Tritium labelling of amino sugars at C-2 by alkaline epimerization in tritiated water

N-Acetyl-D-[2-³H]glucosamine was synthesized from N-acetyl-D-mannosamineby alkaline 2-epimerization in pyridine containing ³H₂O andnickelous acetate. The reaction involves reversible formationof an enol intermediate and therefore also resulted in incorporationof tritium into N-acetylmannosamine. After completed reaction,the two N-acetylhexosamines were separated from other radioactiveproducts and Morgan-Elson chromogens by chromatography on acolumn of Sephadex G-10, which was eluted with 10% ethanol,and were then separated from each other by chromatography onSephadex G-15 in 0·27 M sodium borate (pH 7·8).The location of the incorporated tritium was established bytreatment of the N-acetylhexosamines with borate under the conditionsof the Morgan-Elson reaction, which converts the sugars to Kuhn'schromogen I with concomitant loss of the C-2 hydrogen. As expected,this treatment resulted in the formation of ³H₂O, indicatingthat the tritium was located at C-2. [2-³H]Glucosamine was preparedby acid hydrolysis of the labelled N-acetylglucosamine and wasconverted to [2-³H]glucosamine 6-phosphate by incubation withhexokinase and ATP. The sugar phosphate was used as a substratefor glucosamine 6-phosphate deaminase (isomerase, EC 5.3.1.10 [EC] )in a simple ³H₂O release assay. N-acetyl[2-³H]glucosamine N-acetyl[2-³H]mannosamine [2-³H]glucosamine glucosamine 6-phosphate deaminase [2-³H]mannosamine     

The Effect of Wind and a Reduced Supply of Water on the Growth and Water Relations of Festuca arundinacea Schreb

Festuca arundinacea was grown at high and low wind-speeds attwo levels of soil water. Transpiration was increased at highwind-speed and accompanied by leaf water stress. Growth of leafarea was progressively reduced according to the severity ofthe experimental treatments in the sequence: wet soil and lowwind; dry soil and low wind; wet soil and high wind; dry soiland high wind. The leaf water potential was also reduced inthis sequence. Festuca arundinacea Schreb., transpiration, water stress, wind, water potential     

Transport-limited sucrose utilization and neokestose production by Phaffia rhodozyma

Summary Growth of an astaxanthin hyper-producing strain of Phaffia rhodozyma on sucrose is accompanied by the accumulation of glucose and fructose in the medium due to the limited capacity of the corresponding monosaccharide transport system or systems. This is accompanied by the production of the trisaccharide neokestose by transglycosylation reactions.     

Production of Cellulolytic Enzymes by Botryodiplodia theobromae, Pat.

Carboxymethylcellulase (CM-cellulase) and ß-glucosidaseactivities were induced in cultures of Botryodiplodia theobromae,Pat. Both growth of the fungus and CM-cellulase production werebetter with sodium nitrate as nitrogen source than with eitherammonium nitrate or ammonium chloride. Growth, per unit nitrogensupplied, was greater with glutamic acid and aspartic acid asnitrogen sources than with sodium nitrate; this was probablybecause the fungus utilized the carbon of these amino-acids.Most ß-glucosidase activity was formed with ammoniumchloride or ammonium nitrate as nitrogen source. Glucose inhibited the activity of ß-glucosidase markedly.Low glucose concentration (c. 0.003 M) stimulated CM-cellulaseactivity but concentrations above 0.05 M inhibited. The formationof both enzymic activities was repressed in the presence ofglucose. ß-glucosidase activity was more thermolabilethan that of CM-cellulase.     

Role of Cell-wall-degrading Enzymes in the Development of Leaf Spots Caused by Ascochyta pisi and Mycosphaerella pinodes

