Activation of chitin synthetase from Phycomyces blakesleeanus by calcium and calmodulin

Levels of basal chitin synthetase in cell-free extracts from Phycomyces blakesleeanus were reduced by breakage of cells in the presence of EDTA or EGTA. Addition of Ca²⁺ to these extracts activated chitin synthetase. Maximal activation was obtained after 2 h at a Ca²⁺ concentration of 2–5 mM. Activation by calcium was not reduced by any protease inhibitor tested but benzamidine, whereas the weak proteolytic activity of the extracts was inhibited by antipain. Larger levels of chitin synthetase activation were obtained by the simultaneous addition of calcium and calmodulin in most, but not all extracts. This further activation by calmodulin was prevented by TFP. ATP or cAMP did not stimulate activation by calcium or calcium-calmodulin.Abbreviations EGTA ethylene glycol-bis(B-aminoethylether)-N,NN-tetraacetic acid - GlcNAc N-acetyl-d-glucosamine - PMSF phenylmethylsulfonyl fluoride - SBTI soybean trypsin inhibitor - TFP trifluoperazine - TLCK N-p-tosyl-l-lysine choromethyl ketone - UDPGlcNAc uridine diphosphate N-acetyl-d-glucosamine     

Chitosome-like vesicles from gamma particles of Blastocladiella emersonii synthesize chitin

When gamma particles isolated from the aquatic fungus, Blastocladiella emersonii, were incubated in a supernatant derived from a homogenate of zoospores previously triggered to encyst with 50 mM KCl, they exhibited a three-fold increase in chitin synthetase activity and produced chitosome-like vesicles. Passage of such vesicles through a linear sucrose gradient resulted in a symmetrical distribution of chitin synthetase activity over a broad portion of the gradient, and the specific activity of the peak fraction was seven times greater than that of the gamma particles. After isopycnic sucrose gradient centrifugation, chitin synthetase activity occurred in a band of particles with a peak buoyant density of 1.18 g/cm³. Ultrastructural examination of negatively stained samples from the peak fraction revealed spheroidal, chitosome-like particles 70–120 nm in diameter. Suspension of these particles produced chitin microfibrils when incubated with uridine diphosphate N-acetylglucosamine, the substrate for chitin synthetase.Non Standard Abbreviations Used GlcNAc N-acetylglucosamine - UDP-GlcNAc uridine diphosphate N-acetylglucosamine - PYG agar 1.25 g of peptone, 1.25 g of yeast extract, 3 g of glucose, and 20 of agar per 1000 ml of water, the pH being adjusted to 6.8 with KOH after autoclaving - EGTA ethyleneglycol-bis (-aminoethylether)-N,N-tetraacetic acid     

The recognition of three different epitopes for the H-type 2 human blood group determinant by lections ofUlex europaeus,Galactia tenuiflora andPsophocarpus tetragonolobus (Winged Bean)

The chemical mapping of the regions of H-type 2 human blood group-related trisaccharide (Fuc(1–2)Gal(1–4)GlcNAcMe) that are recognized by three different lectins, the so-called epitopes, are reviewed together with an account of how and why oligosaccharides form specific complexes with proteins as presently viewed in this laboratory. The occasion is used to report the synthesis of the various mono-O-methyl derivatives of the above trisaccharide that were used in these investigations. Also, Fuc(1–2)Gal(1–4)XylMe was synthesized in order to examine whether or not the hydroxymethyl group of the GlcNAc residue participates in the binding reaction.Abbreviations Me methy - Bn benzyl - Ac acetyl - Bz benzoyl - n-Bu n-butyl - NMR nuclear magnetic resonance - the GlcNAc Gal and Fuc residues of the H-type 2 trisaccharide are designated as the a, b and c structural units, respectively. This is paper XV in a series devoted to molecular recognition.     

