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.     

Species Heterogeneity Between Gerbils and Rats: Quinolinate Production by Microglia and Astrocytes and Accumulations in Response to Ischemic Brain Injury and Systemic Immune Activation

Abstract: Quinolinic acid is an excitotoxic kynurenine pathway metabolite, the concentration of which increases in human brain during immune activation. The present study compared quinolinate responses to systemic and brain immune activation in gerbils and rats. Global cerebral ischemia in gerbils, but not rats, increased hippocampus indoleamine-2,3-dioxygenase activity and quinolinate levels 4 days postinjury. In a rat focal ischemia model, small increases in quinolinate concentrations occurred in infarcted regions on days 1, 3, and 7, although concentrations remained below serum values. In gerbils, systemic immune activation by an intraperitoneal injection of endotoxin (1 mg/kg of body weight) increased quinolinate levels in brain, blood, lung, liver, and spleen, with proportional increases in lung indoleamine-2,3-dioxygenase activity at 24 h postinjection. In rats, however, no significant quinolinate content changes occurred, whereas lung indoleamine-2,3-dioxygenase activity increased slightly. Gerbil, but not rat, brain microglia and peritoneal monocytes produced large quantities of [¹³C₆]-quinolinate from l -[¹³C₆]tryptophan. Gerbil astrocytes produced relatively small quantities of quinolinate, whereas rat astrocytes produced no detectable amounts. These results demonstrate that the limited capacity of rats to replicate elevations in brain and blood quinolinic acid levels in response to immune activation is attributable to blunted increases in local indoleamine-2,3-dioxygenase activity and a low capacity of microglia, astrocytes, and macrophages to convert l -tryptophan to quinolinate.     

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,.     

Origin and dynamics of indoleacetic acid under polar transport in Pinus sylvestris

Polarly‐transported IAA is regarded as a long distance correlative signal important in many aspects of plant physiology, including, for example, apical dominance and growth and development of vascular tissues. In this study, we investigated the importance of apical sources in supplying stem tissues with IAA. The current‐yearshoots of 4‐year‐old Pinus sylvestris L. saplings were replaced with a source of [¹³C₆]IAA. Subsequent mass spectrometric analysis showed that most of the IAA present at two positions in the subjacent 1‐year‐old internode consisted of [¹³C₆]IAA, while [¹²C]IAA of endogenous origin formed a minor pool. However, the pool of [¹³C₆]IAA decreased from 90 to 80% of the total free IAA pool [¹³C₆]IAA+[¹²C]IAA) while being transported down the shoot. This dilution with [¹²C]IAA indicates that de novo biosynthesis of IAA occurred. An additional defoliation experiment showed that the synthesis took place in stem tissues rather than in the mature leaves. The results confirm the role of apical shoots as the major source of polarly‐transported IAA, but also indicate that synthesis of IAA takes place in stem tissues. This is important when considering IAA balance at the whole plant level.     

Metabolic Precursors and Compartmentation of Cerebral GABA in Vigabatrin-Treated Rats

Abstract: The metabolic precursors and cerebral compartmentation of the augmented GABA pool induced by vigabatrin, an irreversible inhibitor of GABA transaminase, have been investigated by ¹³C NMR. Adult rats receiving rat chow ad libitum were given either drinking water only or drinking water containing 2.5 g/L vigabatrin for 7 days. Both groups of animals were infused either with [1,2-¹³C₂]acetate (15 µmol/min/100 g body weight), an exclusive precursor of GABA formation through the glial glutamine pathway, or with [1,2-¹³C₂]glucose (15 µmol/min/100 g body weight), a substrate that can produce GABA through the glial glutamine pathway or by direct metabolism in the neurons. The brains were frozen in situ, extracted with perchloric acid, and analyzed by ¹³C NMR. In vigabatrin-treated animals [¹³C]glutamine, a common intermediate for [¹³C]GABA synthesis from glucose or acetate, was accumulated to similar amounts during infusions with [1,2-¹³C₂]glucose or [1,2-¹³C₂]acetate. However, [¹³C]GABA accumulation was sevenfold higher during [1,2-¹³C₂]glucose infusions or twofold higher during [1,2-¹³C₂]acetate infusions. These results show that the direct pathway of GABA formation by neuronal metabolism of glucose predominates over the alternative pathway through glial glutamine. Near-equilibrium relationships of the aminotransferases of GABA and aspartate imply that the observed [¹³C]GABA accumulation occurs initially in the neuronal compartment.     

