydroxy-3-methoxyphenylglycol and 4-hydroxy-3-methoxymandelic acid in man

Abstract: 4-Hydroxy-3-methoxyphenylglycol (HMPG) labelled with ¹⁴C was used to study the metabolic fate of HMPG in six healthy volunteers. Besides conjugation and oxidation to 4-hydroxy-3-methoxymandelic acid (HMMA, VMA) a minor portion, 8.4 ± 1.1% (mean ± SEM) was excreted as ¹⁴C-labelled vantllic acid (VA). To study if VA was formed from HMPG or HMMA (VMA), deuterium-labelled HMPG ([²H₃]HMPG) and HMMA ([²H₆]HMMA) were simultaneously injected intravenously to seven healthy volunteers. The recovery of [²H₃]VA from [²H₃]HMPG was 8.3 ± 2.1% and the recovery of [²H₆]VA from [²H₆]HMMA was 9.0 ± 2.1%. The ²H-labelled VAs were probably formed by a decar boxylation reaction, in the case of HMPG after previous oxidation to HMMA.     

Stimulation of shoot elongation in Salix pentandra by gibberellin A9; activity appears to be dependent upon hydroxylation to GAl via GA20

Cessation of shoot elongation in seedlings of Salix pentandra L. is induced by short photoperiod. Gibbereliin A₉ (GA₉) applied either to the apical bud or injected into a mature leaf, induced shoot elongation under a short photoperiod of 12 h, and GA₉ could completely substitute for a transfer to a long photoperiod. When [³H]GA₉ or [²H₂]GA₉ was injected into a leaf, no [³H]GA₉ was detected in the elongating apex and only traces of [³H]GA₉ were found in the shoot above the treated leaf. By the use of gas chromatography-mass spectrometry (GC-MS), [²H₂]GA₂₀ was identified as the main metabolite of [²H₂]GA₉ in both the shoot and the treated leaf. In addition, [²H₂]GA₁ and [²H₂]GA₂₉ were also identified as metabolites of [²H₂]GA₉. These results are consistent with the hypothesis that exogenous GA, promotes shoot elongation in Salix through its metabolism to GA₂₀ and GA,.     

Development of Synaptic Glycoproteins: Effect of Postnatal Age on the Synthesis and Concentration of Synaptic Membrane and Synaptic Junctional Fucosyl and Sialyl Glycoproteins

Abstract: Synaptic plasma membranes (SPM) and synaptic junctions (SJ) were isolated from the cortices of rats varying in age between 5 and 28 days. Gel electrophoresis of SPM and SJ indicated a marked increase in the concentration of the "PSD protein" (M. W. 52,000) with development. The biosynthesis of glycoproteins was measured following the intracranial injection of [³H]fucose or [³H] N '-acetylmannosamine. The incorporation of [³H]fucose into synaptic fractions decreased two- to threefold between 10 and 28 days whereas little change in the incorporation of [³H] N '-acetylmannosamine occurred over the same period. Gel electrophoretic analyses of labeled synaptic membranes indicated major increases in the relative incorporation of radiolabeled precursors into glycoproteins with apparent molecular weights of 74,000, 65,000, 50,000, and 40,000 with increasing age. Identification of fucosyl and sialyl glycoproteins following reaction with ¹²⁵I-fucose-binding protein or labeling of sialic acid with NaIO₄NaB[³H₄] demonstrated similar increases in the concentrations of these glycoproteins. Synaptic junctions contained three major glycoproteins with apparent molecular weights of 180,000, 130,000 and 110,000. The reaction of these glycoproteins with ¹²⁵I-fucose-binding protein increased one- to twofold between 10 and 28 days but little variation in their relative distribution or synthesis occurred over this period. The reaction of synaptic junctional glycoproteins GP 180 and GP 110 with ¹²⁵I-wheat germ ag-glutinin increased between 10 and 28 days. The results indicate that the molecular composition of the synapse continues to evolve after the initial synaptic contact has been formed.     