Chitinase activity in germinating cells ofMucor rouxii

Chitinase activity in germinating cells (4 h cultures) ofMucor rouxii was studied. The enzyme activity was recovered in a high speed supernatant of cell homogenates. No activity was detected in the mixed membrane fraction or in the cell walls. Maximum activity was observed at pH 7.6 and at 30–35°C using the chromogenic assay with chitin azure. The latter was digested by GS-chitinase in a manner dependent on substrate concentration and time of incubation. As with other chitinases, GS-chitinase was much more effective against nascent than against preformed chitin. The main product of nascent chitin digestion was diacetylchitobiose, although significant amounts of the trimer were also detected in the hydrolyzates. Allosamidin, an insect and fungal chitinase inhibitor, strongly inhibited hydrolysis of nascent chitin but not of chitin azure by GS-chitinase. The drug failed to inhibit the germination and the ensuing growth of the fungus. Results are discussed in terms of the possible role of GS-chitinase in germination.Abbreviations UDP-GlcNAc uridine 5-diphospho-N-acetylglucosamine - GlcNAc N-acetylglucosamine - GlcNAcP N-acetylglucosamine phosphate - PMB phosphate-magnesium buffer - C₂ N, N-diacetylchitobiose - C₃ N, N, N-triacetylchitotriose - RVB remazol brilliant blue - DPC descending paper chromatography - MU(Ch)₃ 4-methylumbelliferyl-N - N N-triacetylchitotriose     

Synthesis of chitin by particulate preparations from Aspergillus flavus

Cell-free extracts from Aspergillus flavus catalyzed the synthesis of chitin from UDP-GlcNAc. Most of the activity was associated with membrane-rich fractions whereas no activity was detected in the cell walls. Chitin synthetase was activated by fungal acid proteases; animal and plant proteases destroyed it. Upon incubation at 0 C and 28 C chitin synthetase was inactivated, probably by the action of proteases present in the particulate preparations. Maximal activity was obtained at pH 6.6–7.1 and 15 C. Arrhenius plot showed a biphasic curve with the transition at 7 C. E values were 3300 Kcal/mole above this temperature and 15500 Kcal/mole below it. The enzyme was activated by GlcNAc and required a divalent metal, the most active being Mg⁺⁺. By plotting v vs UDP-GlcNAc concentration a sigmoidal curve was obtained. Km calculated at high substrate concentrations was 20mm. Chitin synthetase was competitively inhibited by polyoxin D (Ki 6.5 m) and UDP (Ki 1.35mm), the latter giving complex kinetics.This work was supported by grants No. 020 and 847 of the Consejo Nacional de Ciencia y Tecnología, México.Part of this work was carried out while the authors were research visitors at the Department of Plant Pathology, University of California, Riverside.     

Chitin synthetase mutants of Phycomyces blakesleeanus

Mutants resistant to nikkomycin, an inhibitor of chitin biosynthesis, were isolated after exposure of wild-type spores of the fungus Phycomyces blakesleeanus to N-methyl-N-nitro-N-nitrosoguanidine. Genetic analysis revealed that nikkomycin resistance was due to mutations in a single gene, chsA. Mutants and wild type grew equally well in the absence of nikkomycin. In contrast to the wild type, whose spore germination and mycelial growth were inhibited by 5 M nikkomycin, chsA mutants grew reasonably well in the presence of 50 M nikkomycin. Chitin synthesis in vivo was much less affected by the drug in the mutants than in the wild type. Resistance was not due to impaired uptake or detoxification of the drug. Analysis of the kinetics of chitin synthesis in vitro showed that the mutants had a decreased K_a for the allosteric activator, N-acetylglucosamine, and gross alterations in nikkomycin inhibition kinetics. These results indicate that chsA is the structural gene for chitin synthetase, or at least for the polypeptide that bears the catalytic and allosteric sites.     