Enrichment and characterization of a sulfate-reducing toluene-degrading microbial consortium by combining in situ microcosms and stable isotope probing techniques

A toluene-degrading microbial consortium was enriched directly in a BTEX-contaminated aquifer under sulfate-reducing conditions using in situ microcosms consisting of toluene-loaded activated carbon pellets. Degradation of toluene and concomitant sulfide production by the consortium was subsequently demonstrated in laboratory microcosms. The consortium was physiologically and phylogenetically characterized by isotope tracer experiments using nonlabeled toluene, [¹³C]-α-toluene or [¹³C₇]-toluene as growth substrates. Cells incubated with [¹³C]-α-toluene or [¹³C₇]-toluene incorporated 8–15 at.%¹³C and 51–57 at.%¹³C into total lipid fatty acids, respectively, indicating a lower specific incorporation of ¹³C from [¹³C₇]-toluene. In order to identify the toluene-assimilating bacteria, the incorporation of carbon from both [¹³C]-α-toluene and [¹³C₇]-toluene into rRNA was analyzed by stable isotope probing. Time and buoyant density-resolved 16S rRNA gene-based terminal restriction fragment length polymorphism profiles, combined with cloning and sequencing, revealed that an uncultured bacterium (99% sequence similarity) related to the genus Desulfocapsa was the main toluene-degrading organism in the consortium. The ratio of the respective terminal restriction fragments changed over time, indicating trophic interactions within this consortium.     

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.     

Altered cerebral glucose and acetate metabolism in succinic semialdehyde dehydrogenase-deficient mice: evidence for glial dysfunction and reduced glutamate/glutamine cycling

Succinic semialdehyde dehydrogenase (SSADH) catalyzes the NADP-dependent oxidation of succinic semialdehyde to succinate, the final step of the GABA shunt pathway. SSADH deficiency in humans is associated with excessive elevation of GABA and γ-hydroxybutyrate (GHB). Recent studies of SSADH-null mice show that elevated GABA and GHB are accompanied by reduced glutamine, a known precursor of the neurotransmitters glutamate and GABA. In this study, cerebral metabolism was investigated in urethane-anesthetized SSADH-null and wild-type 17-day-old mice by intraperitoneal infusion of [1,6-¹³C₂]glucose or [2-¹³C]acetate for different periods. Cortical extracts were prepared and measured using high-resolution ¹H-[¹³C] NMR spectroscopy. Compared with wild-type, levels of GABA, GHB, aspartate, and alanine were significantly higher in SSADH-null cortex, whereas glutamate, glutamine, and taurine were lower. ¹³C Labeling from [1,6-¹³C₂]glucose, which is metabolized in neurons and glia, was significantly lower (expressed as μmol of ¹³C incorporated per gram of brain tissue) for glutamate-(C4,C3), glutamine-C4, succinate-(C3/2), and aspartate-C3 in SSADH-null cortex, whereas Ala-C3 was higher and GABA-C2 unchanged. ¹³C Labeling from [2-¹³C]acetate, a glial substrate, was lower mainly in glutamine-C4 and glutamate-(C4,C3). GHB was labeled by both substrates in SSADH-null mice consistent with GABA as precursor. Our findings indicate that SSADH deficiency is associated with major alterations in glutamate and glutamine metabolism in glia and neurons with surprisingly lesser effects on GABA synthesis.     

