Importance of proline and other amino acids during honeybee flight

Summary. The levels of proline and other amino acids in the haemolymph and other body parts of honeybee foragers were investigated by HPLC analysis. The concentrations of proline in the blood of glucose-fed or -injected bees finishing their exhaustive tethered flights on a roundabout were significantly reduced compared to bees that were fed and rested for one hour. This indicates some utilization of proline during flight metabolism. The levels of essential amino acids and of the sum of all amino acids except proline remained roughly constant, indicating that the decrease of proline did not result from a changed haemolymph volume. ¹⁴C-labelled proline was injected into bees either shortly before starting their flight or before a resting period of equal duration in an incubator at the same temperature. Bees that rested had incorporated more proline into thorax body protein, and less of the labelled substance was unrecovered ("missing") and considered to be respired or less probably defecated. If the entire amount of missing ¹⁴C-proline is regarded as exhaled, the oxidative breakdown of proline reached higher levels after flight than in rested bees. This is another hint that proline is utilized during flight. Usually the exhaled amount did not exceed 10 μg proline in half an hour of flight. Although our data indicate involvement of proline in flight metabolism, the amount metabolized is low compared to the utilization of carbohydrates. Received December 5, 1998, Accepted February 1, 1999     

Effect of the tetrapeptide RGDS on somatic embryogenesis in Daucus carota

The present work examines the requirement for RGD-binding sites, such as those in the plasma membrane protein integrin during somatic embryogenesis in carrot ( Daucus carota L. cv. Scarlet nantes). It is possible to assay for this requirement by competitively inhibiting binding of the site to the natural RGD-containing ligand by adding an excess of synthetic RGDS. We found that treatment of carrot callus cultures with RGDS (2.7 m M ) inhibited the development of a normal shoot pole in carrot somatic embryos. The structures that formed contained separate zones of highly cytoplasmic and vacuolate cells and no evidence of embryonic organization, although occasionally a root-like structure was observed. If the aspartic acid residue in the peptide was replaced by a chemically similar amino acid (glutamic acid), the resultant somatic embryos were indistinguishable from those developing in untreated cultures. These effects are similar to those reported in animal systems where the protein receptor involved has been identified as integrin. Our results are thus consistent with the conclusion that a binding site for a motif similar to the integrin-binding site in a variety of animal extracellular matrix proteins exists in plants and appears to be important in somatic embryo development in carrot.     

Morphometric measurements of Daucus carota suspension culture cells

Standard stereologic methods have been applied to electron micrographs of Daucus carota L. suspension culture cells. The relative frequencies of the different membrane systems within the cells have been determined and compared with published data form mature leaf cells.     

Pinocytosis and intracellular degradation of exogenous protein: modulation by amino acids

Intracellular degradation of exogenous (serum) proteins provides a source of amino acids for cellular protein synthesis. Pinocytosis serves as the mechanism for delivering exogenous protein to the lysosomes, the major site of intracellular degradation of exogenous protein. To determine whether the availability of extracellular free amino acids altered pinocytic function, we incubated monolayers of pulmonary alveolar macrophages with the fluid-phase marker, [14C]sucrose, and we dissected the pinocytic process by kinetic analysis. Additionally, intracellular degradation of endogenous and exogenous protein was monitored by measuring phenylalanine released from the cell monolayers in the presence of cycloheximide. Results revealed that in response to a subphysiological level of essential amino acids or to amino acid deprivation, (a) the rate of fluid-phase pinocytosis increased in such a manner as to preferentially increase both delivery to and size of an intracellular compartment believed to be the lysosomes, (b) the degradation of exogenously supplied albumin increased, and (c) the fraction of phenylalanine derived from degradation of exogenous albumin and reutilized for de novo protein synthesis increased. Thus, modulation of the pinosome-lysosome pathway may represent a homeostatic mechanism sensitive to the availability of extracellular free amino acids.     

