Carbon distribution in grass (Festuca pratensis L.) during regrowth after cutting—utilization of stored and newly assimilated carbon

Distribution of net assimilated C in meadow fescue (Fectuca pratensi L.) was followed before and after cutting of the shoots. Plants were continuously labelled in a growth chamber with ¹⁴C-labelled CO₂ in the atmosphere from seedling to cutting and with ¹³C-labelled CO₂ in the atmosphere during regrowth after the cutting. Labelled C, both ¹⁴C and ¹³C, was determined at the end of the two growth periods in shoots, crowns, roots, soil and rhizosphere respiration. Distribution of net assimilated C followed almost the same pattern at the end of the two growth periods, i.e. at the end of the ¹⁴C- and the ¹³C-labelling periods. Shoots retained 71–73% of net assimilated C while 9% was detected in the roots and 11–14% was released from the roots, determined as labelled C in soil and as rhizosphere respiration. At the end of the 2nd growth period, after cutting and regrowth, 21% of the residual plant ¹⁴C at cutting (¹⁴C in crowns and roots) was found in the new shoot biomass. A minor part of the residual plant ¹⁴C, 12%, was lost from the plants. The decreases in ¹⁴C in crowns and roots during the regrowth period suggest that ¹⁴C in both crowns and roots was translocated to new shoot tissue. Approximately half of the total root C at the end of the regrowth period after cutting was ¹³C-labelled C and thus represents new root growth. Root death after cutting could not be determined in this experiment, since the decline in root ¹⁴C during the regrowth period may also be assigned to root respiration, root exudation and translocation to the shoots. ei]{gnH}{fnLambers} ei]{gnA C}{fnBorstlap}     

Incorporation of [methyl-3H]thymidine by obligate and facultative anaerobic bacteria when grown under defined culture conditions

Abstract Incorporation of [ methyl -³H]thymidine into bacterial DNA was determined for a range of axenic anaerobic bacterial cultures: fermentative heterotrophs, sulphate-reducing bacteria, purple sulphur bacteria, acetogens and methanogens. Anaerobically growing Bacillus sp. and the obligate aerobe Thiobacillus ferrooxidans were also investigated. Actively growing cultures of sulphate-reducing bacteria belonging to the genera Desulfovibrio, Desulfotomaculum, Desulfobacter, Desulfobotulus and Desulfobulbus , purple sulphur bacteria ( Chromatium vinosum OP2 and Thiocapsa roseopersicina OP1), methanogens ( Methanococcus GS16 and Methanosarcina barkeri ) and an acetogen ( Acetobacterium woodii ) did not incorporate [ methyl -³H]thymidine into DNA. The only obligate anaerobes in which thymidine incorporation into DNA could be unequivocally demonstrated were members of the genus Clostridium . Anaerobically growing Bacillus sp. also incorporated thymidine. These data demonstrate that pure culture representatives of major groups of anaerobic bacteria involved in the terminal oxidation of organic carbon and anoxygenic phototrophs within sediments are unable to incorporate [ methyl -³H]thymidine into DNA, although some obligate and facultative anaerobes can. Variability in thymidine incorporation amongst pure culture isolates indicates that unless existing techniques can be calibrated to take this into consideration then productivity estimates in both aerobic and anaerobic environments may be greatly underestimated using the [ methyl -³H]thymidine technique.     

Interactions Between the Glutamate and Glycine Recognition Sites of the N-Methyl-d-Aspartate Receptor from Rat Brain, as Revealed from Radioligand Binding Studies

Abstract: The N-methyl-d -aspartate (NMDA) receptor possesses two distinct amino acid recognition sites, one for glutamate and one for glycine, which appear to be allosterically linked. Using rat cortex/hippocampus P₂ membranes we have investigated the effect of glutamate recognition site ligands on [³H]glycine (agonist) and (±)4-trans-2-car-boxy-5,7-dichloro-4-[³H]phenylaminocarbonylamino-1,2,3,4-tetrahydroquinoline ([³H]l -689,560; antagonist) binding to the glycine site and the effect of glycine recognition site ligands on l -[³H]glutamate (agonist), dl -3-(2-carboxypiperazin-4-yl)-[³H]propyl-1 -phosphonate ([³H]-CPP; “C-7” antagonist), and cis-4-phosphonomethyl-2-[³H]piperidine carboxylate ([³H]CGS-19755; “C-5” antagonist) binding to the glutamate site. “C-7” glutamate site antagonists partially inhibited [³H]l -689,560 binding but had no effect on [³H]glycine binding, whereas “C-5” antagonists partially inhibited the binding of both radioligands. Glycine, d -serine, and d -cycloserine partially inhibited [³H]CGS-19755 binding but had little effect on l -[³H]-glutamate or [³H]CPP binding, whereas the partial agonists (+)-3-amino-1-hydroxypyrrolid-2-one [(+)-HA-966], 3R-(+)cis-4-methyl-HA-966 (l -687,414), and 1-amino-1-carboxycyclobutane all enhanced [³H]CPP binding but had no effect on [³H]CGS-19755 binding, and (+)-HA-966 and l -687,414 inhibited l -[³H]glutamate binding. The association and dissociation rates of [³H]l -689,560 binding were decreased by CPP and d -2-amino-5-phosphonopentanoic acid (“C-5”). Saturation analysis of [³H]l -689,560 binding carried out at equilibrium showed that CPP had little effect on the affinity or number of [³H]l -689,560 binding sites. These results indicate that complex interactions occur between the glutamate and glycine recognition sites on the NMDA receptor. In addition, mechanisms other than allosterism may underlie some effects, and the possibility of a steric interaction between CPP and [³H]l -689,560 is discussed.     