REGULATION OF ACETYLCHOLINE SYNTHESIS: CONTROL OF CHOLINE TRANSPORT AND ACETYLATION IN SYNAPTOSOMES

Abstract— The high affinity transport of choline (Ch) and the synthesis of acetylcholine (ACh) were measured in synaptosomes by measuring the utilization of [²H₄]Ch. The synthesis of ACh was reduced under several conditions which reduce the availability of acetyl coenzyme A (AcCoA) including no glucose added, replacement of glucose with succinate or impairment of glucose utilization by bromopyr-uvate, NaCN, or pentobarbital. These conditions did not reduce the amount of unacetylated [²H₄]Ch in the synaptosomes indicating that the high affinity transport of Ch is not directly coupled to the synthesis of ACh.     

A survey of surface glycoproteins of four human squamous cell carcinoma lines

The major cell surface glycoprotein components of four new cell lines derived from human squamous cell carcinomas of the head and neck (TR126, TR131, TR138, TR146, Rupniak, H. T. et al., JNCI 75, 621-635, 1985) were identified by three complementary labelling methods. The profile of labelled glycoprotein components was very similar in the four cell lines, although quite large quantitative differences in individual bands were seen. Two galactoproteins, designated GPC-130 and GPC-80 (apparent molecular weight X 10(-3)) were labelled by galactose oxidase/NaB [3H]4 but in all four lines only GPC-130 was prominent. The cell surface galactose and N-Acetylgalactosaminyl residues of glycoproteins were quite highly sialylated, as the galactose oxidase/NaB [3H]4 reaction was increased by between 3- and 6-fold after neuraminidase treatment. The neuraminidase-galactose oxidase/NaB [3H]4 and NaIO4/NaB [3H]4 methods identified a complex profile of glycoprotein components, with very high molecular weight sialogalactoconjugates being prominent. The major sialoglycoproteins were GPC-205, GPC-175, GPC-155, GPC-90 and GPC-70 and in addition, GPC-130 and GPC-80 showed enhanced labelling. Lactoperoxidase catalyzed the iodination of a similar profile of high molecular weight glycoprotein components, with GPC-205 and GPC-175 being prominent in TR126, TR131 and TR146 but less evident in TR138. Overall, the profile of labelled glycoprotein components was similar to the pattern seen in the well differentiated transitional carcinoma lines RT112 and RT4 (Steele, J. G. et al., Biochim. Biophys. Acta. 732, 219-228, 1983).     

Neurites and Somata of Sensory and Sympathetic Neurons in Culture Contain Multiple Species of Ganglio sides

Abstract: We have used tissue culture methods to study the capacity of sensory and sympathetic neurons to synthesize gangliosides. Under appropriate culture conditions, explants of dorsal root or superior cervical ganglia generate an extensive halo of ncurites, which is substantially free of contaminating cells. The cultures incorporate enough [³H]glucosamine into glycolipids to allow biochemical characterization. Gangliosides synthesized by the cells are extracted and freed of radioactive precursors and other lipids by column chromatography. Synthesized material comigrates on (thin-layer chromatograms with the gangliosides, G_Q, G_T, G_D1a, G^D1b, and G_M1, In addition, a substantial amount of unidentified labeled material migrates in a region between the mono- and disialo bands. The ganglioside profiles show that a similar spectrum of gangliosides is found on the neurites and somata of a particular class of neuron. Furthermore, the ganglioside compositions of the two types of neuron studied appear to be similar. We conclude that both sensory and adrenergic autonomic neurons synthesize gangliosides of each of the major classes and that representatives of each class are found in both somata and neurites. Dispersed cell cultures of the superior cervical ganglion synthesize a repertoire of gangliosides similar or identical to that of explant cultures.     