Effect of brassinosteroid on cell division and enlargement in cultured carrot (Daucus carota L.) cells

When added at 3.10^–6 mM to auxin-starved suspension cultured carrot (Daucus carota L.) cells, the steroid hormone 24-epibrassinolide (BR) induces cell enlargement but not cell division.This is at variance from the effect of 2,4-D which, in the same experimental conditions, restores cell division without having any effect on cell enlargement.Furthermore, in the tested experimental conditions, the effect of BR is dominant over the effect of 2,4-D when the two hormones are simultaneously supplied to the auxin-starved culture.Abbreviations BR brassinosteroid, 24-epibrassinolide - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - GA₃ gibberellic acid     

Influence of some exogenous amino acids on the production of maize embryogenic callus and on endogenous amino acid content

The effects of four exogenous amino acids (proline, glycine, asparagine and serine) on the production of maize embryogenic callus and on its endogenous amino acid content have been investigated. For this purpose, an established embryogenic line of Type 1 callus from the inbred W64Ao2 has been used. From the results it may be concluded that a concentration of proline exceeding 6 mM is negative for the production of embryogenic callus. When proline is eliminated from the medium, other amino acids tested in certain concentrations yield a percentage of embryogenic callus production that exceeds or equals that of proline. The endogenous free proline content in embryogenic callus is significantly higher than that in non-embryogenic callus regardless of proline presence in the medium. The only exception are the glycine-containing media, in which endogenous free alanine of embryogenic callus increases at the expense of endogenous free proline. This study suggest a positive role of endogenous free proline or alanine accumulation in the embryogenic callus production which might be related to an adaptation to the metabolic changes produced by in vitro culture and embryogenesis induction. Furthermore, these results indicate that treatments with amino acids that are different from proline can be used to improve the efficiency of embryogenic callus production from well established maize callus cultures.Abbreviations Ala alanine - Asn asparagine - 2,4-d 2,4-dichlorophenoxyacetic acid - EC embryogenic callus - nEC non-embryogenic callus - Gaba gamma-aminobutyric acid - Glu glutamic acid - Gly glycine - Pro proline - Ser serine     

Effect of levorin on the intracellular pool of free amino acids in Ehrlich ascites carcinoma cells

Levorene, a polyenic antibiotic, lowered the concentration of amino acids in the cells of Ehrlich carcinoma. The decrease in the intracellular level of the amino acids was due not only to inhibition of their entrance to the cells but also to their increased leaching from the cells. The effect of levorene on the intracellular level of the neutral amino acids was higher than that on the main amino acids which was associated with different sensitivity of the amino acid transport systems to the antibiotic.     

Studies on the origin of totipotent cells in explants of Daucus carota L.

In an attempt to identify the origin of cells capable of generatingsomatic embryos, hypocotyl explants from carrot seedlings werecultured in the presence of auxin. The various tissues respondedin different ways. The external layers (epidermis, corticalparenchyma) expanded, whereas the provascular cells dividedand expanded. Hence only the latter cells can generate celllines and somatic embryos. A cyto-histological analysis showedthat pro-embryogenic masses, from which embryos develop in theabsence of auxin, are generated from the same provascular cellswhich, after a proper exposure to auxin, undergo an asymmetricdivision. Key words: Somatic embryogenesis, pro-embryogenic masses, Daucus carota, totipotency     

Purification and properties of DNA topoisomerase II from Daucus carota cells

Topoisomerase II was partially purified from Daucus carota cellsby a procedure including ammonium sulphate fractionation, ion-exchange,and affinity chromatography steps. The type II enzyme, identifiedfor its ability to unknot knotted P4 DNA and decatenate Trypanosomacruzi kDNA, requires ATP and Mg²⁺ for activity. The unknottingactivity was sensitive to an inhibitor of the mammalian typeII enzyme, the drug VP16 (IC₅₀ 32 mmol m^–3), whereas inhibitorsof DNA gyrase showed a limited effect on activity. The SDS-PAGEanalysis of the dsDNA cellulose fraction revealed the presenceof four polypeptides of apparent molecular masses of 72, 71,34, and 33 kDa among which only a polypeptide of about 70 kDacrossreacted with antibodies against yeast topoisomerase II.Immunoprecipitation experiments with monoclonal antibodies tothe and ß isoforms of the human enzyme confirmedthe recognition of a polypeptide of 70 kDa. The sedimentationcoefficient (S) of the topoisomerase II in the phosphocellulosefraction, calculated by analytical glycerol gradient, was 6.1corresponding to a molecular mass of about 123 kDa. Resultssuggest the presence in carrot of a protein of molecular massof 70 kDa having the typical properties of an eukaryotic topoisomeraseII and carrying epitopes recognized by MoAbs to both human and ß enzymes. The 70 kDa polypeptide might then representthe monomer of a homodimer enzyme of 123 kDa. Key words: Daucus carota, topoisomerase II, immunoprecipitation     

