Nitrogen uptake in barley after spring incorporation of 15N-labelled Italian ryegrass into sandy soils

A 5-month laboratory incubation experiment was conducted to study the immobilization-mineralization of N in soil to which dried or composted ¹⁵N labelled ryegrass (Lolium italicum L.) had been added. Cellulose was added to dried ryegrass to give a C/N ratio similar to that of composted ryegrass. Exchangeable NH₄ ⁺ and NO₃ ^–, HCl-hydrolyzable N forms, microbial biomass N, NaOH-soluble and insoluble N were monitored during incubation. Dried ryegrass brought about a significant increase in total and labelled exchangeable NH₄ ⁺, while a rapid immobilization and a subsequent slow release of exchangeable NH₄ ⁺ was observed in soil with composted ryegrass, together with a resistance to degradation of the labelled humic substances. Compounds synthesized during the composting process and resistant to microbial decomposition probably caused an increase in the amino-acid fraction of soil. These findings suggest that composting can reduce the risk of N losses.     

Vanillic acid metabolism by the white-rot fungus Sporotrichum pulverulentum

Vanillic acid metabolism was studied in wild-type Sporotrichum pulverulentum and three different mutants. Vanillic acid was found to be oxidatively decarboxylated to methoxyhydroquinone (MHQ) and simultaneously reduced to vanillin and vanillyl alcohol to different degrees depending upon the cultivation conditions. The reducing pathway cannot be utilized unless the fungus has access to an easily metabolized carbon source such as glucose or cellobiose, while decarboxylation takes place in cultures with only vanillic acid present. Polymerization reactions also occurred in the culture solutions. Some evidence for reoxidation of vanillin and vanillyl alcohol was obtained in vivo, and in vitro experiments using horseradish peroxidase.Using vanillic acids labelled in the carboxyl, methoxyl and the aromatic ring it was shown that decarboxylation occures before ring-cleavage, which in turn takes place earlier than the release of ¹⁴CO₂ from O¹⁴CH₃-vanillate. The ¹⁴CO₂ evolution from the methoxyl group is repressed by 1% cellobiose as compared to 0.25% cellobiose, but is stimulated by 26 mM nitrogen (as asparagine plus NH₄NO₃) compared to 2.6 mM nitrogen. Since S. pulverulentum appears to require three hydroxyl groups attached to the benzene ring before ring-cleavage can occur, preparation for ring-cleavage is apparently achieved by hydroxylation rather than by demethylation.A scheme for metabolism of vanillic acid by S. pulverulentum based upon these results is proposed.Non-Standard Abbreviations WT wild type Sporotrichum pulverulentum - MHQ methoxyhydroquinone - MQ methoxyquinone - NKM Norkrans medium - DMS dimethylsuccinate - DHP dehydropolymer of coniferyl alcohol     

DECARBOXYLATION STUDIES OF GLUTAMATE, GLUTAMINE, AND ASPARTATE FROM BRAIN LABELLED WITH [1-14C] ACETATE, l-[U-14C]-ASPARTATE, AND l-[U-14C]GLUTAMATE

Studies in vivo and in vitro of the distribution of label in C-1 of glutamate and glutamine and C-4 of aspartate in the free amino acids of brain were carried out. [1-¹⁴C]-Acetate was used both in vivo and in vitro and l -[U-¹⁴C]aspartate and l -[U-¹⁴C]glutamate were used in vitro.

• 1 The results obtained with labelled acetate and aspartate suggest that CO₂ and a 3-carbon acid may exchange at different rates on a CO_a-fixing enzyme.
• 2 The apparent cycling times of both glutamate and glutamine show fast components measured in minutes and slow components measured in hours.
• 3 With [1-¹⁴C]acetate in vitro glutamine is more rapidly labelled in C-1 than is glutamate at early time points; the curves cross over at about 7 min.
• 4 The results support and extend the concept of metabolic compartmentation of amino acid metabolism in brain.