Identification of exposed surface glycoproteins of four human bladder carcinoma cell lines

Three cell surface protein-specific methods were used to radiolabel the major glycoproteins of four human bladder carcinoma cell lines: The well-differentiated lines RT112 and TR4 and more anaplastic lines T24 and EJ. Five acidic glycoproteins iodinated in all lines by the lactoperoxidase/125I method were designated CP-175/5.8-6.0 (apparent molecular weight X 10(-3)/pl of iodoprotein), GP-155/5.0-5.3, GP-145/4.9-5.2, GP-130/4.8-5.5 and GP-110/4.9-5.3. Another iodinated glycoprotein, GP-200/5.5-6.0, was prominently labelled in RT112 and RT4 but was not detected in T24 or EJ. GP-200 as well as GP-175, GP-155 and GP-145 were not detected by the galactose oxidase/NaB(3H)4 method and were poorly labelled by the neuraminidase-galactose oxidase/NaB(3H)4 and NaIO4/NaB(3H)4 labelling methods. The major sialogalactoproteins identified in the four lines by the neuraminidase-galactose oxidase/NaB(3H)4 and NaIO4/NaB(3H)4 methods were GP-130, and a duplet of GP-90 and GP-80 which were poorly iodinated by lactoperoxidase/125I. The galactose oxidase/NaB(3H)4 reaction was increased by between 4- and 10-fold and many additional glycoproteins were labelled after neuraminidase treatment, indicating that the cell surface galactose and N-acetylgalactosamine residues of glycoproteins are highly sialylated. In cell lines RT112 and RT4 there was prominent labelling of very high molecular weight sialogalactoconjugates that was not present in extracts of T24 and EJ.     

Concanavalin A Receptors Associated with Rat Brain Synaptic Junctions Are High Mannose-Type Oligosaccharides

Abstract: Glycoproteins were isolated from a rat brain synaptic junction fraction by affinity chromatography on Concanavalin A-agarose. The isolated glycoproteins were digested with pronase and radiolabeled with ¹²⁵I-Bolton Hunter reagent, and ¹²⁵I-Concanavalin A-binding glycopeptides were isolated by chromatography on Concanavalin A-agarose. Treatment of the ¹²⁵I-Concanavalin A-binding glycopeptides with either α-mannosidase or endo-β- N -acetylglucosaminidase-C₁₁ abolished their interaction with Concanavalin A. The pronase digest was reacted with endo-β-N-acetylglucosaminidase-C₁₁ and released oligosaccharides were reduced with NaB³H₄. Following affinity chromatography on Concanavalin A-agarose, Concanavalin A-binding [³H]oligosaccharides were chromatographed on Biogel P₄. Two major oligosaccharides corresponding to standard carbohydrates containing eight and five mannose residues were identified. Treatment of these oligosaccharides with α-mannosidase converted them to smaller saccharides having a mobility on Biogel P₄ columns equal to the standard disaccharide mannose-β-1-4- N '-acetylglucosamine. These results demonstrate that the Concanavalin A receptor activity associated with CNS synaptic junctions resides in asparaginelinked oligosaccharides of the high-mannose type.     

Characterization of plasma-membrane glycoproteins from functional domains of the rat hepatocyte.