The ultrastructure of cell wall formation and of gamma particles during encystment of Allomyces macrogynus zoospores

Structural changes during cell wall formation by populations of semisynchronously germinating zoospores were studied in the water mold Allomyces macrogynus. Fluorescence microscopy using Calcofluor white ST (which binds to -1,4-linked glycans) demonstrated that Calcofluor-specific material was deposited around most cells between 2–10 min after the induction of encystment (beginning when a wall-less zoospore retracts its flagellum and rounds up). During the first 15 min of encystment there was a progressive increase in fluorescence intensity. Ultrastructural analysis of encysting cells showed that within 2–10 min after the induction of encystment small vesicles 35–70 nm diameter were present near the spore surface, and some were in the process of fusing with the plasma membrane. The fusion of vesicles with the zoospore membrane was concomitant with the appearance of electron-opaque fibrillar material outside the plasma membrane. Vesicles similar to those near the spore surface were found within the gamma () particles of encysting cells. These particles had a crystalline inclusion within the electron-opaque matrix. During the period of initial cyst cell wall formation numerous vesicles appeared to arise at the crystal-matrix interface. Approximately 15–20 min was required for the cell wall to be formed. We suggest that the initial response of the zoospore to induction of encystment is the formation of a cell wall mediated by the fusion of cytoplasmic vesicles with the plasma membrane.Non-Standard Abbreviations GlcNac N-Acetylglucosamine - DS sterile dilute salts solution - PYG peptone-yeast extract-glucose broth     

Antibiotic tetaine — a selective inhibitor of chitin and mannoprotein biosynthesis in Candida albicans

The antibiotic tetaine inhibits in Candida albicans the biosynthesis of two important cell wall constituents, chitin and mannoprotein. This effect is a consequence of inactivation of the enzyme glucosamine-6-phosphate synthetase. Due to the lack of glucosamine-6-phosphate the effective secretion of mannoprotein enzymes, acid phosphatase and invertase, by Candida albicans spheroplasts is inhibited. In the presence of tetaine, probably a modified mannoprotein, lacking a branched polymannan, is synthesized. The antibiotic action decreases the viability of Candida albicans cells, especially that of mycelial forms of this fungus.Abbreviations GlcNAc N-acetyl-d-glucosamine - GlcN-6-P d-glucosamine-6-phosphate - ManNAc N-acetyl-d-mannosamine - -MM -methylmannoside - EGTA 1,2 di/2-aminoethoxy/ethane - N,N,N,N tetra-acetic acid     

Immunochemical characterization of antisera reactivities toN-acetyl-d-glucosamine oligosaccharides with theβ(1–4)-glycosidic linkage

The (1–4)-linked oligosaccharides ofN-acetyl-d-glucosamine (GlcNAc) isolated from chitin were used to prepare synthetic immunogens and antigens by reductive amination of (GicNAc)_n to bovine serum albumin (BSA). The rabbit antisera produced to the (GlcNAc)_n-BSA conjugates were characterized using an enzyme-linked immunosorbent assay (ELISA) system under conditions that, only the antibodies with carbohydrate specificity were reactive with the solid-phase adsorbed (GlcNAc)_n-BSA antigens. Inhibition assays using the (GlcNAc)_n-BSA, (GlcNAc)_n oligosaccharides, and the reduced oligosaccharides showed a relative specificity of the antisera for the chain length of the (GlcNAc)_n sequences. For example, the anti-(GlcNAc)₅-and anti-(GlcNAc)₄-sera were inhibited best by the longer chain (GlcNAc)_n ologosaccharides with the antibody combining sites directed mainly to the cyclic GlcNAc residues of the (GlcNAc)_n-BSA conjugates. The antibody combining sites were in part directed to the acyclic moiety of the reducing end of the oligosaccharides as shown by the increased inhibitory activities of the reduced (GlcNAc)_n oligosaccharides particularly, with the anti-(GlcNAc)₂-and anti-(GlcNAc)₃-sera. The best hapten inhibitors for the anti-(GlcNAc)₂-BSA and anti-(GlcNAc)₁-BSA sera were theN-butylamine derivatives of (GlcNAc)₂ and (GlcNAc)₁, respectively, indicating that the antibodies were also reactive with the secondary amine formed between the reducing end of the oligosaccharides and the -amino groups of lysine.Abbreviations ELISA enzyme-linked immunosorbent assay - BSA bovine serum albumin - GicNAc N-acetyl-d-glucosamine - (GlcNAc)_n Oligosaccharides containing GlcNAc in 1–4 linkages - (GlcNAc)₂ DGlcNAc(1–4)-d-GlcNAc - (GlcNAc)₃ (GlcNAc)₄ and (GlcNAc)₅, the homologous oligosaccharides of (GlcNAc)₂ - PBS phosphate buffered saline (0.01 M sodium phosphate, pH73 containing 0.15%M (NaCl) - PBSA PBS containing 1% BSA and 0.1% Tween-20 - ONPG o-nitrophenyl--d-galactopyranoside     