Lindane Administration to the Rat Induces Modifications in the Regional Cerebral Binding of [3H]Muscimol, [3H]-Flunitrazepam, and t-[35S]Butylbicyclophosphorothionate: An Autoradiographic Study

Abstract: The effect of lindane administration on the specific binding of ligands to different sites on the GABA_A receptor-ionophore complex was studied in the rat brain by receptor mapping autoradiography. [³H]Muscimol (Mus), [³H]flunitrazepam (Flu), and t -[³⁵S]butylbicyclophosphorothionate (TBPS) were used as specific ligands of GABA, benzodiazepine, and picrotoxinin binding sites, respectively. Rats received a single oral dose of 30 mg/kg lindane and they were classified into two groups according to the absence or presence of convulsions. Vehicle-treated groups acted as controls. The effect of the xenobiotic on ligand binding was measured in different brain areas and nuclei 12 min or 5 h after its administration. Lindane induced a generalized decrease in [³⁵S]TBPS binding, which was present shortly after dosing. In addition, [³H]Flu binding was increased in lindane-treated animals, this modification also appearing shortly after administration but diminishing during the studied time. Finally, lindane induced a decrease in [³H]Mus binding, which became more evident over time. These modifications were observed both in the presence and in the absence of convulsions. However, an increase in [³H]-Mus binding was detected shortly after lindane-induced convulsions. The observed decrease in [³⁵S]TBPS binding is in agreement with the postulated action of lindane at the picrotoxinin binding site of the GABA_A receptor chloride channel. The effects observed on the binding of [³H]Flu and [³H]Mus may be secondary to the action of lindane as an allosteric antagonist of the GABA_A receptor.     

Further Evidence for Multiple Forms of an N-Methyl-d-Aspartate Recognition Domain in Rat Brain Using Membrane Binding Techniques

Abstract— Pretreatment with sulfhydryl-reactive agents, such as N-ethylmaleimide and p-chloromercuriphenylsul-fonic acid, invariably resulted in marked inhibition of the binding of dl -(E)-2-amino-4-[³H]propyl-5-phosphono-3-pentenoic acid ([³H]CGP 39653), a competitive antagonist at an N-methyl-d -aspartate (NMDA)-sensitive subclass of central excitatory amino acid receptors, in brain synaptic membranes extensively washed and treated with Triton X-100, but did not significantly affect the binding of L-[³H]-glutamic acid ([³H]Glu), an endogenous agonist. The pre-treatment was effective in reducing the binding of [³H]-CGP 39653 at equilibrium, without altering the initial association rate, and decreased the affinity for the ligand. Pretreatment with sulfhydryl-reactive agents also enhanced the potencies of NMDA agonists to displace [³H]-CGP 39653 binding and attenuated those of NMDA antagonists, but had little effect on the potencies of the agonists and antagonists to displace [³H]Glu binding. The binding of both [³H]CGP 39653 and [³H]Glu was similarly sensitive to pretreatment with four different proteases in Tritontreated membranes, whereas pretreatment with phospho-lipase A₂ or C markedly inhibited [³H]CGP 39653 binding without altering [³H]Glu binding. Moreover, both phospho-lipases not only induced enhancement of the abilities of NMDA agonists to displace the binding of [³H]CGP 39653 and [³H]Glu, but also caused diminution of those of NMDA antagonists. These results suggest that both sulfhydryl-reactive agents and phospholipases may predominantly interfere with radiolabeling of the NMDA recognition domain in a state favorable to an antagonist by [³H]CGP 39653, with concomitant facilitation of that in an agonist-preferring form by [³H]Glu. The possible presence of multiple forms of the NMDA recognition domain is further supported by these data.     

