Internal lipid synthesis and vesicle growth as a step toward self-reproduction of the minimal cell

One of the major properties of the semi-synthetic minimal cell, as a model for early living cells, is the ability to self-reproduce itself, and the reproduction of the boundary layer or vesicle compartment is part of this process. A minimal bio-molecular mechanism based on the activity of one single enzyme, the FAS-B (Fatty Acid Synthase) Type I enzyme from Brevibacterium ammoniagenes, is encapsulated in 1-palmitoyl-2oleoyl-sn-glycero-3-phosphatidylcholine (POPC) liposomes to control lipid synthesis. Consequently molecules of palmitic acid released from the FAS catalysis, within the internal lumen, move toward the membrane compartment and become incorporated into the phospholipid bilayer. As a result the vesicle membranes change in lipid composition and liposome growth can be monitored. Here we report the first experiments showing vesicles growth by catalysis of one enzyme only that produces cell boundary from within. This is the prototype of the simplest autopoietic minimal cell.     

The calcium requirement for functional vesicle development and nitrogen fixation by Frankia strains EAN1pec and CpI1

A calcium requirement was shown for both vesicle development and nitrogenase activity by Frankia strains EAN1_pec and CpI1. Washing cells with EGTA or EDTA inhibited both vesicle development and nitrogenase activity. The inhibition of both was reversed by the addition of calcium. A variety of agents known to affect calcium-dependent biological processes, such as a Ca-ATPase inhibitor, Ca-channel blockers, Ca-ionophores, calmodulin antagonists and the local anaesthetics, tetracaine and dibucaine, inhibited nitrogenase activity. Respiratory studies showed that a CN-insensitive respiration process occurred only under nitrogen derepressing conditions. Respiration by NH₄Cl-grown cells was completely inhibited by KCN while N₂-grown cells were inhibited by only 70%. Removal of calcium ions by EGTA or by the addition of dibucaine or tetracaine blocked the CN-insensitive respiration. This CN-insensitive respiration may be involved in protecting nitrogenase inside the vesicles from oxygen.Abbreviations EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis-( amino-ethyl ether) N,N¹-tetraacetic acid - GI germination inhibitor - MOPS 3-[N-morpholino] propane sulfonic acid - PCMBS p-chloromercuribenzene sulphonate - TMB 8,8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate     

Comparative physiology of nitrogenase activity and vesicle development for Frankia strains CpI1, ACN1 ag,EAN1pec and EUN1f

The relationship between nitrogen fixation and development of a specialized cell structure, called the vesicle, was studied using four Frankia isolates. Nitrogenase activity was repressed in all four strains during growth with ammonia. Strain CpI1 formed no vesicles during NH₄ growth. Strains ACN1 ^ag , EAN1_pec and EUN1_f produced low numbers of vesicles in the presence of ammonia. Following transfer to nitrogen-free media, a parallel increase in nitrogenase activity and vesicle numbers occurred with all four isolates. Appearance of nitrogenase activity was more rapid in those strains that possessed some vesicles at the time of shift to N₂ as a nitrogen source. The ratio of vesicle numbers to level of nitrogenase activity varied widely among the four strains and in response to different growth conditions and culture age of the individual strains. Optimum conditions of temperature, carbon and energy source, nitrogen source and availability of iron and molybdenum were different for each of the four strains. Those conditions that significantly reduced nitrogenase activity were always associated with decreased numbers of vesicles.     

Intracellular compartmentation of two enzymes of berberine biosynthesis in plant cell cultures

Out of the eight enzymes involved in the biosynthesis of the isoquinoline alkaloid berberine, at least, two enzymes, berberine bridge enzyme and (S)-tetrahydroprotoberberine oxidase, are exclusively located in a vesicle with a specific gravity of =1.14 g·cm^–3 as shown by direct enzymatic assay as well as immunoelectrophoresis. Electronmicroscopic examination of the enzyme-containing particulate preparation from Berberis wilsoniae var. subcaulialata cultured cells demonstrated that it is composed mainly of membranous vesicles. The protein composition of this preparation reveals the presence of only about 20 separable proteins, of which two major ones are berberine bridge enzyme and (S)-tetrahydroprotoberberine oxidase. Incubation of these vesicles with the substrate (S)-reticuline in the presence and absence of S-adenosyl-l-methionine leads to the formation of a red product which was identified as dehydroscoulerine. If the cytoplasmic enzyme S-adenosyl-l-methionine:(S)-scoulerine-9-O-methyltransferase is added to the vesicle preparation in the presence of (S)-reticuline and S-adenosyl-l-methionine, not dehydroscoulerine but columbamine, the immediate precursor of berberine is formed. Some of the quaternary alkaloids are located inside the vesicles; fusion of these vesicles leads to vacuoles containing the quaternary alkaloids. These vesicles are the first highly specific and unique compartment serving only alkaloid biosynthesis; they are found in members of four different plant families and in cell cultures as well as in differentiated tissue.Abbreviations BBE berberine bridge enzyme - STOX (S)-tetrahydroprotoberberine oxidase Dedicated to Professor Karl Decker, Freiburg, on the occasion of his 60^th birthday     

Chloroplast and extrachloroplastic starch-degrading enzymes in Pisum sativum L.