Extracts of limited and spreading lesions caused by Mycosphaerellapinodes on detached pea leaflets contained proteolytic, cellulolytic,and pectolytic enzymes although only in spreading lesions wasthere much degradation of cell walls. The brown tissue fromlimited M. pinodes lesions was resistant to maceration by enzymesfrom spreading lesions. Limited lesions contained water-soluble,95 per cent ethanol insoluble, partially dialysable, inhibitorsof pectin transeliminase which is probably the macerating enzyme. Green, spreading M. pinodes lesions developed only on leafletsfloating on water. Growth of these lesions was accompanied bycontinous loss of phenolic substances to the water while thephenol content in infected tissue remained similar to that inuninoculated controls. In contrast, the phenol content in mature,limited M. pinodes lesions on leaflets suspended just abovethe water level was about four times that in healthy tissue.It is suggested that loss of phenolics from floating leafletsprevents tissue browning and the development of resistance ofthe cell walls to maceration. But this type of resistance doesnot appear to be a major factor in the limitation of lesionson suspended tissue. Extracts of limited Ascochyta pisi lesions on leaflets floatingon water contained pectolytic and hemicellulolytic enzymes.Some cellulase (Cx) activity was detected although there waslittle evidence of cellulose degradation in cell walls in infectedtissue. The nature of the macerating factor remains uncertainbut it was found that extracts from lesions contained inhibitorsof pectic enzymes and that tissue just beyond that colonizedby the fungus was resistant to maceration; this resistance isprobably important in restricting the growth of the pathogenin the leaf.     

The influence of cell size on the growth rate of Thalassiosira weissflogii

Growth rate and average cell volume were measured throughoutauxospore formation in two populations of Thalassiosira weissflogii(Hustedt). In both cases, the entire population shifted fromrelatively small (800 µm³) to large cells (2800 µm³)over a 5 day interval. This shift was accompanied by a dramaticincrease in the average growth rate of the populations from1.6 to 3.4 doublings/day.     

Possible Roles of Methyl Glucoside andMyo-inositol in the Opening of Cut Rose Flowers

Methyl glucoside andmyo-inositol are present in all organs ofrose (Rosa hybridaL.). To investigate the possible role of thesecarbohydrates in the opening of cut roses, flowers with a 10,20 or 40-cm-long stem and a single flower bud (about 1.5 cmin diameter) were placed in water and flower opening and changesin sugar content in flowers and stems examined for 7 d. Thelonger the stem of the cut flower, the larger was the flowerdiameter. In stems, the concentration of carbohydrates, includingmethyl glucoside andmyo-inositol markedly decreased before floweropening. In petals, contents of glucose, methyl glucoside andmyo-inositolalso decreased before flower opening, but those of fructose,sucrose and xylose did not. When glucose and methyl glucosidewere added to the vase water (4%) flower opening was clearlypromoted; this was accompanied by an increase in methyl glucosideand fructose concentrations in petals. On the contrary,myo-inositolinhibited flower opening, and this was accompanied by an increaseinmyo-inositol and xylose concentrations in petals. These resultssuggest that methyl glucoside and/or its metabolites are transportedinto the petal cells, thereby lowering the osmotic water potentialand promoting flower opening.Myo-inositol is not readily metabolized,and exogenousmyo-inositol given at a high concentration mayact as an extracellular osmolyte, which inhibits water uptakeand flower opening.Copyright 1999 Annals of Botany Company Cut flowers, methyl glucoside,myo-inositol,Rosa hybrida,soluble carbohydrate.     

Uptake and Metabolism of Sugars by Suspension Cultured Catharanthus roseus Cells

The uptake and metabolism of sugars by suspension-cultured Catharanthusroseus cells were investigated. Substantially all the sucrosein the culture medium was hydrolyzed to glucose and fructosebefore being taken up by the cells. The activity of invertasebound to cell walls, determined in situ, was high at the earlystage of culture. Glucose was more easily taken up by the cellsthan was fructose. Tracer experiments using [U-¹⁴C]glucose and[U-¹⁴C]fructose indicated that glucose is a better precursorfor respiration than fructose, while fructose is preferentiallyutilized for the synthesis of sucrose, especially in the earlyphase of cell growth. Possible metabolic routes of sugar insuspension-cultured Catharanthus roseus cells are discussedin the context of these results. Catharanthus roseus, Madagascar periwinkle, suspension culture, sucrose, glucose, fructose, metabolism, glycolysis     

Some Properties of the Arginine Decarboxylase in Vicia faba Leaves

Growth of Vicia faba seedlings is accompanied by a rapid increasein arginine decarboxylase (EC 4.1.1.19 [EC] ) in the leaves and epicotyl.Increased enzyme activity was observed under saline conditionsin the presence of NaCl and with osmotic stress by mannitol.The partially purified enzyme (about 86-fold) readily decarboxylatedL-arginine, while D-arginine, L-homoarginine, L-ornithine andL-lysine were decarboxylated very slowly, and L-citrulline andL-glutamic acid were not decarboxylated. The K_m value was 5.8?10^–4M for L-arginine. The optimal pH and temperature for activitywere 8.5 and 45?C, respectively. p-Chloromercuribenzoate andN-ethylmaleimide were effective inhibitors of the enzyme. Inhibitionby spermidine, putrescine and agmatine suggested a possiblefeed-back mechanism in the pathway of polyamine biosynthesis. (Received October 11, 1983; Accepted February 24, 1984)     