Metabolism of [U-13C5]Glutamine in Cultured Astrocytes Studied by NMR Spectroscopy: First Evidence of Astrocytic Pyruvate Recycling

Abstract: Metabolism of [U-¹³C₅]glutamine was studied in primary cultures of cerebral cortical astrocytes in the presence or absence of extracellular glutamate. Perchloric acid extracts of the cells as well as redissolved lyophilized media were subjected to nuclear magnetic resonance and mass spectrometry to identify ¹³C-labeled metabolites. Label from glutamine was found in glutamate and to a lesser extent in lactate and alanine. In the presence of unlabeled glutamate, label was also observed in aspartate. It could be clearly demonstrated that some [U-¹³C₅]glutamine is metabolized through the tricarboxylic acid cycle, although to a much smaller extent than previously shown for [U-¹³C₅]glutamate. Lactate formation from tricarboxylic acid cycle intermediates has previously been demonstrated. It has, however, not been demonstrated that pyruvate, formed from glutamate or glutamine, may reenter the tricarboxylic acid cycle after conversion to acetyl-CoA. The present work demonstrates that this pathway is active, because [4,5-¹³C₂]glutamate was observed in astrocytes incubated with [U-¹³C₅]glutamine in the additional presence of unlabeled glutamate. Furthermore, using mass spectrometry, mono-labeled alanine, glutamate, and glutamine were detected. This isotopomer could be derived via the action of pyruvate carboxylase using ¹³CO₂ produced within the mitochondria or from labeled intermediates that had stayed in the tricarboxylic acid cycle for more than one turn.     

ACETOACETATE AND d-(-)-BETA-HYDROXYBUTYRATE AS PRECURSORS FOR STEROL SYNTHESIS BY CALF OLIGODENDROCYTES IN SUSPENSION CULTURE: EXTRAMITOCHONDRIAL PATHWAY FOR ACETOACETATE METABOLISM

Abstract— Oligodendroglia prepared from calf cerebral white matter were incubated with [3^-14C]aceto-acetate (AcAc), d -[3^-14C](-)-beta-hydroxybutyrate (OHbut) or ³H₂O. Addition to the medium of the ATP-rate oxaloacetate lyase inhibitor (-)-hydroxycitrate diminished sterol labelling from OHbut and ³H₂O, but caused 4-fold stimulation of sterol labelling from AcAc. The discrepancy between results with the two ketone bodies indicates the existence in oligodendroglia of an extrarnitochondrial pathway for conversion of AcAc but not OHbut to acetyl CoA. Acetoacetyl CoA synthetase, the first enzyme in this pathway, was more than 20-fold enriched in oligodendroglia over whole brain.     

Identification, Development, and Regional Distribution of Thymidylate Synthetase in Adult Rabbit Brain

Abstract: The development and regional distribution of thymidylate synthetase (TS) (EC 2.1.1.45) in rabbit brain were determined. After optimization of the assay for brain, TS activity in brain was measured by a nonspecific (³H₂O release) and specific method. The specific method involved the conversion of [6-³H]deoxyuridine monophosphate (dUMP) to [³H]thymidine phosphate and the subsequent identification of [³H]thymidine. The specific activity of the enzyme in whole brain of newborn rabbits declined from 10.35 ± 1.17 units/mg protein to 0.71 ± 0.09 units/mg protein at 10–12 weeks of age. Two-year-old rabbits had 0.81 ± 0.04 units/mg protein. The decline in specific activity with age was not due to an inhibitor of TS activity or a change in the K_m for dUMP. The K_m for dUMP of the unpurified enzyme in the brains of both 10-day-old and young adult rabbits was 0.8 μ m . In young adult rabbits (3 months) the specific activity of TS was similar in the various regions of the brain tested except for the cerebellum, which had 40% higher specific activity than the whole brain. The results show that TS is widely distributed in adult rabbit brain, and, although the activity declines with age, it stabilizes at adult levels at 3 months of age.     