Effect of exogenous amino acids on Cu uptake and translocation in maize seedlings

This study aimed to assess the effects of four contrasting proteinogenic amino acids on copper (Cu) uptake and translocation in maize (Zea mays L.) seedlings grown in a modified Hoagland solution. Glycine, aspartic acid and lysine at three concentrations (10, 25 and 100 μM) did not have any significant effect on Cu uptake and translocation in maize seedlings over a two-day experimental period. However, cysteine (a reductive amino acid) at the three concentrations increased very significantly (P < 0.01) Cu accumulations in the root symplast and the shoots of maize seedlings in comparison to the control. Cu uptake in the whole plant and Cu translocation from root to shoot were also increased in the cysteine treatments. In the 25 μM cysteine treatment, where cysteine was in moderate excess, the Cu uptake in the whole plant and Cu translocation from root to shoot were significantly (P < 0.01) higher than those of the 10 or 100 μM cysteine treatments, where the concentration of cysteine was equivalent to that of Cu(II) or in great excess according to the stoichiometry of the redox reaction of cysteine with Cu(II). It is hypothesized that the cysteine-induced oxidation state alteration from Cu(II) to Cu(I) could be responsible for the increased Cu uptake and Cu translocation, on the ground that Cu(I), as free cuprous ion or cysteine cuprous complex, may be more available to maize roots than Cu(II).     

Stimulation of glycogen synthesis in hepatocytes by added amino acids is related to the total intracellular content of amino acids

Katz et al. [Katz, J., Golden, S. & Wals, P.A. (1976) Proc. Natl Acad. Sci. USA 73, 3433-3437] were the first to report that in hepatocytes isolated from fasted rats and incubated with either dihydroxyacetone, glucose or other sugars, glycogen synthesis was greatly accelerated by addition of amino acids. We have looked for possible mediators responsible for this effect and have tested the effect of alanine, proline, asparagine, glutamine or a combination of ammonia with either pyruvate or lactate in activating glycogen synthesis from dihydroxyacetone. The following observations were made. 1. Stimulation of glycogen synthesis by alanine, proline or asparagine does not require production of glutamine since the effect also occurs in periportal hepatocytes which lack glutamine synthetase. 2. Under various conditions, stimulation of glycogen synthesis by added amino acids directly correlated with increases in the intracellular content of amino acids, expressed in osmotic equivalents. 3. 3-Mercaptopicolinic acid, the inhibitor of phosphoenolpyruvate carboxykinase, further enhances stimulation of glycogen synthesis by amino acids because it increases the intracellular accumulation of aspartate and glutamate. 4. The previously reported enhancement by leucine of the stimulation of glycogen synthesis by glutamine [Chen. K. S. & Lardy, H. A. (1985) J. Biol. Chem. 260, 14683-14688] can be ascribed to inhibition of urea synthesis by leucine which results in accumulation of glutamate and of ammonia, the essential activator of glutaminase. It is concluded that activation of glycogen synthesis by added amino acids is due to an increase in intracellular osmolarity following their uptake and the accumulation of intracellular catabolites. This results in an increase in hepatic volume which stimulates glycogen synthesis [Baquet, A., Hue, L., Meijer, A. J., van Woerkom, G. M. & Plomp, P. J. A. M. (1990) J. Biol. Chem. 265, 955-959].     

Fluctuations in proline and other free amino acids during the mitotic cycle of the myxomycete Physarum polycephalum.

1. During synchronous growth of the acellular slime mould Physarum polycephalum the free amino pool had two maxima, one of 650 units [nmol/plasmodium dry weight (mg)] at metaphase and the other of 780 units in mid G2 with minima of 550 units before and after mitosis. 2. Proline formed 20--25% of the total pool with aspartic acid, glutamic acid, threonine, valine, leucine, lysine and arginine making up 55% of the pool. 3. The fluctuation of proline during the mitotic cycle was quite different from that of the other amino acids and was transiently very low during telophase.     