     

Absorption of short-chain fatty acids across ruminal epithelium of sheep

Investigations on the absorption of shortchain fatty acids across ruminal epithelium of sheep were performed both in vitro (Ussing chamber technique, using propionic acid representatively for shortchain fatty acids) and in vivo (washed, isolated reticulorumen). A pH-induced, nearly tenfold increase in the concentration of undissociated propionate led to an only twofold increase in mucosal-to-serosal flux of propionate (in vitro). Neither amiloride (1 mmol·l^-1, in vitro) nor theophylline (10 mmol·l^-1, in vivo), inhibitors of the ruminal Na⁺/H⁺ exchanger, exerted any significant influence on propionate fluxes or short-chain fatty acids absorption, respectively. Total replacement of luminal Na⁺ (by choline) did not alter short-chain fatty acids absorption (in vivo). Mucosal 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (0.1 mmol·l^-1) or mucosal nitrate (40 mmol·l^-1) markedly reduced propionate net flux (in vitro). Increasing mucosal Cl^- concentration brought about a significant drop in mucosal-to-serosal flux of propionate (in vitro) and in short-chain fatty acids net absorption (in vivo), respectively. The results obtained suggest that short-chain fatty acids are absorbed both as anions and as undissociated acids across ruminal epithelium of sheep. It is concluded that short-chain fatty acids anions either compete with Cl^- for binding sites at a common anion-exchange mechanism or that they are absorbed by an short-chain fatty acids anion/HCO ₃ ^- exchanger indirectly coupled to a Cl^-/HCO ₃ ^- exchanger via intracellular bicarbonate.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - DMSO dimethylsulfoxide - G _t tissue conductance - HSCFA protonated - SCFA i.e. undissociated form - J _ms mucosal-to-serosal flux - J _sm serosal-to-mucosal flux - J _net net flux - I _sc short-circuit current - MOPS (3-[N-morpholino]propanesulfonic acid) - mu mucosal - Prop Propionate - SCFA ^- SCFA anions, i.e. dissociated form - SCFA short-chain fatty acids - SEM standard error of mean     

The genetic control of natural killer cell activity and its association with in vivo resistance against a moloney lymphoma isograft

Spleens of normal young mice of certain strains contain lymphocytes that can kill strain A-derived YAC-1 lymphoma cells in a⁵¹Cr release cytotoxic assay in vitro. We have previously classified mouse genotypes as high or low reactors, according to their responses in this test. In vivo resistance to small numbers of YAC ascites lymphoma cells is correlated with in vitro cytolytic activity. In vitro and in vivo tests were carried out on the same individual (A x C57BL)F₁ x A backcross mice. Natural in vitro killer cell activity appeared to be under polygenic control, including a strong H-2-linked factor. No linkage was found with five different isozyme loci, with theIg-l locus or with C5 serum activity. Also in vivo resistance showed strong linkage with theH-2 complex. In (A x CBA)F₁ x A backcross mice, a weak linkage was found with the coat color locusC. There was a correlation between in vitro killer activity and in vivo resistance in the same backcross mice. In vivo resistance was particularly strong in mice that combined theH-2 ^b -linked resistance factor with a high cytolytic activity in vitro.     

The role of selenium in thyroid hormone metabolism and effects of selenium deficiency on thyroid hormone and iodine metabolism

Selenium deficiency impairs thyroid hormone metabolism by inhibiting the synthesis and activity of the iodothyronine deiodinases, which convert thyroxine (T₄) to the more metabolically active 3,3′-5 triiodothyronine (T₃). Hepatic type I iodothyronine deiodinase, identified in partially purified cell fractions using affinity labeling with [¹²⁵I]N-bromoacetyl reverse triiodothyronine, is also labeled with⁷⁵Se by in vivo treatment of rats with⁷⁵Se-Na₂SeO₃. Thus, the type I iodothyronine 5′-deiodinase is a selenoenzyme. In rats, concurrent selenium and iodine deficiency produces greater increases in thyroid weight and plasma thyrotrophin than iodine deficiency alone. These results indicate that a concurrent selenium deficiency could be a major determinant of the severity of iodine deficiency.     