Plasma-membrane glycoproteins from the three different functional domains of the rat hepatocyte were radioactively labelled by oxidation with NaIO4, followed by reduction with NaB3H4. Analysis of the radioactively labelled glycoproteins by polyacrylamide-gel electrophoresis revealed the presence of at least 12 major sialoglycoproteins in each different region of the hepatocyte surface. The Mr-110 000 component was homogeneously distributed over the plasma membrane, whereas the Mr-90 000 polypeptide was only located at the sinusoidal face. These radiolabelled glycoproteins were solubilized in 1% Triton X-100, and the soluble fraction was subjected to affinity chromatography on Sepharose-conjugated wheat-germ agglutinin (WGA). The labelled glycoproteins were poorly bound to WGA. Membrane glycoproteins were also labelled by the galactose oxidase/NaB3H4 method. The results show that the polypeptides with apparent Mr 170 000 from the sinusoidal, 230 000 from the canalicular and 170 000 from the lateral membranes were specifically labelled. When the membranes were treated with neuraminidase and galactose oxidase/NaB3H4, the electrophoretic patterns showed changes in the apparent Mr values of the glycoproteins, owing to loss of sialic acid, and a clear increase in labelling in the sinusoidal and canalicular membranes compared with the lateral membranes. When these labelled membranes were solubilized in 1% Triton X-100 and subjected to affinity chromatography on Sepharose-conjugated Ricinus communis agglutinin and/or Lens culinaris agglutinin, the results showed that the former columns efficiently bound the radiolabelled glycoproteins, whereas the latter columns bound poorly. The results show that there is a differential distribution of glycoproteins along the hepatocyte's surface.     

N-Methyl-d-Aspartate-Sensitive Glutamate Receptors Induce Calcium-Mediated Arachidonic Acid Release in Primary Cultures of Cerebellar Granule Cells

Abstract: In primary cultures of cerebellar granule cells, glutamate, aspartate, and N -methyl-d-aspartate (NMDA) induced a dose-dependent release of [³H]arachidonic acid ([³H]AA) which was selective for these agonists and was inhibited by NMDA receptor antagonists. The agonist-induced [³H]AA release was reduced by quinacrine at concentrations that inhibited phospholipase A₂ (PLA₂) but affected neither the activity of phospholipase C (PLC) nor the hydrolysis of phosphoinositides induced by glutamate or quisqualate. Thus, the increased formation of AA was due to the receptor-mediated activation of PLA₂ rather than to the action of PLC followed by diacylglycerol lipase. The receptor-mediated [³H]AA release was dependent on the presence of extracellular Ca²⁺ and was mimicked by the Ca²⁺ ionophore ionomycin. Pretreatment of granule cells with either pertussis or cholera toxin failed to inhibit the receptor-mediated [³H]AA release. Hence, in cerebellar granule cells, the stimulation of NMDA-sensitive glutamate receptors leads to the activation of PLA₂ that is mediated by Ca²⁺ ions entering through the cationic channels functioning as effectors of NMDA receptors. A coupling through a toxin-sensitive GTP-binding protein can be excluded.     

INCORPORATION OF RADIOACTIVE SULPHATE INTO SULPHATIDE DURING MYELINATION IN CULTURES OF RAT CEREBELLUM

Abstract— Explants of rat cerebellum obtained 12-24 h after birth were maintained in culture in Maximow assemblies. About 90 per cent of the cultures myelinated after 10–12 days in vitro . Cultures maintained for varying periods of time were exposed to [³⁵S]Na₂SO₄; labelled sulphatide was recovered from the total homogenate. Preparation of a subcellular fraction with density properties corresponding to those of myelin indicated that labelled sulphatide appeared in this fraction. Cultures which were poorly-myelinated always exhibited a lower rate of inccrpomtion than well-myelmated cultures from littermate animals, but the distribution of labelled sulphatide into the 'myelin' fraction was similar in the two groups. The rate of incorporation of [³⁵S]Na₂SO₄ into total sulphatide increased with the duration of the culture, with a low level of incorporation until the seventh day in vitro , followed by a sharp increase in rate up to the 21st day. This pattern resembles that observed for rat cerebellum in vivo .     

Evidence for the direct 2beta- and 3beta-hydroxylation of [2H2]GA20-13-O-[6'-2H2]glucoside in seedlings of Phaseolus coccineus

[17-²H₂]GA₂₀-13-O-[6'-²H₂]glucoside was synthesized and applied to seedlings of Phaseolus coccineus L. After incubation for 72 h the conjugate metabolites were purified and shown by LC-ESI-tandem-MS and GC-MS to be [17-²H₂]GA₁-13-O-[6'-²H₂]glucoside and [17-²H₂]GA₂₉-13-O-[6'-²H₂]glucoside. This is the first evidence for the conversion of intact GA-O-glucosides, and represents an additional metabolic pathway of the gibberellin metabolism in P. coccineus L. The results indicate that intact GA-O-glucosides are accepted by 2- and 3-oxidases in the plant.     

