Studies on the Growth Effects of the Canaline-Urea Cycle Amino Acids with Lemna minor L

The aquatic microphyte, Lemna minor L., was utilized to assess the relative toxicity and general growth effects of canavanine, canaline, ureidohomoserine (UHS), and canavaninosuccinate (CSA). These amino acids are constituents of the canaline-urea cycle and structural analogues of the ornithine-urea cycle amino acids.     

Regulation of Sulfate Assimilation by Nitrogen Nutrition in the Duckweed Lemna minor L

The effect of nitrate and ammonium on the extractable activity of two enzymes of assimilatory sulfate reduction, ATP sulfurylase (EC 2.7.7.4) and adenosine 5′-phosphosulfate sulfotransferase (APSSTase), was examined in Lemna minor L. cultivated under steady state conditions. Nitrate reductase (EC 1.6.6.1) was measured for comparison. Low nitrate concentrations (0.2 and 0.04 millimolar) caused a decrease in the specific activity of all three enzymes measured. Twenty-four hours after transfer to medium without a nitrogen source, the specific activity of APSSTase and nitrate reductase was at less than 30% of the original level, whereas ATP sulfurylase was still at about 80%. NH₄⁺ added to the nutrient solution caused a 50 to 100% increase in the specific activity of APSSTase within 24 hours, followed by a slow decrease. After 72 hours with NH₄⁺, the specific activity was still 25% higher than originally. During the same period, the extractable protein increased by 30% on a fresh weight basis, and total protein by 55 to 60%. Nitrate reductase activity decreased to less than 5%. After omission of NH₄⁺ from the nutrient solution extractable APSSTase activity rapidly decreased to the level of cultures with NO₃⁻ as a nitrogen source. Using [³⁵S]SO₄²⁻ as a sulfur source, an increased incorporation of label into the protein fraction could be detected when NH₄⁺ was added to the nutrient solution. This indicated that more sulfate was assimilated and used for protein synthesis. The higher extractable activity of APSSTase with NH₄⁺ may be a regulatory mechanism involved in the formation of sufficient sulfur amino acids during a period of increased protein synthesis.     

Amino Acid Metabolism of Lemna minor L. : IV. N-Labeling Kinetics of the Amide and Amino Groups of Glutamine and Asparagine

A serious limitation to the use of N(O,S)-heptafluorobutyryl isobutyl amino acid derivatives in the analysis of ¹⁵N-labeling kinetics of amino acids in plant tissues, is that the amides glutamine and asparagine undergo acid hydrolysis to glutamate and aspartate, respectively, during derivatization. This led us to consider an alternative procedure (G Fortier et al. [1986] J Chromatogr 361: 253-261) for derivatization of glutamine and asparagine with N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide in pyridine. Gas chromatography-mass spectrometry (electron ionization) yielded fragment ions (M-57) of mass 417 and 431 for the [¹⁴N]asparagine and [¹⁴N]glutamine derivatives, respectively, suitable for monitoring unlabeled, single-¹⁵N- and double-¹⁵N-labeled amide species from the ion clusters at mass to charge ratio (m/z) 415 to 423 for asparagine, and m/z 429 to 437 for glutamine. From separate analyses of the specific isotope abundance of the amino-N groups of asparagine and glutamine as their N-heptafluorobutyryl isobutyl derivatives, the specific amide-[¹⁵N] abundance of these amino acids was determined. We demonstrate that this approach to ¹⁵N analysis of the amides can yield unique insights as to the compartmentation of asparagine and glutamine in vivo. The ratios of unlabeled:single-¹⁵N:double-¹⁵N-labeled species are highly diagnostic of the relative sizes and turnover of metabolically active and inactive pools of the amides and their precursors. Kinetic evidence is presented to indicate that a significant proportion (approximately 10%) of the free asparagine pool may be metabolically inactive (vacuolar). If the amide group of asparagine is derived exclusively from glutamine-amide, then asparagine must be synthesized in a compartment of the cell in which both glutamine-amide and aspartate are more heavily labeled with ¹⁵N than the bulk pools of these amino acids. This compartment is presumably the chloroplast. The transaminase inhibitor aminooxyacetate is shown to markedly inhibit amino acid synthesis; several amino acid pools accumulated in the presence of aminooxyacetate and [¹⁵N]H₄⁺ are ¹⁴N-enriched and must be derived primarily from protein turnover.     