A simple technique for producing 13C or 14C-labelled fronds of Lemna gibba and their use in soil incubation investigations

The duckweed Lemna gibba required light and a suitable energy source such as sucrose, glucose or fructose, for maximum growth in culture. The requirement for light was relatively unimportant and the plants grew well in a photon flux density of only 52 μmol m^-2s^-1 PAR. The uptake and incorporation of uniformly labelled ¹⁴C-glucose into fronds was related only to the concentration of the sugar. When incubated with soil, labelled L. gibba behaved in a manner similar to that of labelled ryegrass roots which had been produced by a more elaborate technique using a ¹⁴CO₂ labelled atmosphere. During incubation with soil for 224 days the L. gibba material (specific activity 6133 Bq mg^-1 d. wt) lost 64% of its radioactivity as ¹⁴CO₂ and ryegrass (specific activity 6634 Bq mg^-1 d. wt) lost 49%. Alkaline extracted humic and fulvic acids from soil had specific activities for the L. gibba incubation of 3409 and 407 Bq mg^-1 solid and for ryegrass roots of 4609 and 546 Bq mg^-1 solid respectively. The production of ¹³C or ¹⁴C-labelled L. gibba can be undertaken using only simple equipment producing material the specific radioactivity of which can be controlled by adjusting the activity of the sugar energy source.     

Postendocytic vitellin processing in ovarian follicles of the stick insect Carausius morosus (Br.)

Newly laid eggs of the stick insect Carausius morosus contain two native vitellins (Vit A and Vit B). Under denaturing conditions, these vitellins resolved into 3 (A₁, A₂, and A₃) and 2 (B₁ and B₂) polypeptides. All of these polypeptides had counterparts in the female hemolymph from which they were shown to be derived by in vivo labelling. During ovarian development, the 2 vitellins changed both in charge and polypeptide composition. In EV and LV follicles, Vit A resolved into 4 distinct vitellin polypeptides (A₀, A₁, A₂ and A₃). Using a panel of monoclonal antibodies, polypeptide A₀ proved to be immunologically related to polypeptide A₂. In follicles about to begin choriongenesis, polypeptide A₃ was gradually replaced by a lower M_r polypeptide. Over the same time period, polypeptide B₁ changed in charge, but not in M_r. To confirm the existence of a polypeptide processing in C. morosus, ovarian follicles of different developmental stages were exposed in vivo to [³⁵S]-methionine from 6 to 72 h. Data showed that A₀ and B₁ were the polypeptides most heavily labelled after short time exposures to the radioisotope. Polypeptides B₂ and A₃ were also labelled to some extent. With progressively longer exposures, polypeptides A₁ and A₂ also became labelled. In vivo exposure to [³H]-GlcNAc caused all vitellin polypeptides to become heavily labelled. Autoradiographic analysis of ovarian follicles labelled this way showed that, during development, radioactivity was gradually transferred from newly formed yolk spheres in the cortical ooplasm to the central ooplasm. Data were interpreted as suggesting a causal relationship between polypeptide processing and progressive yolk sphere fusion to yield the central ooplasm. © 1993 Wiley-Liss, Inc.     

Changes in whole body concentrations of thyroid hormones and cortisol in metamorphosing conger eel

Summary To clarify the hormonal regulation of metamorphosis of the conger eel (Conger myriaster), changes in whole body concentrations of thyroid hormones, thyroxine (T₄) and triiodothyronine (T₃), and cortisol during metamorphosis were examined, as well as the changes in the histological activity of the thyroid gland. In larvae before metamorphosis, T₄ and T₃ levels were less than 5 and 0.15 ng·g^-1 respectively. Levels of T₄ increased to about 30 ng·g^-1 during early metamorphosis, and decreased subsequently. Levels of T₃ increased gradually in early metamorphosis, and then increased abruptly to about 2.0 ng·g^-1 in late metamorphosis. Before metamorphosis, cortisol levels of the leptocephali less than 11 cm in total length were greater than 200 ng·g^-1. Cortisol levels decreased rapidly in larger premetamorphic leptocephali, and low levels were maintained throughout the metamorphic period. Histological observation revealed an activation of the thyroid gland in early metamorphosis; thyroid follicle epithelial cells became columnar and their nuclei larger. Active uptake of colloid by these cells and intensive vascularization of the gland were also observed. By the end of metamorphosis, follicle epithelial cells became squamous, indicating a low level of glandular activity. These results suggest that thyroid hormone plays an important role in regulation of conger eel metamorphosis.Abbreviations AL anal length - TL total length - T ₃ triiodothyronine - T ₄ thyroxine     

Autotrophe Gewebekulturen von Ruta graveolens und deren 14CO2-Markierungsprodukte