Changes in proline level in response to osmotic stress and exogenous amino acids in Raphanus sativus L. seedlings

The changes in endogenous proline levels of Raphanus sativus L. seedlings was monitored in presence of exogenous amino acids in normal and osmotically stressed seedlings. In unstressed seedlings, proline uptake was detected only at higher (1 mM) concentration of applied L-proline. however, proline uptake was promoted at all (1 μM to 1000 μM) concentrations of applied L-proline under osmotic stress conditions. Amongst other exogenous amino acids, L-leucine, L-glutamic acid, L-alanine, and L-histidine enhanced endogenous levels of proline, while exogenous hydorxyproline and γ-amino butyric acid reduced it.     

Mitochondrial DNA diversity and male sterility in natural populations of Daucus carota ssp carota

Mitochondrial variability was investigated in natural populations of wild carrot (Daucus carota ssp carota) in different regions: South of France, Greece, and various sites in the Mediterranean Basin and Asia. Total DNA was digested with two restriction endonucleases (EcoRV and HindIII) and probed with three mitochondrial DMA-specific genes (coxI, atp6, and coxII). Twenty-five different mitochondrial types were found in 80 analyzed individuals. Thirteen mitotypes were found among the 7 French populations studied. On average, 4.4 different mitotypes were observed per population, and these mitotypes were well-distributed among the populations. All of the mitochondrial types were specific to a single region. However, the proportion of shared restriction fragments between 2 mitotypes from different regions was not particularly lower than that which occurred among mitotypes from a single region. On the basis of the sexual phenotype [male-sterile (MS) or hermaphrodite] of the plants studied in situ and that of their progeny, 2 mitotypes were found to be highly associated with male sterility. Eighty percent of the plants bearing these mitotypes were MS in situ, and all of these plants produced more than 30% MS plants in their progeny. This association with male sterility was consistent in several populations, suggesting an association with a cytoplasmic male-sterility system. Moreover, these two mitotypes had very similar mitochondrial DNA restriction patterns and were well-differentiated from the other mitotypes observed in wild plants and also from those observed in the two CMS types already known in the cultivated carrot. This suggests that they correspond to a third cytoplasmic sterility.     

Effect of exogenous quercetin on the level of carbohydrates and amino acids in Lycopersicon esculentum fruit

Low doses of the exogenous flavonoid quercetin increased in contents of sugars in tomato fruits of various cultivars. The content of glucose in tomato fruits of cv. Ukrainskii teplichnyi increased from 3.62 to 11.24% per unit dry weight. Increases in the content of glucose were found in all tomato cultivars examined. An analysis of qualitative and quantitative compositions of amino acids showed that their levels were markedly decreased in fruits of quercetin-treated plants. Our studies and data found in the literature suggest that this effect is due to the synthesis of sugars from amino acids by gluconeogenesis. The reverse process of sugar hydrolysis does not occur because exogenous quercetin inhibits the activity of pyruvate kinase.     

Methotrexate and aminopterin effects on growth and regeneration in Daucus carota

Daucus carota L., callus was cultured on various levels of the folate analogs, methotrexate (4-amino-10-methylfolic acid, amethopterin) and aminopter in (4-aminofolic acid). Callus growth was inhibited as analog concentrations were increased from 0.01 M to 10 M. Methotrexate concentrations in excess of 10 M were lethal. In contrast, concentrations of aminopterin in the range of 10 to 100 M resulted in renewed growth and somatic embryogenesis leading to plant regeneration. This plant regeneration occurred even in the presence of 5.0 mg/l 2,4-D or NAA (concentrations up to fifty times higher than that required to maintain callus growth). These observations reveal that aminopterin at high concentrations, but not methotrexate, triggers somatic embryogenesis in the presence of auxin. All tested levels of aminopterin permitted regeneration in the absence of auxin.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthylacetic acid - DHFR EC 1.5.1.3, dihydrofolate reductase or 5,6,7,8,- tetrahydrofolate: NADP⁺ oxidoreductase - C1 single carbon - dTMP deoxyribothymidine-5 — monophosphate Technical Article No. 21590 from Texas Agricultural Experiment Station     

Glucose and Pyruvate Metabolism in Daucus carota Cells: The Effect of the Ammonium Ion

In Daucus carota cells cultivated in vitro, the ammonium ionstimulates the incorporation of radioactivity from labelledglucose and labelled pyruvate into CO₂ and into the residueinsoluble in 60 per cent (v/v) ethanol. There is a higher ¹⁴CO₂production from [6-¹⁴C₂] glucose than from [6-¹⁴C] glucose.These results suggest a possible stimulation of glycolysis bythe ammonium ion.