香菇蛋白质氨基酸的分析

分析结果表明,香菇的菌柄与菌盖中蛋白质氨基酸均为18种,缺少谷氨酰胺(Gln)和天冬酰胺(Asn),其中,含量最高的是谷氨酸(菌柄为11.03 mg/gDW,菌盖为12.57 mg/gDW)。菌盖与菌柄均含必需氨基酸、半必需氨基酸10种,其中精氨酸含量(菌柄为10.73 mg/gDW,菌盖为11.84 mg/gDW)。必需氨基酸的含量十分接近于非必需氨基酸的含量(比率为1.00∶1.04),无论总氨基酸含量还是必需氨基酸含量,菌盖中的皆略高于菌柄中的。     

Identification of Gibberellins in Norway Spruce (Picea abies [L.] Karst.) by Combined Gas Chromatography-Mass Spectrometry

Gibberellins A₁ (GA₁), A₃ and A₉ were identified from extracts of shoots of 6-month old Norway spruce (Picea abies) seedlings by the use of sequential reverse and normal phase high performance liquid chromatography (HPLC), bioassay, radioimmunoassay (RIA) and combined gas chromatography-mass spectrometry (GC-MS). The bioassay and RIA were used after fractionation by HPLC to detect the GA-containing fractions, which were then examined by GC-MS. The GAs identified are considered to be endogenous.     

Translocation Profiles of [11C] and [13N]-Labelled Metabolites after Assimilation of 11CO2 and [13N]-Labelled Ammonia Gas by Leaves of Helianthus annuus L. and Lupinus albus L.

Tracer amounts of atmospheric [¹³N]-Iabelled ammonia gas, wereabsorbed by leaves of Lupinus albus and Helianthus annuus inboth the light and the dark. Exogenous [¹³N]-ammonia was onlyabsorbed in the dark when the feeding occurred shortly aftera period of illumination and the tissue was not depleted ofits carbohydrate reserves (e.g. starch). Incorporation of the[¹³N]-ammonia appeared to occur via the leaf glutamine synthetase/glutamatesynthase (GS/GOGAT) cycle since 2.0 mol m^–3 MSX, an inhibitorof the GS reduced uptake in both the light and dark. Photosyntheticincorporation of ¹¹CO₂ was not affected by this treatment Therate of movement of [¹³N]-assimilates in the petiole of attachedleaves of Helianthus and Lupinus was similar to that of the¹¹Cl-photo assimilates. Export of both [¹³N] and [¹¹C]-Iabelledassimilates from the leaf and movement in the petiole in boththe light and the dark was inhibited by source leaf anoxia (i.e.nitrogen gas). Translocation was re-established at the samerate when the feed leaf was exposed to gas containing more than2% O₂ which permitted dark respiration to proceed. After aninitial feeding of either ¹¹CO₂ or [¹³N]-ammonia at ambient(21%) O₂ exposure of the source leaf to 2% O2, or 50% O² didnot alter the rates of translocation, indicating that changesin photosynthetic activity in the source leaf due to photorespiratoryactivity need not markedly alter, at least during the shortperiod, the loading and translocation of either [¹¹C ] or [¹³N]-labelledleaf products. Key words: Translocation, CO₂, NH₃, Leaves, Helianthus annuus, Lupinus albus     

Diatom ecology in the phyllosphere of the common duckweed (Lemna minor L.)