Summary It is possible to obtain autotrophic callus cultures by inhibiting cell respiration. During a first passage of four weeks the cultures synthesized chlorophyll on an agar-medium with a minimum of organic substances such as sugar, amino acids and vitamins. In the second passage these cultures were kept on the same medium but were aerated with a mixture of 99% N₂ and 1% CO₂. In the third and last passage the medium contained only mineral substances and the same mixture of N₂ and CO₂ was used for aeration. This pure mineral medium was supplemented with the Hoagland's solution.These autotrophic callus cultures were grown for about two years under these conditions and showed a growth quotient of ten.Three different groups of tissues were taken for the ¹⁴CO₂-fixation. The first group was grown for four weeks on a heterotrophic medium and aerated with O₂. This is the socalled respirating group. The second and third group were both aerated with the mixture of N₂/CO₂ but they were grown on different mediums. One of these groups was grown on a heterotrophic medium for four weeks: these are heterotrophic photosynthesizing tissues. The third group was grown on a pure mineral medium, and these are the autotrophic photosynthesizing callus tissues.Respirating tissues are different from photosynthesizing cultures in respect to the quantity of light-induced CO₂-fixation.The thin-layer chromatograms reveal the difference between heterotrophic and autotrophic tissues. In the light dependent ¹⁴CO₂-incorporation the difference is in the amounts of the labelled amino acids glycine and serine. In the dark dependent incorporation the difference is found in the amount of the labelled amino acid aspartic acid. The more autotrophic these tissues are, the higher the level of the CO₂-fixation in these amino acids is.

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Mit Hilfe der Deutschen Forschungsgemeinschaft.     

Triiodothyronine bound to red blood cells is not available for transport through the blood-brain barrier

Many steroid and thyroid hormones and some drugs are bound by circulating red cells. Red cell-bound ligand may not be physiologically inert, as recent studies show that red cell-bound drug is available for uptake by brain. To investigate whether triiodothyronine (T₃) is available for uptake by brain in vivo from the circulating red cell pool, the present investigations measure the effects of human erythrocytes on rat brain uptake of [¹²⁵I]T₃ in vivo. The fraction of circulating T₃ available for uptake in vivo in the presence of 0, 2, 5, 10, 22, or 44% red cells was essentially identical to the fraction of [¹²⁵I]T₃ unbound in vitro. Therefore, [¹²⁵I]T₃ bound to red cells obtained from normal volunteers is not available for uptake by brain in vivo.     

DEPENDENCE OF FAST AXOPLASMIC TRANSPORT IN NERVE ON OXIDATIVE METABOLISM

—A crest of labelled activity moving down the sciatic nerve at 401 ± 35 mm/day after injection of the L7 dorsal root ganglion of the cat with L-[³H]leucine characterizes fast axoplasmic transport of materials and has been studied with regard to its dependence on oxidative metabolism. Transport of labelled materials in vitro occurred if the nerve was supplied with O₂ or 95 % O₂+ 5 % CO₂. Transport was not dependent upon continuity of the fibres with the ganglionic soma. Asphyxiation (N₂) rapidly blocked fast transport in vitro. Likewise NaCN or dinitrophenol in an O₂ atmosphere both effectively block fast transport within 15 min. Tetrodotoxin and procaine, agents which block excitation of the membrane, had no effect on fast transport. The inference is that oxidative metabolism supplies the energy required by the molecular mechanism underlying fast axoplasmic transport.     

In vitro percutaneous absorption of boron as boric acid, borax, and disodium octaborate tetrahydrate in human skin: a summary

Literature from the first half of this century reports concern for toxicity from topical use of boric acid, but assessment of percutaneous absorption has been impaired by lack of analytical sensitivity. Analytical methods in this study included inductively coupled plasmamass spectrometry which now allows quantitation of percutaneous absorption of¹⁰B in¹⁰B-enriched boric acid, borax and disodium octaborate tetrahydrate (DOT) in biological matrices. In vitro human skin percent doses of boric acid absorbed were 1.2 for a 0.05% solution, 0.28 for a 0.5% solution, and 0.70 for a 5.0% solution. These absorption amounts translated into flux values of, respectively, 0.25, 0.58, and 14.58 μg/cm²/h, and permeability constants (K _p ) of 5.0 x 10^-4, 1.2 x 10^-4, and 2.9 x 10^-4 cm/h for the 0.05%, 0.5%, and 5.0% solutions. The above in vitro doses were at infinite, 1000 μL/cm² volume. At 2 μL/cm² (the in vivo dosing volume), flux decreased some 200-fold to 0.07 μg/cm²/h andK _p of 1.4 x 10^-6 cm/h, while percent dose absorbed was 1.75%. Borax dosed at 5.0%/1000 μL/cm² had 0.41 percent dose absorbed, flux at 8.5 μg/cm²/h, andK _p was 1.7 x 10^-4 cm/h. Disodium octaborate tetrahydrate (DOT) dosed at 10%/1000 μL/cm² was 0.19 percent dose absorbed, flux at 7.9 μg/cm²/h, andK _p was 0.8 x ICH cm/h. These in vitro results from infinite doses (1000 μL/cm²) were a 1000-fold greater than those obtained in the companion in vivo study. The results from the finite (2 μL/cm²) dosing were closer (10-fold difference) to the in vivo results. General application of infinite dose percutaneous absorption values for risk assessment is questioned by these results.     