INCORPORATION OF PHENYLALANINE AS A SINGLE UNIT INTO RAT BRAIN PROTEIN: RECIPROCAL INHIBITION BY PHENYLALANINE AND TYROSINE OF THEIR RESPECTIVE INCORPORATIONS

Abstract— Of seven amino acids studied, glutamic acid and phenylalanine were incorporated in highest amounts into the hot-TCA-insoluble material of the 100,000 g supernatant fraction of rat brain homogenate. The system for incorporation of phenylalanine was RNase-insensitive and required ATP (apparent K_m = 0.64 m m ), KC1 (apparent K_m = 14 m m ) and MgCl₂ (optimal concentration range 4-15 m m ). The apparent K_m for phenylalanine was 2.9 m m . [¹⁴C]Phenylalanine did not undergo modification before incorporation. Tyrosine and phenylalanine inhibited the incorporation, respectively, of [¹⁴C]phenylalanine and [¹⁴C]tyrosine when incubated simultaneously or successively. The K_m and K_t (3.3 m m ) values for phenylalanine in the incorporation reaction and as inhibitor of the incorporation of [¹⁴C]tyrosine were similar. We suggest that both the enzyme and the acceptor for the incorporation of these two amino acids are the same. [¹⁴C]Phenylalanine and [¹⁴C]tyrosine entered into COOH-terminal positions in the reactions described. Brain exhibited a 25- to 100-fold higher capacity to incorporate phenylalanine than that of liver, kidney or thyroid. The acceptor capacity in rat brain rapidly decreased from day 5 to day 15 of postnatal age and then slowly until age 150 days.     

In vivo neurochemical profiling of rat brain by 1H-[13C] NMR spectroscopy: cerebral energetics and glutamatergic/GABAergic neurotransmission

The quantification of excitatory and inhibitory neurotransmission and the associated energy metabolism is crucial for a proper understanding of brain function. Although the detection of glutamatergic neurotransmission in vivo by ¹³C NMR spectroscopy is now relatively routine, the detection of GABAergic neurotransmission in vivo has remained elusive because of the low GABA concentration and spectral overlap. Using ¹H-[¹³C] NMR spectroscopy at high magnetic field in combination with robust spectral modeling and the use of different substrates, [U-¹³C₆]-glucose and [2-¹³C]-acetate, it is shown that GABAergic, as well as glutamatergic neurotransmitter fluxes can be detected non-invasively in rat brain in vivo .     

Metabolism of [5-3H]Kynurenine in the Rat Brain In Vivo: Evidence for the Existence of a Functional Kynurenine Pathway

Abstract: The incorporation of tritium label into quinolinic acid (QUIN), kynurenic acid (KYNA), and other kynurenine (KYN) pathway metabolites was studied in normal and QUIN-lesioned rat striata after a focal injection of [5-³H]KYN in vivo. The time course of metabolite accumulation was examined 15 min to 4 h after injection of [5-³H]KYN, and the concentration dependence of KYN metabolism was studied in rats killed 2 h after injection of 1.5–1,500 µ M [5-³H]KYN. Labeled QUIN, KYNA, 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid, and xanthurenic acid (XA) were recovered from the striatum in every experiment. Following injection of 15 µ M [5-³H]KYN, a lesion-induced increase in KYN metabolism was noted. Thus, the proportional recoveries of [³H]KYNA (5.0 vs. 1.8%), [³H]3-HK (20.9 vs. 4.5%), [³H]XA (1.5 vs. 0.4%), and [³H]QUIN (3.6 vs. 0.6%) were markedly elevated in the lesioned striatum. Increases in KYN metabolism in lesioned tissue were evident at all time points and KYN concentrations used. Lesion-induced increases of the activities of kynurenine-3-hydroxylase (3.6-fold), kynureninase (7.6-fold), kynurenine aminotransferase (1.8-fold), and 3-hydroxyanthranilic acid oxygenase (4.2-fold) likely contributed to the enhanced flux through the pathway in the lesioned striatum. These data provide evidence for the existence of a functional KYN pathway in the normal rat brain and for a substantial increase in flux after neuronal ablation. This method should be of value for in vivo studies of cerebral KYN pathway function and dysfunction.     