Epiphyton associated with thick, floating mats of the common duckweed (Lemna minor L.) was studied at four sites in western Canada between 1985 and 1988. Maximum epiphyton abundance generally occurred in spring as biomass of the duckweed mat was increasing. Epiphytic biomass was low during summer and increased at some sites in autumn with mat decomposition. The community was composed mostly of diatoms and, during summer, photosynthetic bacteria. Species richness of the diatom flora was low, suggesting that duckweed mats are environments to which few species are adapted. Photosynthesis - irradiance curves indicated that duckweed epiphyton was not adapted to low light levels that occurred in the mat (< 1 % of ambient), suggesting they may survive via other means of nutrition. The mat phyllosphere was also characterized by wide spatial and temporal variation in temperature, and sharp vertical profiles of dissolved oxygen and nutrients.     

Synthesis and biological activity of N(alpha)-[4-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]-N-propargylamino]phenylacetyl]-L-glutamic acid

2-Deamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583) is a potent inhibitor of thymidylate synthase. Its analogue, N(alpha)-[4-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]-N-propargylamino]phenylacetyl]-L-glutamic acid, containing p-aminophenylacetic acid residue substituting p-aminobenzoic acid residue, was synthesized. The new analogue exhibited a moderately potent thymidylate synthase inhibition, of linear mixed type vs. the cofactor, N(5,10)-methylenetetrahydrofolate. The Ki value of 0.34 microM, determined with a purified recombinant rat hepatoma enzyme, was about 30-fold higher than that reported for inhibition of thymidylate synthase from mouse leukemia L1210 cells by ICI 198583 (Hughes et al., 1990, J. Med. Chem. 33, 3060). Growth of mouse leukemia L5178Y cells was inhibited by the analogue (IC50 = 1.26 mM) 180-fold weaker than by ICI 198583 (IC50 = 6.9 microM).     

Comparison of [3H] phencyclidine ([3H] PCP) and [3H] N-[1-(2-thienyl) cyclohexyl] piperidine ([3H] TCP) binding properties to rat and human brain membranes

The investigation of [3H] PCP and [3H] TCP binding properties to rat cerebrum and cerebellum resulted in the demonstration of multiple binding sites for the two drugs. In the two tissue preparations PCP had a lower affinity than TCP. In membranes from the cerebrum an equal number of high affinity binding sites were present for [3H] PCP and [3H] TCP. However, low affinity binding sites were two times more numerous for [3H] PCP than for [3H] TCP. In the cerebellum, the number of high and low affinity sites labeled by the two radioligands was identical, but the number of high affinity sites was about 7 fold lower than in the cerebrum. Taken together these results may indicate that in the cerebrum [3H] PCP labels other sites than NMDA/PCP receptor(s), maybe sigma receptors and/or the dopamine uptake complex. In human cerebral cortex samples [3H] TCP also bound to two different sites. The number of high and low affinity sites were 12 and 3 times, respectively, less abundant than in the rat cerebrum. Low affinity sites were of higher affinity (5 times) than corresponding sites in the rat brain. In the human cerebellum [3H] TCP binding parameters were identical to those measured in the same region in the rat.     

Synthesis of [N]glutamate from [N]h(4) and [N]glycine by mitochondria isolated from pea and corn shoots

Metabolically competent mitochondria were isolated from pea and corn shoots on Percoll discontinuous density gradients. Rates of synthesis of [¹⁵N]glutamate were measured by gas chromatography-mass spectrometry after the incubation of mitochondria with either 2 millimolar [¹⁵N] H₄⁺ or [¹⁵N]glycine in the presence of 1 millimolar citrate as the respiratory substrate. When [¹⁵N]H₄⁺ was provided, mitochondria isolated from light-grown pea shoots synthesized [¹⁵N]glutamate with a rate of 2.64 nanomoles per hour per milligram mitochondrial protein. Corn mitochondria produced [¹⁵N]glutamate at a rate approximately 11 times greater than the pea mitochondria. Dark treatment during growth for the last 24 hours caused a slight reduction in the rate of synthesis in both species. When [¹⁵N]glycine was used, pea mitochondria synthesized [¹⁵N]glutamate with a rate of 6.32 nanomoles per hour per milligram protein. Rapid disappearance of [¹⁵N]glycine and synthesis of [¹⁵N]serine was observed with a molar ratio of 2 glycine to 0.78 serine. The rate of glutamate synthesis was only 0.2% that of serine, due in part to the dilution of [¹⁵N]H₄⁺ by the [¹⁴N]H₄⁺ pool in the mitochondria. The majority of the [¹⁵N]H₄⁺ released from glycine appears to have been released from or remains unmetabolized in the mitochondria. Corn mitochondria showed no apparent disappearance of [¹⁵N]glycine and little synthesis of [¹⁵N]serine, indicating that our preparation originated primarily from mesophyll cells. Under our conditions of glycine/serine conversion, [¹⁵N]glutatmate was synthesized at a rate of 7% of that of [¹⁵N]serine synthesis by corn mitochondria.     