Studies on the Growth in Culture of Plant Cells: XXIV. EFFECTS OF 2, 4-DICHLOROPHENOXYACETIC ACID AND LIGHT ON THE GROWTH AND METABOLISM OF ACER PSEUDOPLATANUS L. SUSPENSION CULTURES

Omission of 2,4-dichlorophenoxyacetic acid (2,4-D) from batchcultures of sycamore (AcerpseudoplatanusL.) caused growth cessationafter an initial period of exponential growth. The presenceof white light reduced the amount of growth after 2,4-D withdrawal.Growth cessation, in the absence of 2,4-D, was accompanied bythe accumulation of the free amino acid, serine. This accumulationbegan before the cessation of growth and was rapidly reversedby the re-addition of 2,4-D.     

Carbon Source and Micronutrient Requirements of the Aquatic Phycomycete, Catenaria anguillulae Sorokin

The requirements of the aquatic Phycomycete, Catenaria anguillulaewere analysed in liquid, shake cultures using a standardizedzoospore inoculum. Growth was determined by measuring mycelialdry weight and rate of production of titratable acid. D-glucose was the best carbon source and had an optimum concentrationof 166 mM of carbon for the medium used. When other carbon sourceswere supplied, only those related to glucose (fructose and mannose)or composed of glucose units with an alpha-linkage (maltose,glycogen, and starch) were readily utilized. Lactic acid wasdetermined qualitatively as an end-product of carbon metabolism. The optimum level of phosphate was 1.0 mM. The optimum concentrationof EDTA was 0.032 mM. Of the chelated cations included in themedium only the omission of iron, zinc, calcium, or magnesiumreduced growth. Concentrations of calcium below 0.4 mM and ofmagnesium below 0.2 mM were limiting; whereas, concentrationsof both ions up to 1 mM were non-toxic.     

The effect of mannosamine on the formation of lipid-linked oligosaccharides and glycoproteins in canine kidney cells

Madin-Darby canine kidney (MDCK) cells normally form lipid-linked oligosaccharides having mostly the Glc3Man9GlcNAc2 oligosaccharide. However, when MDCK cells are incubated in 1 to 10 mM mannosamine and labeled with [2-3H]mannose, the major oligosaccharides associated with the dolichol were Man5GlcNAc2 and Man6GlcNAc2 structures. Since both of these oligosaccharides were susceptible to digestion by endo-beta-N-acetylglucosaminidase H, the Man5GlcNAc2 must be different in structure than the Man5GlcNAc2 usually found as a biosynthetic intermediate in the lipid-linked oligosaccharides. Methylation analysis also indicated that this Man5GlcNAc2 contained 1----3 linked mannose residues. Since pulse chase studies indicated that the lesion was in biosynthesis, it appears that mannosamine inhibits the in vivo formation of lipid-linked oligosaccharides perhaps by inhibiting the alpha-1,2-mannosyl transferases. Although the lipid-linked oligosaccharides produced in the presence of mannosamine were smaller in size than those of control cells and did not contain glucose, the oligosaccharides were still transferred in vivo to protein. Furthermore, the oligosaccharide portions of the glycoproteins were still processed as shown by the fact that the glycopeptides were of the complex and hybrid types and were labeled with [3H]mannose or [3H]galactose. In contrast, control cells produced complex and high-mannose structures but no hybrid oligosaccharides were detected. The inhibition by mannosamine could be overcome by adding high concentrations of glucose to the medium.     

Photoinhibition of Glucose Uptake in Chlorella

In colorless mutant cells of Chlorella vulgaris (M125), endogenousrespiration in the dark was not affected by 30-min preilluminationwith white light (9,000 mW?m^–2), while exogenous respirationof glucose or fructose was inhibited significantly by the sametreatment in air, but not under N₂. This light effect on exogenousrespiration was accompanied by an inhibition of hexose uptake. When autotrophically grown wild-type cells of Chlorella vulgaris(211-11h) were incubated in glucose medium with DCMU, lightalso greatly inhibited glucose uptake and growth. Blue lightwas very effective, while red light had only a slight effect.This photoinhibitory effect was also observed in algal cellsthat had been grown in a glucose-containing medium in the dark. Using SDS-gel electrophoresis, a new protein peak with a molecularweight of 35–40 kDa was detected in plasma membrane-richcell wall fractions when Chlorella vulgaris (211-11h) cellswere transferred to a glucose-containing medium. This peak disappearedafter the algal cells were returned to the glucose-free medium.These findings suggest that this protein includes the hexose-carrierprotein. Blue light significantly inhibited the formation ofthis protein during incubation in a glucose-containing medium. ¹ Present address: Laboratory of Chemistry, Faculty of PharmaceuticalSciences, Teikyo University, Sagamiko, Kanagawa 199-01, Japan. (Received July 31, 1986; Accepted March 12, 1987)     