Carbon metabolism of Listeria monocytogenes growing inside macrophages

The intracellular metabolism of Listeria monocytogenes was studied by ¹³C-isotopologue profiling using murine J774A.1 macrophages as host cells. Six hours after infection, bacteria were separated from the macrophages and hydrolyzed. Amino acids were converted into tert-butyl-dimethylsilyl derivatives and subjected to gas chromatography/mass spectrometry. When the macrophages were supplied with [U-¹³C₆]glucose prior to infection, but not during infection, label was detected only in Ala, Asp and Glu of the macrophage and bacterial protein with equal isotope distribution. When [U-¹³C₆]glucose was provided during the infection period, ¹³C label was found again in Ala, Asp and Glu from host and bacterial protein, but also in Ser, Gly, Thr and Val from the bacterial fraction. Mutants of L. monocytogenes defective in the uptake and catabolism of the C₃-metabolites, glycerol and/or dihydroxyacetone, showed reduced incorporation of [U-¹³C₆]glucose into bacterial amino acids under the same experimental settings. The ¹³C pattern suggests that (i) significant fractions (50–100%) of bacterial amino acids were provided by the host cell, (ii) a C₃-metabolite can serve as carbon source for L. monocytogenes under intracellular conditions and (iii) bacterial biosynthesis of Asp, Thr and Glu proceeds via oxaloacetate by carboxylation of pyruvate.     

Rat Brain and Plasma Norepinephrine Glycol Metabolites Determined by Gas Chromatography-Mass Fragmentography

Abstract: A gas chromatographic-mass fragmentographic (GC-MF) procedure is described for the simultaneous quantitation of 3,4-dihydroxyphenyl-ethyleneglycol (DHPG) and 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) in brain tissue and plasma. DHPG and MHPG were assayed as their respective acetyl-trifluoroacyl esters, using [²H₂]DHPG and [²H₃]MHPG as internal standards. Assay sensitivities of at least 1 ng per sample were attainable for the quantitation of free glycols, whereas for determination of total DHPG, assay sensitivity was 2.5 ng. Whole rat brain total (99.2 ±4.11 ng/g) and free (13.0 ± 1.14 ng/g) DHPG concentrations were similar to respective total (86.0 ± 3.70 ng/g) and free (12.3 ± 0.41 ng/g) MHPG levels. Total DHPG concentrations exceeded total MHPG levels in hypothalamus (3.0:1), midbrain (1.4:1), pons plus medulla (1.3:1), and hippocampus (1.5:1), whereas in other brain regions the levels of these metabolites were similar. In plasma, however, total DHPG levels were only 20% as high as MHPG concentrations. In mouse brain, DHPG and MHPG occurred almost entirely in free form (>90%), but total DHPG levels were only 50% as high as respective MHPG concentrations. These results emphasize the substantial formation of DHPG compared with MHPG in rat and mouse brain and suggest that DHPG formation and eMux may be of equal or greater importance than MHPG in the metabolic clearance of CNS norepinephrine in some species.     