The organelles of soybean (Glycine max (L.) Merr.) protoplasts were separated using a recently developed procedure which allows rapid (3-h) recovery of a fraction enriched for coated vesicles (CVs). As determined by marker-enzyme enrichment and ultrastructural analysis of isolated membrane fractions, endoplasmic reticulum, Golgi membranes, glucan-synthase-II (EC 2.4.1.34)-containing membranes (putative plasma membrane), mitochondria, and CVs were enriched in separate fractions in a sucrose density gradient. Glucan synthase I (EC 2.4.1.12) had the highest specific activity in the Golgi-enriched and CV-enriched fractions and was found to comigrate with CVs upon rate-zonal centrifugation of a CV-enriched fraction. For further elucidation of the role of these latter organelles in cell-wall regeneration, freshly isolated protoplasts were pulsed with [³H]glucose for 20 min, and the disappearance of label from the organelles was followed for the ensuing 1 h. Although a CV-enriched fraction contained glucan synthase I, it contained very small amounts of labelled polysaccharide during the period of study. Pulse-chase experiments with [³H]glucose helped to confirm the role of the Golgi apparatus in secretion of matrix polysaccharides by protoplasts.Abbreviations CV(s) coated vesicle(s) - Da dalton - ER endoplasmic reticulum - GSI,II glucan synthase I and II, respecitively Two whom correspondence should be directed. Address after February 1986:Department of Biology, Texas A&M University. College Station, TX 77843-3258, USA     

Characterisation of chloride transport at the tonoplast of higher plants using a chloride-sensitive fluorescent probe

The characteristics of Cl^– transport in isolated tonoplast vesicles from red-beet (Beta vulgaris L.) storage tissue have been investigated using the Cl^–-sensitive fluorescent probe, 6-methoxy-1-(3-sulfonatopropyl)-quinolinium (SPQ). The imposition of (inside) positive diffusion potentials, generated with K⁺ and valinomycin, increased the initial rate of Cl^– transport, demonstrating that Cl^– could be electrically driven into the vesicles. Chloride influx was unaffected by SO ₄ ^2- , but was competitively blocked by NO ₃ ^– , indicating that both Cl^– and NO ₃ ^– may be transported by the same porter. In some preparations, increases in free-Ca²⁺ concentration from 10^–8 to 10^–5 mol·dm^–3 caused a significant decrease in Cl^– influx, which may indicate that cytosolic Ca²⁺ concentration has a role in controlling Cl^– fluxes at the tonoplast. However, this effect was only seen in about 50% of membrane preparations and some doubt remains over its physiological significance. A range of compounds known to block anion transport in other systems was tested, and some partially blocked Cl^– transport. However, many of these inhibitors interfered with SPQ fluorescence and so only irreversible effects could be tested. The results are discussed in the context of recent advances made using the patch-clamp technique on isolated vacuoles.Abbreviations and Symbols BTP 1,3-bis[tris(hydroxymethyl)-methylamino]propane - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - membrane potential - pH pH gradient - SPQ 6-methoxy-1-(3-sulfonatopropyl)quinolinium - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl] glycine     

Special features of phosphatidylcholine vesicles as seen in cryo-transmission electron microscopy

Vesicles of egg yolk phosphatidylcholine (EYPC) were studied by cryo-transmission electron microscopy. The electron micrographs indicate that, despite the rapidity of cooling, membrane undulations are flattened and some vesicles change their shapes before the samples freeze. These artefacts are attributed to the action of the lateral tension that results from the membrane area contraction associated with the temperature drop. Other micrographs represent grainy membranes and angular vesicles. We regard them as the first direct evidence for the superstructure and optically invisible roughness which were recently postulated for these membranes.     

Synaptic Vesicle Recycling in Cultured Cerebellar Granule Cells: Role of Vesicular Acidification and Refilling

Abstract: The role of the transvesicular protonmotive force in synaptic vesicle recycling was investigated in cultured cerebellar granule cells. The vesicular V-ATPase was inhibited by 1 µ M bafilomycin A1; as an alternative, the pH component of the gradient was selectively collapsed by equilibration of the cells with 10 m M methylamine and monitored with the fluorescent probe Lysosensor Green. Electrical field-evoked exocytosis of d -[³H]aspartate was inhibited by bafilomycin A1 but not by methylamine, indicating that a transvesicular membrane potential rather than pH gradient is required for transmitter retention within vesicles. In contrast, neither compound affected the field-evoked uptake, recycling, or destaining of the vesicle-specific dye FM2-10; thus, vesicles whose lumens were neutral and/or depleted of transmitter could still recycle in the nerve terminal. No exhaustion of d -[³H]aspartate exocytosis was observed when cells were subjected to six consecutive trains of field stimuli (40 Hz/10 s separated by 10 s). In contrast, the release of preloaded FM2-10 was reduced by ∼50%, with each stimulus indicating that unlabeled vesicles with accumulated d -[³H]aspartate were competing with labeled vesicles for exocytosis. As d -[³H]aspartate was accumulated rapidly across the vesicle membrane from the large cytoplasmic pool, the transmitter-loaded but unlabelled vesicles may represent refilled recycling vesicles. FM2-10 destaining and d -[³H]aspartate exocytosis were reduced in parallel at low frequencies, challenging a role for transient vesicle fusion.     

菌根真菌在红壤中对柑桔吸收磷肥的研究

接种一种球囊霉 Glomus sp.编号 CC-1于红壤中,使酸桔和枳实生苗的根系感染形成泡囊丛枝内生菌根。应用放射性同位素~(32)P 标记的可溶性磷肥和难溶性磷肥来研究菌根真菌对柑桔吸收磷肥的作用。放射性宏观自显影和显微自显影表明菌根真菌加强了柑桔对两类磷肥的吸收。放射性测量和化学分析证明有菌根柑桔实生苗的地上部含磷量以及从红壤中和从肥料中吸收的磷量比无菌根的增多,有菌根实生苗的生长反应也较好。