Accumulation of N-Arachidonoylethanolamine (Anandamide) into Cerebellar Granule Cells Occurs via Facilitated Diffusion

Abstract: N -Arachidonoylethanolamine (anandamide, AEA) is a putative endogenous ligand of the cannabinoid receptor. Intact cerebellar granule neurons in primary culture rapidly accumulate AEA. [³H]AEA accumulation by cerebellar granule cells is dependent on incubation time ( t _1/2 of 2.6 ± 0.8 min at 37°C) and temperature. The accumulation of AEA is saturable and has an apparent K _m of 41 ± 15 µ M and a V _max of 0.61 ± 0.04 nmol/min/10⁶ cells. [³H]AEA accumulation by cerebellar granule cells is significantly reduced by 200 µ M phloretin (57.4 ± 4% of control) in a noncompetitive manner. [³H]AEA accumulation is not inhibited by either ouabain or removal of extracellular sodium. [³H]AEA accumulation is fairly selective for AEA among other naturally occurring N -acylethanolamines; only N -oleoylethanolamine significantly inhibited [³H]AEA accumulation at a concentration of 10 µ M . The ethanolamides of palmitic acid and linolenic acid were inactive at 10 µ M . N -Arachidonoylbenzylamine and N -arachidonoylpropylamine, but not arachidonic acid, 15-hydroxy-AEA, or 12-hydroxy-AEA, compete for AEA accumulation. When cells are preloaded with [³H]AEA, temperature-dependent efflux occurs with a half-life of 1.9 ± 1.0 min. Phloretin does not inhibit [³H]AEA efflux from cells. These results suggest that AEA is accumulated by cerebellar granule cells by a protein-mediated transport process that has the characteristics of facilitated diffusion.     

Chronic Exposure to Adenosine Receptor Agonists and Antagonists Reciprocally Regulates the A1 Adenosine Receptor-Adenylyl Cyclase System in Cerebellar Granule Cells

Abstract: Chronic treatment with the adenosine receptor antagonist caffeine evokes an up-regulation of A₁ adenosine receptors and increased coupling of the receptor to G proteins in rat brain membranes. However, chronic agonist exposure has not been explored. Primary cultures of cerebellar granule cells were exposed chronically to A₁ adenosine receptor agonists and antagonists. Exposure to the A₁ adenosine receptor agonist N ⁶-cyclopentyladenosine resulted in (1) a time- and concentration-dependent reduction in the density of receptors labeled by 1,3-[³H]dipropyl-8-cyclopentylxanthine, (2) an enhanced ability of guanyl nucleotides to decrease the fraction of A₁ adenosine receptor sites displaying high affinity for 2-chloroadenosine, and (3) a functional uncoupling of receptors from adenylyl cyclase (EC 4.6.1.1). The adenosine antagonists caffeine and 8- p -sulfophenyltheophylline produced alterations in A₁ adenosine receptor homeostasis that were antipodal to those associated with agonist treatment. Antagonist exposure (1) increased the density of A₁ adenosine receptors in cerebellar granule cell membranes, (2) blunted the effect of guanyl nucleotides on receptor coupling to G proteins, and (3) increased the functional coupling of receptors to adenylyl cyclase inhibition. Forskolin treatment of cerebellar granule cells did not affect receptor density, suggesting that cyclic AMP is not involved in the regulation of A₁ adenosine receptor expression.     