动态吸附与气相色谱-质谱（GC-MS）联用分析密蒙花（醉鱼草科）的挥发性花香成分

采用气相色谱-质谱联用技术对动态吸附法收集的密蒙花（Buddleja officinalis）花香成分进行了分析,并用气相色谱面积归-化法对各成分进行了定量。从密蒙花中分离出16个挥发性成分,定性定量出其中的11个,占挥发性成分总量的95．44％。其中丁基醋酸乙酯（81．57％）、苯甲醛（4．92％）、3-已烯-1-醇（3．26％）、欧洲丁香醛（2．34％）和芳樟醇（1．05％）为主要成分。该研究阐明了自然条件下密蒙花的花香成分及组成,其结果为今后定向创新醉鱼草属新香型观赏品种提供了科学依据。     

薄皮甜瓜果实不同部位中的挥发性酯类物质与氨基酸的关系

结合固相微萃取法（SPME）和气质联用仪（GC—Ms）检测薄皮甜瓜果实成熟时期不同部位中挥发性酯类物质和游离氨基酸的种类及含量的结果表明,果实中有25种挥发性酯类物质,脐果肉、中果肉、蒂果肉以及瓜瓤中的酯类物质种类分别为17种、13种、7种和13种,相对含量分别为71．67％、61．45％、51．46％和68．76％。果肉中共检测到12种游离氨基酸,脐果肉、中果肉和蒂果肉中游离氨基酸含量依序呈递减趋势,与总酯类物质和支链酯类物质的种类及含量趋势变化相一致。     

Analysis of structure-activity relationships for the 'A-region' of N-(4-t-butylbenzyl)-N'-[4-(methylsulfonylamino)benzyl]thiourea analogues as TRPV1 antagonists

The structure-activity relationships for the 'A-region' of N-(4-t-butylbenzyl)-N'-[4-(methylsulfonylamino)benzyl]thiourea analogues have been investigated as TRPV1 receptor antagonists. The 2-halogen analogues showed enhanced antagonism compared to the prototype antagonist.     

Analysis of structure-activity relationships for the 'B-region' of N-(4-t-butylbenzyl)-N'-[4-(methylsulfonylamino)benzyl]-thiourea analogues as TRPV1 antagonists

The structure-activity relationships for the 'B-region' of N-(4-t-butylbenzyl)-N'-[4-(methylsulfonylamino)benzyl]thiourea analogues have been investigated as TRPV1 receptor antagonists. A docking model of potent antagonist 2 with the sensor region of TRPV1 is proposed.     

Regulation of the xanthophyll cycle pool size in duckweed (Lemna minor) plants

Duckweed ( Lemna minor L.) plants grown under high light are characterized, when compared to low light acclimated plants, by a higher xanthophyll cycle (VAZ) pool content, but also by a higher proportion of photoconvertible violaxanthin and a superior ability to synthesize VAZ pigments. When duckweed plants were transferred to a high light environment a general response was the quick adjustment of the carotenoid composition, mainly xanthophyll cycle pigments. These changes resulted from a balance between a process of continuous light-independent carotenoid degradation and a light-induced accumulation. The use of norflurazon, an inhibitor of carotenogenesis, allowed us to demonstrate that the observed light induced increase of the VAZ pool was mainly caused by de novo synthesis through carotenogenesis. The extent of light-induced carotenogenesis was proportional to the light treatment and also to the operation of the VAZ cycle since it was partly abolished by treatments leading to a low activity of the VAZ cycle, such as low light, DTT or DCMU. These results suggest that not only the light itself, but also a mechanism triggered by a factor associated with the de-epoxidation state of the VAZ cycle controls carotenogenesis at some point before phytoene formation in the terpenoid biosynthesis pathway.     