Cerebral Metabolic Compartmentation as Revealed by Nuclear Magnetic Resonance Analysis of D-[1-13C]Glucose Metabolism

Abstract: Nuclear magnetic resonance (NMR) was used to study the metabolic pathways involved in the conversion of glucose to glutamate, γ-aminobutyrate (GABA), glutamine, and aspartate. d -[1^-13C]Glucose was administered to rats intraperitoneally, and 6, 15, 30, or 45 min later the rats were killed and extracts from the forebrain were prepared for ¹³C-NMR analysis and amino acid analysis. The absolute amount of ¹³C present within each carbon-atom pool was determined for C-2, C-3, and C-4 of glutamate, glutamine, and GABA, for C-2 and C-3 of aspartate, and for C-3 of lactate. The natural abundance ¹³C present in extracts from control rats was also determined for each of these compounds and for N-acetylaspartate and taurine. The pattern of labeling within glutamate and GABA indicates that these amino acids were synthesized primarily within compartments in which glucose was metabolized to pyruvate, followed by decarboxylation to acetyl-CoA for entry into the tricarboxylic acid cycle. In contrast, the labeling pattern for glutamine and aspartate indicates that appreciable amounts of these amino acids were synthesized within a compartment in which glucose was metabolized to pyruvate, followed by carboxylation to oxaloacetate. These results are consistent with the concept that pyruvate carboxylase and glutamine synthetase are glia-specific enzymes, and that this partially accounts for the unusual metabolic compartmentation in CNS tissues. The results of our study also support the concept that there are several pools of glutamate, with different metabolic turnover rates. Our results also are consistent with the concept that glutamine and/or a tricarboxylic acid cycle intermediate is supplied by astrocytes to neurons for replenishing the neurotransmitter pool of GABA. However, a similar role for astrocytes in replenishing the transmitter pool of glutamate was not substantiated, possibly due to difficulties in quantitating satellite peaks arising from ¹³C-¹³C coupling.     

Support for Radiolabeling of a Glycine Recognition Domain on the N-Methyl-d-Aspartate Receptor Ionophore Complex by 5,7-[3H]Dichlorokynurenate in Rat Brain

Abstract: Pretreatment with Triton X-100 more than doubled the binding of radiolabeled 5,7-dichlorokynurenic acid (DCKA), a proposed antagonist at a glycine (Gly) recognition domain on the N-methyl-d -aspartate (NMDA) receptor ionophore complex, in rat brain synaptic membranes. The binding exhibited an inverse temperature dependency, reversibility, and saturability, the binding sites consisting of a single component with a high affinity (27.5 nM) and a relatively low density (2.87 pmol/mg of protein). The binding of both [³H]DCKA and [³H]Gly was similarly displaced by numerous putative agonists and antagonists at the Gly domain in a concentration-dependent manner at a concentration range of 100 nM to 0.1 mM. Among the 24 putative ligands tested, DCKA was the second most potent displacer of the binding of both radioligands with no intrinsic affinity for the binding of [³H]kainic acid and α-amino-3-hydroxy-5-[³H]methylisoxazole-4-propionic acid (AMPA) to the non-NMDA receptors. In contrast, the other proposed potent Gly antagonist, 5,7-dinitroquinoxaline-2,3-dione, was active in displacing the binding of [³H]glutamic ([³H]Glu) and D,L-(E)-2-amino-4-[³H]propyl-5-phosphono-3-pentenoic acids to the NMDA recognition domain with a relatively high affinity for the non-NMDA receptors. In addition, the proposed antagonist at the AMPA-sensitive receptor, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline, not only displaced weakly the binding of both [³H]- Gly and [³H]DCKA, but also inhibited the binding of (+)-5-[³H]methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine ([³H]MK-801) to an ion channel associated with the NMDA-sensitive receptor in the presence of added Glu alone in a manner sensitive to antagonism by further added Gly. Clear correlations were seen between potencies of the displacers to displace [³H]DCKA binding and [³H]Gly binding, in addition to between the potencies to displace [³H]-DCKA or [³H]Gly binding and to potentiate or inhibit [³H]MK-801 binding. All quinoxalines tested were invariably more potent displacers of [³H]DCKA binding than [³H]Gly binding, whereas kynurenines were similarly effective in displacing the binding of both [³H]Gly and [³H]-DCKA. These results undoubtedly give support to the proposal that [³H]DCKA is one useful radioligand available in terms of its high selectivity and affinity for the Gly domain in the brain. Possible multiplicity of the Gly domain is suggested by the differential pharmacological profiles between the binding of [³H]Gly and [³H]DCKA.     