[3H]LY303870, a Novel Nonpeptide Radioligand for the NK-1 Receptor

Abstract: We synthesized a potent and selective antagonist radioligand for the neurokinin (NK)-1 receptor and characterized its binding to guinea pig striatal membranes. ( R ) - N - [2 - [Acetyl[³H₃][(2 - methoxyphenyl) - methyl]amino] - 1 - (1 H - indol - 3 - ylmethyl)ethyl][1,4' - bipiperidine]-1'-acetamide ([³H]LY303870) binds to a single class of sites with an equilibrium K _D of 0.22 n M and a B _max of 723 fmol/mg of protein. Unlabeled LY303870 potently inhibited the binding with an IC₅₀ of 0.56 n M , whereas the less active ( S )-enantiomer (LY306155) was substantially less potent. The nonpeptide NK-1 antagonists (±)-CP96,345 and (±)-RP 67580 had IC₅₀ values of 0.74 and 49 n M , respectively. Substance P (SP) was also a potent inhibitor with with an IC₅₀ of 3.1 n M . The inhibition by SP could be separated into two components: a high-affinity component with a K _i of 0.53 n M and a lower-affinity component with a K _i of 155 n M . Addition of 100 µ M guanylyl 5'-imidodiphosphate [Gpp(NH)p] in the incubation increased the relative amount of the low-affinity agonist state of the receptor. Consistent with the antagonist properties of LY303870, the dissociation rate of [³H]LY303870 was not changed by the presence of 100 µ M Gpp(NH)p. The distribution of [³H]LY303870 binding sites in the guinea pig brain closely matched the distribution of NK-1 receptors labeled by [³H]SP. Therefore, [³H]LY303870 is a potent and selective antagonist radioligand for NK-1 receptors in guinea pig brain. In addition, regulation of NK-1 agonist affinity by guanine nucleotides is similar to that seen for monoaminergic receptors.     

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.     

Growth and development of Brassica genotypes differing in endogenous gibberellin content. II. Gibberellin content, growth analyses and cell size

Three rapid cycling Brassica rapa genotypes were grown in greenhouse conditions to investigate the possible relationships between endogenous gibberellin (GA) content and shoot growth. Endogenous GA₁ GA₃ and GA₂₀ were extracted from stem samples harvested at 3 weekly intervals and analyzed by gas chromatography-mass spectrometry with selected ion monitoring, using [²H₂]-GA₁ and [²H₂]-GA₂₀ as quantitative internal standards. During the first 2 weeks, GA levels of the dwarf, rosette ( ros ), averaged 36% of levels in normal plants (on a per stem basis). Levels in the tall mutant, elongated internode (ein) , were consistently higher, averaging 305% of levels in normal plants.
Differences in shoot height across the genotypes resulted from varying internode length which resulted from epidermal cell length and number being increased in ein and decreased in ros relative to the normal genotype. The exogenous application of GA₃ to normal plants increased cell length while the application of paclobutrazol (PP333), a triazole plant growth retardant, reduced cell size. Thus, exogenous GA manipulations mimicked the influence of the mutant genes ros and ein. The dwarf, ros , had reduced shoot dry weights and relative growth rates compared to the other genotypes. Total dry weights were similar in ein and the normal genotype but stem weights were increased in ein , compensating for decreased leaf weights. Thus, the gibberellin-deficiency of ros resulted in generally reduced shoot growth. The overproduction of endogenous GA by ein did not result in enhanced shoot growth but rather a specific enhancement of internode elongation and stem growth at the expense of leaf size.     

RAPIDLY LABELED AND SECRETED PROTEINS OF THE CHICK BRAIN

Abstract— The extracellular and cerebrospinal fluids (ECF) of the chick brain were found to contain a distinctive group of rapidly labeled proteins. Gel staining patterns suggest that most ECF protein bands correspond with components also found in either the homogenized whole brain cytoplasmic fraction or the blood serum. The valine-incorporation profiles of these three fractions, however, were entirely distinctive. Comparisons were carried out using a sensitive double-labeling method, in which ECF proteins from chicks labeled for 1 h with [³H]valine were comigrated on SDS-polyacrylamide gels with the cytoplasmic or serum proteins from a ¹⁴C-labeled animal. Analyses of the ³H- and ¹⁴C-labeling profiles from these gels showed that certain newly-synthesized proteins are heavily enriched in the ECF relative to the other two fractions. Most prominently, material with an apparent molecular weight of # 17,000 was found to incorporate nearly one-third of all the radioactivity appearing in the ECF proteins, but was not heavily labeled in either the cytoplasmic or serum fractions. The effects of a simple training experience on the pattern of chicks' brain protein synthesis were also examined.