The crystal and molecular structures of dichloro [N,O-(D,L)-diaminopropionic acid] platinum(II) and Dichloro [N,O-(L)-lysine] platinum(II) monohydrate

K₂PtCl₄ reacts with L-lysine and with D,L-diaminiopropionic acid (Dap) forming the neutral complexes [PtCl₂(N,O-Lys)]·H₂0 (1) and [PtCl₂(N,O-Dap)], (2) respectively.Compound 1 is monoclinic, space group P2₁ with a = 11.262(3), b = 11.041(2), c = 9.690(2) Å, β = 102.07(5)°, V = 1178(1) ų and Z = 4. Compound 2 is monoclinic, space group P2₁/n with a = 8.777(1), b = 10.615(2), c = 7.947(1) Å, β = 94.98(3)°, V = 738(1) ų and Z = 4. In both compounds, the zwitterionic ligands form an N,O-five membered chelate with the platinum atom. Structures 1 and 2 were refined to R values of 3.3% and 6.3% respectively.     

Efficiency of Nitrogen Assimilation by N(2)-Fixing and Nitrate-Grown Soybean Plants (Glycine max [L.] Merr.)

Nodulated and non-nodulated (not inoculated) soybeans (Glycine max [L.] Merr. cv Wells) were grown in controlled environments with N₂ or nonlimiting levels of NO₃⁻, respectively, serving as sole source of nitrogen. The efficiency of the N₂-fixing plants was compared with that of the nitrate-supplied plants on the basis of both plant age and plant size. Efficiency evaluations of the plants were expressed as the ratio of moles of carbon respired by the whole plant to the moles of nitrogen incorporated into plant material.

Continuous 24-hour CO₂ exchange measurements on shoot and root systems made at the beginning of flowering (28 days after planting) indicated that N₂-fixing plants respired 8.28 moles of carbon per mole of N, fixed from dinitrogen, while nitrate-supplied plants respired only 4.99 moles of carbon per mole of nitrate reduced. Twenty-one-day-old nitrate-supplied plants were even more efficient, respiring only 3.18 moles of carbon per mole of nitrate reduced. The decreased efficiency of the N₂-fixing plants was not due to plant size since, on a dry weight basis, the 28-day-old N₂-fixing plants were intermediate between the 28- and 21-day-old nitrate-supplied plants.

The calculated efficiencies were predominantly a reflection of root-system respiration. N₂-fixing plants lost 25% of their daily net photosynthetic input of carbon through root-system respiration, compared with 16% for 28-day-old nitrate-supplied plants and 12% for 21-day-old nitrate-supplied plants. Shoot dark respiration was similar for all three plant groups, varying between 7.9% and 9.0% of the apparent photosynthate.

The increased respiratory loss by the roots of the N₂-fixing plants was not compensated for by increased net photosynthetic effectiveness. Canopy photosynthesis expressed on a leaf area basis was similar for 28-day-old N₂-fixing plants (15.5 milligrams CO₂ square decimeter per hour) and 21-day-old nitrate-supplied plants (14.5 milligrams CO₂ square decimeter per hour). Both were similar in total canopy leaf area. The larger nitrate-supplied plants (28-day-old) had lower photosynthetic rates (12.5 milligrams CO₂ square decimeter per hour), presumably due to self-shading of the leaves.

These data indicate that, during the early stages of plant development, dependence solely on N₂-fixation is an expensive process compared to nitrate reduction in nitrate-supplied plants, since the N₂-fixing plants retained 8% to 12% less of their photosynthate as dry matter.