Effects of Desiccation on Phosphorus and Potassium Acquisition by a Desiccation-tolerant Moss and Lichen

The hypothesis that desiccation-tolerant mosses and lichensmay be more responsive to nutrient inputs accompanying intermittentdesiccation than mesophytic forest species was investigatedemploying species from semi-arid grassland in Hungary. Shootapices of the moss Syntrichia ruralis and marginal lobes ofthe lichen Cladonia convoluta were maintained for 7 weeks undercontrolled conditions. They were cultivated with or withouta weekly application of the major inorganic macronutrients,and either under constant hydration or with one or two 24 hperiods of desiccation each week. Growth of S. ruralis was stimulatedby nutrient additions, but lower weight increments were achievedwith increasing frequency of desiccation. All samples of thelichen showed positive growth, and no significant treatmenteffects were detected. A large net uptake of P occurred in nutrient-treatedmaterial of both species that was unaffected by the impositionof desiccation treatments. A smaller net uptake of K into theintracellular fraction was also observed when nutrients wereapplied, but in the moss this was against a baseline of decreasingK content. In contrast, more of the original K content was retainedin C. convoluta. In neither species was any clear evidence foundfor inhibition of nutrient uptake by the desiccation episodes.It is suggested that the lack of growth response in the lichenarises from an inability to bring together the additional nutrients,presumably mainly absorbed by the mycobiont, with photosynthateproduced by the photobiont. Copyright 2000 Annals of BotanyCompany Cladonia convoluta, Syntrichia ruralis, desiccation tolerance, mineral nutrition, phosphorus, potassium     

Studies on the Physiology of Nodule Formation: IX. The Influence of Combined Nitrogen, Glucose, Light Intensity and Day Length on Root-hair Infection in Clover

Small amounts of nitrate or nitrite salts (10 µg N/plant)in the root medium of Trifolium glomeratum or T. repens delayednodulation, prolonged the initial rapid phase of root infectionand slightly stimulated lateral root formation, whereas equivalentquantities of ammonium sulphate or urea did not. Growth of rootsand root hairs was unaffected by any of these substances at10 µg N/plant. Altering the carbohydrate status of the clover seedlings byadding glucose to the root medium, or by changing day lengthor light intensity, influenced neither the stimulation of root-hairinfection nor the delay in nodulation induced by nitrate at10 fig N/plant, except that plants grown in total darkness hadfewer hairs infected when the root medium contained small amountsof nitrate. The nitrogenous compounds at 100 µg to 1,000 µg N/plant generally delayed and decreased nodulation,increased lateral root formation, slowed hair infection, andincreased root growth.     

Studies on the Nutrition of Coprinus lagopus Fr., especially as affecting Fruiting

Growth and fruiting of Coprinus lagopus in pure culture havebeen studied on a medium containing glucose, dl--alanine, mineralsalts and thiamin, and the effects of changes in concentrationof certain components of this medium and of various substitutionsinvestigated. It is demonstrated that fruiting led to withdrawalof materials from the whole of the colony. No evidence was obtainedof the existence of a minimal quantity of substrate for fruitingto take place. The distribution of mature sporophores on a colonywas affected by an internal mechanism. A theory as to its possiblenature is suggested.     