Clearance Rates of Crustacean Macrofiltrators: The Nature of in situ Rate Depressions in a Fertilized Oligotrophic Lake in the Kuokkel Area,Northern Sweden

Lake seston labelled by ¹⁴CO₂, ¹⁴C-acetate and ³²-PO₄ in parallel was used in zooplankton feeding experiments and checked for size distribution of the labelled particulates. The in situ experiments were made to find out if depressed Eudiaptomus and Holopedium clearance rates, in an artificially fertilized lake where seston was dominated by μ-algae, were due to low retention of such algae. Addition of a labelled reference alga (Chlamydomonas sp.) was used to find out if high food concentrations (above the Incipient Limiting Concentration) or toxins caused the depressions. Clearance rates mostly ranked highest for CO₂-labelled seston followed by ³²P and ¹⁴C-acetate labelled seston, reflecting approximately the size distributions of the labelled food. Clearance rates for the added reference alga were high and similar in the fertilized lake and in an oligotrophic reference lake. It was concluded that the detected “rate depression” could be explained purely by poor retention of small particles (passive size selection) by the crustaceans studied, but that active food selection offers optional explanations to the experimental results.     

Labelling of 5-Hydroxytryptamine3 Receptors with a Novel 5-HT3 Receptor Ligand, [3H]RS-42358–197

Abstract: RS-42358–197{(S)-N-(1-azabicyclo[2.2.2]oct-3-yl)-2,4,5,6-tetrahydro-1H-benzo[de]isoquinolin-1-one hydrochloride} displaced the prototypic 5-hydroxytryptamine₃ (5-HT₃) receptor ligand [³H]quipazine in rat cerebral cortical membranes with an affinity (pK_i) of 9.8 ± 0.1, while having weak affinity (pK_i < 6.0) in 23 other receptor binding assays. [³H]RS-42358–197 was then utilized to label 5-HT₃ receptors in a variety of tissues. [³H]RS-42358–197 labelled high-affinity and saturable binding sites in membranes from rat cortex, NG108–15 cells, and rabbit ileal myenteric plexus with affinities (K_D) of 0.12 ± 0.01, 0.20 ± 0.01, and 0.10 ± 0.01 nM and densities (B_max) of 16.0 ± 2.0, 660 ± 74, and 88 ± 12 fmol/mg of protein, respectively. The density of sites labelled in each of these tissues with [³H]RS-42358–197 was similar to that labelled with [³H]GR 65630, but was significantly less than that found with [³H]-quipazine. The binding of [³H]RS-42358–197 had a pharmacological profile similar to that of [³H]quipazine, as indicated by the rank order of displacement potencies: RS-42358–197 > (S)-zacopride > tropisetron > (R)-zacopride > ondansetron > MDL72222 > 5-HT. However, differences in 5-HT₃ receptors of different tissues and species were detected on the basis of statistically significant differences in the affinities of phenylbiguanide, and 1-(m-chlorophenyl)biguanide when displacing [³H]RS-42358-197 binding. [³H]RS-42358–197 also labelled a population (B_max= 91 ± 17 fmol/mg of protein) of binding sites in guinea pig myenteric plexus membranes, with lower affinity (K_D= 1.6 ± 0.3 nM) than those in the other preparations. Moreover, the rank order of displacement potencies of 15 5-HT₃ receptor ligands in guinea pig ileum was found not to be identical to that in other tissues. Binding studies carried out with [³H]RS-42358–197 have detected differences in 5-HT₃ receptor binding sites in tissues of different species and further underscore the unique nature of the guinea pig 5-HT₃ receptor.     