Maternal-embryonic relationships in the goodeid teleost,Xenoophorus captivus

Summary The endodermal trophotaenial epithelium in goodeid embryos acts as a placental exchange site. Fine structural and cytochemical data indicate that the trophotaenial absorptive cells are endocytotically highly active. To test their micropinocytotic capacity and characterize the cellular mechanisms involved in membrane, solute and ligand movements, living embryos of Xenoophorus captivus were incubated in saline media containing horseradish peroxidase (HRP) and/or cationized ferritin (CF) in vitro, and the uptake of these tracer proteins examined by both time sequence analysis and pulse-chase procedures. In some embryos, the effects of prolonged exposure to CF injected into the ovarian cavity, was also investigated.Labelling of the free cell surface was detectable with CF only, but interiorization of both probes was quick from all incubation media. Adsorptive pinocytosis of CF and fluid-phase uptake of HRP sequentially labelled pinocytic vesicles, endosomes, and lysosome-like bodies. In addition, CF-molecules were sequestered within apical tubules and small vesicles. HRP was largely excluded from both organelles and ended up in the lysosomal compartment. For CF, two alternative pathways were indicated by the pulse-chase experiments; transcellular passage and regurgitation of tracer molecules to the apical cell surface. The latter procedure involves membrane and receptor recycling, in which apical tubules are thought to mediate.In double-tracer experiments, using an 81 excess of HRP, external labelling with CF was light or lacking after 1–3 min, and the initial uptake-phase produced pinocytic vesicles and endosomes that mainly contained HRP-reaction product. Prolonged incubation, however, resulted in densely CF-labelled plasmalemmal invaginations and pinocytic vesicles that predominantly carried ferritin granules. After 60 min, the vacuoles of the endosomal compartment contained either high concentrations of HRP-reaction product, both tracers side by side, or virtually exclusively CF.     

Effect of light intensity on ammonia assimilation in maize leaves

The effect of light on the metabolism of ammonia was studied by subjecting detached maize leaves to 150 or 1350 mol m^–2 s^–1 PAR during incubation with the leaf base in 2 mM ¹⁵NH₄Cl. After up to 60 min, leaves were extracted. Ammonia, glutamine, glycine, serine, alanine, and aspartate were separated by isothermal distillation and ion exchange chromatography. ¹⁵N enrichments were analyzed by emission spectroscopy. The uptake of ammonium chloride did not influence CO₂ assimilation (8.3 and 17.4 mol m^–1 s^–1 at 150 and 1350 mol m^–2 s^–1 PAR, respectively). Leaves kept at high light intensity contained more serine and less alanine than leaves from low light treatments. Within 1 h of incubation the enrichment of ammonia extracted from leaves rose to approximately 20% ¹⁵N. In the high light regime the amino acids contained up to 15% ¹⁵N, whereas in low light ¹⁵N enrichments were small (up to 6%). The kinetics of ¹⁵N incorporation indicated that NH₃ was firstly assimilated into glutamine and then into glutamate. After 15 min ¹⁵N was also found in glycine, serine and alanine. At high light intensity nearly half of the ¹⁵N was incorporated in glycine. On the other hand, at low light intensity alanine was the predominant ¹⁵N sink. It is concluded that light influences ammonia assimilation at the glutamine synthetase reaction.     