Hypoxia induces apoptosis via two independent pathways in Jurkat cells: differential regulation by glucose

Glucose uptakeand metabolism inhibit hypoxia-induced apoptosis in a varietyof cell types, but the underlying molecular mechanisms remain poorlyunderstood. In the present study, we explore hypoxia-mediated celldeath pathways in Jurkat cells in the presence and absence ofextracellular glucose. In the absence of extracellular glucose, hypoxiacaused cytochrome c release, caspase 3 andpoly(ADP-ribose)polymerase cleavage, and DNA fragmentation; thisapoptotic response was blocked by the caspase 9 inhibitorz-LEHD-FMK. The presence of extracellular glucose during hypoxiaprevented cytochrome c release and activation of caspase 9 but did not prevent apoptosis in Jurkat cells. In theseconditions, overexpression of the caspase 8 inhibitor v-FLIP preventedhypoxia-mediated cell death. Thus hypoxia can stimulate twoapoptotic pathways in Jurkat cells, one dependent on cytochrome c release from mitochondria that is prevented by glucoseuptake and metabolism, and the other independent of cytochromec release and resulting from activation of the deathreceptor pathway, which is accelerated by glucose uptake and metabolism.

     

Utilization of Carbohydrates by Rhynchosporium secalis. I. Growth and Sporulation on Glucose, Galactose, and Galacturonic acid

The fungus Rhynchosporium secalis (Oud.) Davis grew and speculated in liquid nutrient media that contained glucose, galactose or galacturonic acid, or a pair of those substances, as the sole carbon source. Sporulation was inhibited by high concentrations of glucose and galacturonic acid. Growth and sporulation were greatest on glucose, and least on galactose. Growth was increased when glucose and galacturonic acid were mixed. Nitrogen concentration affected sporulation but not growth.     

Degradation of Extracellular beta-(1,3)(1,6)-d-Glucan by Botrytis cinerea

During growth on glucose, Botrytis cinerea produced extracellular beta-(1,3)(1,6)-d-glucan (cinerean), which formed an adhering capsule and slime. After glucose was exhausted from the medium, cinereanase activity increased from <0.4 to 30 U/liter, effecting a striking loss in the viscosity of the culture. Cinerean was cleaved into glucose and gentiobiose. Gentiobiose was then hydrolyzed to glucose. While cinereanase activity was strongest in the culture supernatant, gentiobiase activity was located mainly in the cell wall fraction. The addition of extra glucose or cycloheximide prevented the cinerean degradation caused by an effect on cinereanase formation. Cinerean degradation was accompanied by microconidiation and sclerotium formation. B. cinerea was found to grow on cinerean with the latter as its single carbon and energy source. In this case, cinerean degradation occurred during hyphal growth, and no microconidiation or sclerotium formation was observed. Growth experiments with various carbon sources indicated that cinerean had a positive effect on the formation of cinerean-degrading enzymes.     

Inhibition of glycosyl-phosphatidylinositol biosynthesis in Plasmodium falciparum by C-2 substituted mannose analogues.

Mannose analogues (2-deoxy-D-glucose, 2-deoxy-2-fluoro-D-glucose and 2-amino-2-deoxy-D-mannose) have been used to study glycosylphosphatidylinositol (GPtdIns) biosynthesis and GPtdIns protein anchoring in protozoal and mammalian systems. The effects of these analogues on GPtdIns biosynthesis and GPtdIns-protein anchoring of the human malaria parasite Plasmodium falciparum were evaluated in this study. At lower concentrations of 2-deoxy-D-glucose and 2-deoxy-2-fluoro-D glucose (0.2 and 0.1 mm, respectively), GPtdIns biosynthesis is inhibited without significant effects on total protein biosynthesis. At higher concentrations of 2-deoxy-D-glucose and 2-deoxy-2-fluoro-D-glucose (1.5 and 0.8 mm, respectively), the incorporation of [3H]glucosamine into glycolipids was inhibited by 90%, and the attachment of GPtdIns anchor to merozoite surface protein-1 (MSP-1) was prevented. However, at these concentrations, both sugar analogues inhibit MSP-1 synthesis and total protein biosynthesis. In contrast to 2-deoxy-2-fluoro-D-glucose and 2-amino-2-deoxy-D-mannose (mannosamine), the formation of new glycolipids was observed only in the presence of tritiated or nonradiolabelled 2-deoxy-D-glucose. Mannosamine inhibits GPtdIns biosynthesis at a concentration of 5 mm, but neither an accumulation of aberrant intermediates nor significant inhibition of total protein biosynthesis was observed in the presence of this analogue. Furthermore, the [3H]mannosamine-labelled glycolipid spectrum resembled the one described for [3H]glucosamine labelling. Total hydrolysis of mannosamine labelled glycolipids showed that half of the tritiated mannosamine incorporated into glycolipids was converted to glucosamine. This high rate of conversion led us to suggest that no actual inhibition from GPtdIns biosynthesis is achieved with the treatment with mannosamine, which is different to what has been observed for mammalian cells and other parasitic protozoa.