Formation and metabolism of glycolate in the cyanobacterium Coccochloris peniocystis

The formation and metabolism of glycolate in the cyanobacterium Coccochloris peniocystis was investigated and the activities of enzymes of glycolate metabolism assayed. Photosynthetic ¹⁴CO₂ incorporation was O₂ insensitive and no labelled glycolate could be detected in cells incubated at 2 and 21% O₂. Under conditions of 100% O₂ glycolate comprised less than 1% of the acid-stable products indicating ribulose 1,5 bisphosphate (RuBP) oxidation only occurs under conditions of extreme O₂ stress. Metabolism of [1-¹⁴C] glycolate indicated that as much as 62% of ¹⁴C metabolized was released as ¹⁴CO₂ in the dark. Metabolism of labelled glycolate, particularly incorporation of ¹⁴C into glycine, was inhibited by the amino-transferase inhibitor amino-oxyacetate. Metabolism of [2-¹⁴C] glycine was not inhibited by the serine hydroxymethyltransferase inhibitor isonicotinic acid hydrazide and little or no labelled serine was detected as a result of ¹⁴C-glycolate metabolism. These findings indicate that a significant amount of metabolized glycolate is totally oxidized to CO₂ via formate. The remainder is converted to glycine or metabolized via a glyoxylate cycle. The conversion of glycine to serine contributes little to glycolate metabolism and the absence of hydroxypyruvate reductase confirms that the glycolate pathway is incomplete in this cyanobacterium.Abbreviations AAN aminoacetonitrile - AOA aminooxyacetate - DIC dissolved inorganic carbon - INH isonicotinic acid hydrazide - PEP phosphoenolpyruvate - PEPcase phosphoenolpyruvate carboxylase - PG phosphoglycolate - PGA phosphoglyceric acid - PGPase phosphoglycolate phosphatase - PR photorespiration - Rubisco ribulose-1,5-bisphosphate carboxylase oxygenase - TCA trichloroacetic acid - RuBP ribulose-1,5-bisphosphate     

Mass-spectrometric evidence for the double-carboxylation pathway of malate synthesis by Crassulacean acid metabolism plants in light

Phyllodia of the Crassulacean acid metabolism (CAM) plant Kalanchoë tubiflora were allowed to fix ¹³CO₂ in light and darkness during phase IV of the diurnal CAM cycle, and during prolongation of the regular light period. After ¹³CO₂ fixation in darkness, only singly labelled [¹³C]malate molecules were found. Fixation of ¹³CO₂ under illumination, however, produced singly labelled malate as well as malate molecules which carried label in two, three or four carbon atoms. When the irradiance during ¹³CO₂ fixation was increased, the proportion of singly labelled malate decreased in favour of plurally labelled malate. The irradiance, however, did not change either the ratio of labelled to unlabelled malate molecules found in the tissue after the ¹³CO₂ application, or the magnitude of malate accumulation during the treatment with label. The ability of the tissue to store malate and the labelling pattern changed throughout the duration of the prolonged light period. The results indicate that malate synthesis by CAM plants in light can proceed via a pathway containing two carboxylation steps, namely ribulose-1,5-bisphosphate-carboxylase/oxygenase (EC 4.1.1.39) and phosphoenolpyruvate carboxylase (EC 4.1.1.31) which operate in series and share common intermediates. It can be concluded that, in light, phosphoenolpyruvate carboxylase can also synthesize malate independently of the proceeding carboxylation step by ribulose-1,5-bisphosphate carboxylase/oxygenase.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase (EC 4.1.1.31) - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) - TMS trimethylsilyl     

The development of potential new fluorine-18 labelled radiotracers for imaging the GABAA receptor

Positron emission tomography (PET) using the tracer [¹¹C]Flumazenil has shown changes in the distribution and expression of the GABA_A receptor in a range of neurological conditions and injury states. We aim to develop a fluorine-18 labelled PET agent with comparable properties to [¹¹C]Flumazenil. In this study we make a direct comparison between the currently known fluorine-18 labelled GABA_A radiotracers and novel imidazobenzodiazepine ligands. A focussed library of novel compound was designed and synthesised where the fluorine containing moiety and the position of attachment is varied. The in vitro affinity of twenty-two compounds for the GABA_A receptor was measured. Compounds containing a fluoroalkyl amide or a longer chain ester group were eliminated due to low potency. The fluorine-18 radiochemistry of one compound from each structural type was assessed to confirm that an automated radiosynthesis in good yield was feasible. Eleven of the novel compounds assessed appeared suitable for in vivo assessment as PET tracers.     