Formation of 2-hydroxy-4-methylpentanoic acid from l-leucine by Clostridium butyricum

Abstract Five Clostridium butyricum strains of different origin were grown in trypticase-yeast extract-hemin medium with or without d-glucose (TGYH or TYH medium, respectively) and in a synthetic basal medium with d-glucose (BMG medium). 2-Hydroxy-4-methylpentanoic acid was detected by gas chromatography-mass spectrometry (GC-MS) for the five strains whether grown in TGYH or TYH medium (270 or 170 μM, respectively). In BMG medium supplemented with l-leucine (10 mM), the concentration of this metabolite was strongly increased (2.8 mM versus 10 μM in the control). After culture in TGYH or TYH medium supplemented with l-( methyl -²H₃)leucine, 2-hydroxy-4-([²H₃]methyl)pentanoic acid was detected by GC-MS. This observation demonstrates that C. butyricum is able to convert l-leucine into the corresponding 2-hydroxy acid and opens a new aspect in the study of C. butyricum metabolism.     

Influence of Growth Hormone and Thyroxine On Cell Kinetics In the Proximal Tibial Growth Plate of Snell Dwarf Mice

Abstract. In Snell dwarf mice, the influence of short-term treatment with human growth hormone (hGH) or thyroxine on the proliferative and sulphation activity of the proximal tibial growth plate was studied. By autoradiographic methods, the [³H]methylthymidine incorporation after a single injection was measured, after 2 hr incorporation time. the labelling index was calculated and the number of labelled mitoses was counted. In addition, the distribution of the labelled nuclei over the proliferating and degenerating zones was determined by continuous labelling for 25 and 73 hr.
In untreated dwarf mice after [³H]-methylthymidine administration, the number of labelled nuclei in the growth plate is low. Labelling occurs, as expected, mainly in the cells of the proliferative zones. the number of labelled nuclei in control dwarf mice was similar after 25 and 73 hr continuous labelling. This suggests that many cells are in a resting G_o or prolonged G₁ phase. Both hGH and T₄ treatment induce a significant increase of the number of labelled nuclei per growth plate and of the number of mitoses. Since hormonal treatment induces a small number of mitoses after 2 hr incorporation of the label, the minimal G₂ phase of the cell cycle is less than 2 hr. In addition, treatment with hGH and T₄ stimulates chondrocytes in the zone of proliferative and hypertrophic cells to actively incorporate [³⁵S]-sulphate.     

Exposure of major neutral glycolipids in red cells to galactose oxidase. Effect of neuraminidase

The exposure of several major red-cell glycolipids to galactose oxidase was studied by oxidizing the cells with the enzyme and reducing them with NaB2H4. After isolation, the deuterium label was detected by mass fragmentography. 60-70% globoside in human and porcine erythrocytes was exposed as measured by this method. In contrast, asialo-GM2 in guinea-pig erythrocytes and Forssman glycolipid in sheep erythrocytes were mainly in a cryptic state. Neuraminidase treatment increased the incorporation of deuterium label to asialo-GM2 4-8-fold. A similar effect was seen in Forssman glycolipid when sheep red cells were labeled with the neuraminidase/galactose oxidase/NaB3H4 method. In contrast, the increase in labeling was only about 10-40% in porcine and human globosides, which were efficiently exposed to galactose oxidase already in native red cells.     

Differences between the carbohydrate units of cell-surface glycoproteins of moust B- and T-lymphocytes.