Cycloheximide-induced inhibition of chitin synthesis, and decay of apical vesicles and chitosomes in Phycomyces blakesleeanus

Addition of cycloheximide rapidly inhibited protein synthesis in Phycomyces blakesleeanus. In contrast, chitin biosynthesis decreased with biphasic kinetics displaying a slow and a rapid decay phases. Electron microscopic studies revealed a decrease in the number of apical vesicles and chitosomes after cycloheximide addition; and no change in wall thickness. It is proposed that the slow phase of decay in chitin biosynthesis represents the exhaustion of the pool of chitosomes which transport the chitin synthase necessary to maintain apical wall growth; whereas the second one corresponds to inactivation of the enzyme, which is short lived in vivo. Data also rule out a change in the polarization of wall synthesis induced by cycloheximide, as suggested in other systems.     

Chitin synthetase activity is bound to chitosomes anf to the plasma membrane in protoplasts of Saccharomyces cerevisiae

The sub-cellular distribution of chitin synthetase was studied in homogenates of Saccharomyces cerevisiae protoplasts. Use of a mild disruption method minimized rupture of vacuoles and ensuing contamination of subcellular fractions by vacoular proteinases. After fractionation of whole or partially purified homogenates through an isopycnic sucrose gradient chitin synthetase activity was found to be distributed between two distinct particulate fractions with different buoyant density and particle diameter. When whole homogenates were used, about 52% of the chitin synthetase loaded was localized in a microvesicular population identified as chitosomes (diameter 40–110 nm; bouyant density (d) = 1.146 g/cm³). Another vesicular population containing 26% of the activity was identified as plasma membrane vesicles because of its large mean diameter (260 nm), its high buoyant density (d = 1.203 g/cm³) and by the presence of the vanadate-sensitive ATPase activity. Moreover, after surface labeling of protoplasts with ³H-concanavalin A, the label cosedimented with the presumed plasma membrane vesicles. There was a negligible cross-contamination of the chitosome fraction by yeast plasma membrane markers. In both the plasma membrane and the chitosome fractions, the chitin synthetase was stable and essentially zymogenic. Activation of the chitosome fraction produces microfibrils 100–250 nm in length. Our results support the idea that chitosomes do not originate by plasma membrane vesiculation but are defined sub-cellular organelles containing most of the chitin synthetase in protoplasts of Saccharomyces cerevisiae.     

Effects of polyoxin D on germination,morphological development and biosynthesis of the cell wall of Trichoderma viride

When polyoxin D is added to a spore suspension of Trichoderma viride at a concentration from 50–100 g/ml, it inhibits from 40–60% of germination. This percentage increases if dimethylsulfoxide (DMSO) is added.Mycelium growing in the presence of polyoxin D becomes irregular and loses its rigidity, showing several bulges along the hypha. Under the electron microscope the features of the cell wall and cytoplasmic content are apparently normal. Nevertheless, after incubation with different lytic systems or with (¹⁴C)glucose, it can be seen that polyoxin D partially inhibits the biosynthesis of -(1-3)glucan and the biosynthesis of chitin to a greater extent attaining inhibition of 83% at 100 g/ml of the antibiotic concentration.Regenerating protoplasts are less affected by polyoxin D. They do regenerate slower but the percentage of regeneration is more than 80%. Aberrant tubes synthesized by these protoplasts are not affected, they manifest their usual morphology and lack of chitin is confirmed in their composition.List of Abbreviations CECT Colecci Española de Cultivos Tipo - GAE Glucose, Asparagine and yeast Extract - DMSO Dimethylsulfoxide - PPO Diphenyloxazole - POPOP 1,4-bis-(4-methyl-5-phenyloxazol-2-yl)     