Elicitor induction of the synthesis of a novel lectin-like arabinosylated hydroxyproline-rich glycoprotein in suspension cultures of Phaseolus vulgaris L.

A novel lectin-like glycoprotein which accumulates in response to fungal elicitor action has been characterised in endomembranes from suspension cultures of French bean (Phaseolus vulgaris L.). The lectin, which has specificity towards N-acetylglucosamine oligomers, consists of a polypeptide of apparent molecular weight (M_r) 31 000 which is rich in glycine and contains 6.7% hydroxyproline O-linked to arabinose-containing oligosaccharides to give a glycoprotein of M_r 42500. A dual-labelling technique has been used to identify changes in the synthesis of the glycoprotein in cells exposed to fungal elicitor molecules. Thus, incorporation of [¹⁴C]proline into membranes in vivo and of [1^-3H]arabinose from uridine 5-diphosphate [1^-3H]arabinose in vitro and analysis by isoelectric focussing-polyacrylamide gel electrophoresis gave absolute correspondence of the labelled isoform of the glycoprotein. Having established the absence of contaminating polypeptides, subsequent analysis of microsomal fractions bysodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the peak of sythesis of the M_r-42500 glycoprotein occurred 4 h after the addition of fungal elicitor. The changes in the level of incorporation into the glycoprotein monomers were concomitant with increases in the activity of prolyl hydroxylase (EC 1.14.11.2)Incorporation of [¹⁴C]proline and its subsequent post-translational modification to hydroxyproline in microsomal polypeptides was followed by rapid transfer into the wall with an average t _1/2 of about 7 min. The M_r-42500 glycoprotein was rapidly transferred out of the endomembrane fraction with a t _1/2 of 2 min and could be detected in wall fractions where it became progressively less extractable. The glycoprotein, which clearly differs from bean extensin, accounts for up to 40% of the hydroxyproline newly exported in response to elicitor action. The lectin, which resembles those found in the Solanaceae and which is coinduced with enzymes of phytoalexin synthesis, may play some role in disease resistance.Abbreviations HRGP hydroxyproline-rich glycoprotein - IEF isoelectric focussing - M_r apparent molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate     

Nitrogen and phosphorus cycling in relation to stand age of Eucalyptus regnans F. Muell

Lupins, canola, ryegrass and wheat fertilized with Na₂ ³⁵SO₄ and either ¹⁵NH₄Cl or K¹⁵NO₃(N:S=10:1), were grown in the field in unconfined microplots, and the sources of N and S (fertilizer, soil, atmosphere, seed) in plant tops during crop development were estimated. Modelled estimates of the proportion of lupin N derived from the atmosphere, which were obtained independently of reference plants, were used to calculate the proportion of lupin N derived from the soil. Total uptake of N and S and uptake of labelled N and S increased during crop development. Total uptake of S by canola was higher than lupins, but labelled S uptake by lupins exceeded uptake by canola. The form of N applied had no effect on uptake of labelled and unlabelled forms of N or S. Ratios of labelled to unlabelled S and ratios of labelled to unlabelled N derived from soil sources decreased during growth, and were less for S than for N for each crop at each sampling time. Although ratios of labelled to unlabelled soil-derived N were similar between crops at 155, 176 and 190 days after sowing, ratios of labelled to unlabelled S for lupins were higher than for the reference crops and declined during this period. The ratios of labelled to unlabelled S in lupins and the reference plants therefore bore no relationship either to ratios of labelled to unlabelled soil-derived N in the plants, or to total S uptake by the plants. Therefore the hypothesis that equal ratios of labelled N to unlabelled soil-derived N in legumes (Rleg) and reference plants (Rref) would be indicated by equal ratios of labelled to unlabelled S was not supported by the data. The results therefore show that the accuracy of reference plant-derived values of Rleg cannot be evaluated by labelling with ³⁵S.