Isolation and chemical characterization of lipopolysaccharides from four Mycoplana species (M. bullata,M. segnis,M. ramosa and M. dimorpha)

Lipopolysaccharides (LPS), isolated from four Mycoplana species, i.e. the type strains of M. bullata, M. segnis, M. ramosa and M. dimorpha, were characterized onto their chemical composition and their respective lipid A-types. Those of M. bullata and M. segnis showed on DOC-PAGE an R-type character and had lipid A's of the Lipid A_DAG-type which exclusively contained 2,3-diamino-2,3-dideoxy-d-glucose as lipid A sugar. LPS's of M. ramosa and M. dimorpha showed, although only weakly expressed, ladder-like patterns on DOC-PAGE indicating some S-type LPS's and lipid A of the d-glucosamine type (Lipid A_GlcN). M. bullata LPS contained mannose and glucose in major amounts and additionally l-glycero-d-mannoheptose, whereas M. segnis LPS was composed of rhamnose, mannose and glucose together with both, d-glycero-d-manno- and l-glycero-d-manno-heptoses in a molar ratio of 1:2. All LPS's contained 2-keto-3-deoxy-octonic acid (Kdo), phosphate and an unidentified acidic component X. In addition to X, M. segnis LPS contained glucuronic and galacturonic acids, whereas M. ramosa LPS contained only galacturonic acid. Acetic acid hydrolysis of the LPS resulted in splitting off lipid A moieties, very rich in 3-hydroxy fatty acids, in particular in 3-OH-12:0 (in Lipid A_DAG), or in 3-OH-14:0 (in Lipid A_GlcN). Analysis of the 3-acyloxyacyl residues revealed major amounts of amide-linked 3-OH(3-OH-13:0)12:0 in lipid A of M. bullata and 3-OH(12:0)12:0 in lipid A of M. segnis. The rare 4-oxo-myristic acid (4-oxo-14:0) was observed only in M. bullata LPS, where it is ester-linked. Amide linked diesters could not be traced in M. ramosa and M. dimorpha. All four lipid A's lacked erster-bound acyloxyacyl residues.Non-standard abbreviations DAG 2,3-diamino-2,3-dideoxy-d-glucose - Kdo 2-keto-3-deoxy-octonate - LPS lipopolysaccharide - PITC phenyl isothiocyanate - NANA N-acetyl neuraminic acid     

Hectogram-scale synthesis of [Aib8, Arg34]-GLP-1 (7–37) by liquid-phase fragment condensation

Based on small-scale synthesis (0.3 g), a 100-g scale-up synthesis of crude [Aib⁸, Arg³⁴]-glucagon-like peptide-1 (GLP-1) (7–37) was completed. The crude [Aib⁸, Arg³⁴]-GLP-1 (7–37) was purified using a dynamic axial compression column 200 (DAC-200). Approximately 61 g of [Aib⁸, Arg³⁴]-GLP-1 (7–37) with a purity of >99% was obtained through one-step reverse-phase chromatography. The purification yield was approximately 92%. The yield from the total reaction was approximately 60%. In summary, we developed an economical and environmentally friendly route to the synthesis and purification of crude [Aib⁸, Arg³⁴]-GLP-1 (7–37), laying a foundation for subsequent industrial production.     

High resolution radiocarbon spike confirms tree ring dating with low sample depth

Radiocarbon (¹⁴C) has been used to date carbon-rich objects in Earth science, archeology, and history since the 1940s. New methods, using spikes in ¹⁴C caused by solar proton events, can be used to annually date wood when crossdating is not possible, such as when sample size is low, samples are floating in time, or external disturbances lead to insecure dates. Here, we use a spike in radiocarbon during a solar energetic particle (SEP) event in 774/775 CE to confirm crossdating of a poorly-replicated King Billy pine (Athrotaxis selaginoides) chronology. Low sample depth between 1498 and 1523 CE (two trees) prevented confident dating of the early period of the chronology. Three core samples with strong correlation with the master chronology that likely included the 774/775 CE Miyake SEP event were identified for radiocarbon isotope analysis. We sectioned segments centered on the estimated 774/775 CE date and then isolated the holocellulose in each sample. Samples were sent to an accelerator mass spectrometry (AMS) for radiocarbon measurements. The AMS data confirmed the crossdating accuracy of the tree ring series and reinforces the applicability of this technique to anchor poorly dated tree ring series in time. In addition, we found sample processing with a microtome proved superior for holocellulose extractions and yielded more accurate ¹⁴C measurements. We recommend sampling with a microtome, processing at least three samples per year, and including sample masses greater than 100 ug C to confirm dating using radiocarbon spikes.