Calcium transport by pea root membranes

Calcium transport has been studied using purified endomembrane vesicles from dark-grown roots of Pisum sativum L. Membranes from a mixed microsomal (non-mitochondrial) fraction showed ATP-dependent calcium uptake which was released by the ionophore A 23187, had a pH optimum of 7.2 and required Mg²⁺ for uptake. Membranes were further purified using a rapid sucrosedensity-gradient technique yielding vesicles suitable for transport studies, and were identified using marker enzymes. Uptake by plasma membrane, tonoplast, endoplasmic reticulum and Golgi apparatus was indicated. Uptake by membranes of low density (predominantly tonoplast) had a pH optimum of 7.2–7.4 and nucleotide specificity ATP> guanosine 5-triphosphate>inosine 5-triphosphate>ADP>, while that by high-density membranes had a pH optimum of 7.5–7.9 and less specificity for ATP. The importance of regulating sucrose concentrations in calcium transport studies was demonstrated.Abbreviations ER endoplasmic reticulum - GTP guanosine 5-triphosphate - IDPase inosine diphosphatase - IIP inosine 5-triphosphate     

Synthesis and proteolytic activation of chitin synthetase in Phycomyces blakesleeanus Burgeff

The chitin synthetase of Phycomyces blakesleeanus mycelium is a particulate enzyme sedimenting mostly at 1000xg. The activity in crude extracts or cellular fractions can be increased more than tenfold by mild trypsin treatment. Plotting the reaction velocity versus UDP-N-acetylglucosamine concentration yields a sigmoidal curve. N-acetylglucosamine, which greatly stimulates the enzyme, changes the kinetics to an almost normal hyperbolic relationship.The enzyme is nearly absent in dormant spores and is synthesized de novo in germinating spores (from 4 h germination on). Trypsin treatment of extracts from germinating spores to assay the synthesis of the proenzyme did not reveal an earlier synthesis of the zymogen, which therefore might have some activity of its own.Abbreviations Used UDP-GlcNAc Uridinediphosphate-N-acetylglucosamine - GlcNAc N-acetylglucosamine - Chitin synthetase UDP-2-acetylamino-deoxyglucosyltransferase (EC 2.4.1.16)     

Identification of a GDP-Fuc:Galβ1–3GalNAc-R (Fuc to Gal)α1–2 fucosyltransferase and a GDP-Fuc:Galβ1–4GlcNAc (Fuc to GlcNAc)α1–3 fucosyltransferase in connective tissue of the snailLymnaea stagnalis

Connective tissue of the freshwater pulmonateLymnaea stagnalis was shown to contain fucosyltransferase activity capable of transferring fucose from GDP-Fuc in 1–2 linkage to terminal Gal of type 3 (Gal1–3GalNAc) acceptors, and in 1–3 linkage to GlcNAc of type 2 (Gal1–4GlcNAc) acceptors. The 1–2 fucosyltransferase was active with Gal1–3GalNAc1-OCH₂CH=CH₂ (K _m=12 mM,V _max=1.3 mU ml^–1) and Gal1–3GalNAc (K _m=20 mM,V _max=2.1 mU ml^–1), whereas the 1–3 fucosyltransferase was active with Gal1–4GlcNAc (K _m=23 mM,V _max=1.1 mU ml^–1). The products formed from Gal1–3GalNAc1-OCH₂CH=CH₂ and Gal1–4GlcNAc were purified by high performance liquid chromatography, and identified by 500 MHz¹H-NMR spectroscopy and methylation analysis to be Fuc1–2Gal1–3GalNAc1-OCH₂CH=CH₂ and Gal1–4(Fuc1–3)GlcNAc, respectively. Competition experiments suggest that the two fucosyltransferase activities are due to two distinct enzymes.Abbreviations 2Fuc-T 1–2 fucosyltransferase - 3Fuc-T 1–3 fucosyltransferase - MeO-3Man 3-O-methyl-D-mannose - MeO-3Gal 3-O-methyl-D-galactose     

Chitin synthesis in Spodoptera frugiperda wing imaginal discs: I. Chlorfluazuron,diflubenzuron, and teflubenzuron inhibit incorporation but not uptake of [14C]N-acetyl-D-glucosamine