Carbohydrate units of cell-surface glycoproteins of mouse B- and T-lymphocytes, labelled in their sialic acid residues by the periodate/NaB3H4 method and in their galactose residues by the galactose oxidase/NaB3H4 method after neuraminidase treatment, have been studied. Glycopeptides were prepared from the labelled cells by Pronase digestion and fractionated by concanavalin A affinity chromatography into two fractions (A and B). Alkali-labile oligosaccharides were isolated after mild NaOH/NaBH4 treatment by gel filtration. The alkali-labile oligosaccharides were further analysed by t.l.c. To study the relative proportion of neutral mannose-rich carbohydrate units (fraction C) in lymphocyte glycoproteins, glycopeptides were also prepared from unlabelled cells and subjected to concanavalin A affinity chromatography after N-[3H]acetylation of their peptide moiety. The major alkali-labile oligosaccharide component of both cell types was identified as galactosyl-(beta 1 leads to 3)-N-acetylgalactosaminitol. T-Lymphocytes were characterized by a high proportion of this oligosaccharide and a lower proportion of alkali-stable fraction A glycopeptides, whereas the opposite was observed for B-lymphocytes. The relative proportions of the concanavalin A-binding fractions B and C were similar in both cell types. The differences observed may correlate with the different surface properties of B- and T-lymphocytes.     

Quinolinic Acid In Vivo Synthesis Rates, Extracellular Concentrations, and Intercompartmental Distributions in Normal and Immune-Activated Brain as Determined by Multiple-Isotope Microdialysis

Abstract: Quinolinic acid (QUIN) kills neurons by activation of NMDA receptors that are accessed via the extracellular fluid (ECF). In vivo microdialysis was employed to quantify the dynamics of ECF QUIN levels. [¹³C₇]QUIN was perfused through the probe for in vivo calibration to accurately quantify ECF QUIN concentrations. Osmotic pumps infused [²H₃]QUIN subcutaneously to quantify blood contributions to ECF and tissue levels. Local QUIN production rates and influx and efflux rates across the blood-brain barrier were calculated from the extraction fraction of [¹³C₇]QUIN, probe geometry, tissue diffusion coefficients, the extracellular volume fraction, and [²H₃]QUIN/QUIN ratios in blood and dialysates. In normal brain, 85% of ECF QUIN levels (110 n M ) originated from blood, whereas 59% of tissue homogenate QUIN (130 pmol/g) originated from local de novo synthesis. During systemic immune activation (intraperitoneal injection of endotoxin), blood QUIN levels increased (10.2-fold) and caused a rise in homogenate (10.8-fold) and ECF (18.5-fold) QUIN levels with an increase in the proportions of QUIN derived from blood. During CNS inflammation (local infusion of endotoxin), increases in brain homogenate (246-fold) and ECF (66-fold) QUIN levels occurred because of an increase in local synthesis rate (146-fold) and a reduction in efflux/influx ratio (by 53%). These results demonstrate that brain homogenate measures are a reflection of ECF concentrations, although there are quantitative differences in the values obtained. The mechanisms that maintain ECF QUIN levels at low values cannot do so when there are large increases in local brain synthesis or when there are large elevations in blood QUIN concentrations.     

Links between methanotroph community composition and CH4 oxidation in a pine forest soil

The main gap in our knowledge about what determines the rate of CH₄ oxidation in forest soils is the biology of the microorganisms involved, the identity of which remains unclear. In this study, we used stable-isotope probing (SIP) following ¹³CH₄ incorporation into phospholipid fatty acids (PLFAs) and DNA/RNA, and sequencing of methane mono-oxygenase ( pmoA ) genes, to identify the influence of variation in community composition on CH₄ oxidation rates. The rates of ¹³C incorporation into PLFAs differed between horizons, with low ¹³C incorporation in the organic soil and relatively high ¹³C incorporation into the two mineral horizons. The microbial community composition of the methanotrophs incorporating the ¹³C label also differed between horizons, and statistical analyses suggested that the methanotroph community composition was a major cause of variation in CH₄ oxidation rates. Both PLFA and pmoA -based data indicated that CH₄ oxidizers in this soil belong to the uncultivated 'upland soil cluster α'. CH₄ oxidation potential exhibited the opposite pattern to ¹³C incorporation, suggesting that CH₄ oxidation potential assays may correlate poorly with in situ oxidation rates. The DNA/RNA-SIP assay was not successful, most likely due to insufficient ¹³C-incorporation into DNA/RNA. The limitations of the technique are briefly discussed.