The purpose of this study was to determine the effects of potent inhibitors of chitin synthesis on an organ culture test system as a basis for determining the mode of action of such compounds. Consequently, we investigated the action of chlorfluazuron (CFA), diflubenzuron (DFB), and teflubenzuron (TFB) on uptake and incorporation into chitin of [¹⁴C]N-acetyl-D-glucosamine ([¹⁴C]GlcNAc) in wing imaginal discs cultured in vitro. Spodoptera frugiperda wing imaginal discs provided a highly responsive test system for studying the inhibition of ecdysteroid-dependent chitin synthesis in a target tissue in vitro. All three inhibitors blocked ecdysteroid-dependent [¹⁴C]GlcNAc incorporation into chitin by the wing imaginal discs. The effectiveness of the inhibitors was not affected by the time of their application, i.e., exposures before, during, or after 20-hydroxyecdysone treatment were equally effective in inhibiting chitin synthesis. Thus, exposure of freshly dissected discs to CFA for periods as short as 15 min inhibited approximately 90% of the chitin synthesis measured 72 h later. In contrast to previous in vivo studies all three inhibitors were similar in their effectiveness in vitro. However, while all three compounds inhibited [¹⁴C]GlcNAc incorporation in a similar dose-dependent manner, only DFB and TFB reduced but did not block uptake of GlcNAc. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Observations on the ultrastructure ofFrankia sp. in root nodules ofDatisca cannabina L.

Summary The fine structures of the microsymbiont inside the root nodules ofDatisca cannabina have been studied by light, by transmission- and by scanning-electron microscopy. The endophyte is prokaryotic and actinomycetal in nature. The hyphae are septate and branched, diameter 0.3–0.5 m. The tips of hyphae are swollen to form electron-dense, clubshaped to filamentous vesicles, ranging in diameter: 0.4–1.4 m. The endophyte penetrates through walls of the cortial cells. The infected zone is kidney shaped and confined to one side of the acentric stele. The orientation of infection is reversed from other actinorhizae exceptCoriaria. The hyphae are near the host cell wall and vesicles are directed towards the central vacuole. Vesicles are aseptate and no collapsing of the vesicle cell wall (void area) has been observed. Vesicle clusters structures are globular with an opening at one side of the cluster. The host cell is multinucleate or contains a lobed nucleus. Groups of mitochondria are located in between the hyphae, suggesting a strong association between the host and the endophyte for energy supply and amino acid production. The consequences of the inability to separate the mitochondria from the vesicle clusters in nodule homogenates in physiological studies have been discussed.Isolated vesicles clusters showed dehydrogenase activity, indicated by the presence of formazan crystals, after incubation with NADH and NBT. Strongest reducing activity was found within the vesicles. The possible role of filamentous vesicles in nitrogen fixation has been discussed.     

In-vivo control of valine and leucine synthesis in the duckweed Spirodela polyrhiza

Duckweed colonies were grown on 1 l of nutrient solution supplied with 10 M l-[¹⁴C]leucine or with 25 M l-[¹⁴C]valine. Under these conditions the exogenously supplied amino acid did not inhibit growth, but caused in the plants a moderately increased pool of that amino acid, which remained essentially constant during the culture period. The effect of the increased pool of valine or leucine on the biosynthesis of these amino acids was determined from isotope dilution in the protein-bound valine and-or leucine. An increase in the leucine pool from 1.1 to 5.0 nmol mg^–1 dry weight resulted in a 21% reduction of metabolite flow through the common part of the valine-leucine biosynthetic pathway; leucine synthesis was reduced by 35%, but valine synthesis by only 5% and isoleucine synthesis was apparently unaffected. An increase in the valine pool from 3.2 to 6.6 nmol mg^–1 dry weight reduced the metabolite flow through the valine-leucine pathway by 48%, valine synthesis by 70%, and leucine synthesis from pyruvate by 29%, which was compensated by leucine synthesis from exogenous valine, whereas the synthesis of isoleucine was not changed. It is concluded that the biosynthesis of valine and leucine is mainly controlled by feedback inhibition of acetohydroxyacid synthetase. In vivo, the feedback inhibition can be exerted in such a way that synthesis of acetolactate (the precursor of valine and leucine) is appreciably reduced, whereas synthesis of acetohydroxybutyrate (the isoleucine precursor) is not inhibited.