An investigation of the role of the anther tapetum during microspore development using genetic cell ablation

The effects on anther development of a fusion of the Arabidopsis anther-specific apg gene promoter to a ribonuclease (barnase) in transgenic tobacco plants were examined. Contrary to expectations, viable pollen grains were produced by these plants despite the demonstration that ribonuclease expression in the microspores and tapetum caused targeted cell ablation. Transformed plants were reduced in male fertility due to ablation of a proportion of pollen dependent on apg-barnase locus number. Plants were otherwise phenotypically normal and fully female fertile, confirming the anther-specific nature of the apg promoter. In microspores inheriting an apg-barnase locus following meiosis, loss of cell viability, as judged by fluorescein diacetate staining, occurred during mid to late microspore development. Microspores not inheriting a transgene went on to mature into viable pollen grains. Premature degeneration of the tapetum was also observed as a result of apg-barnase expression, but this did not appear to disrupt the subsequent microspore and pollen developmental programmes. This was substantiated by observations of microspore development in plants in which the tapetum was rescued from ablation by crossing in a second transgene encoding a tapetum-specific inhibitor of the ribonuclease. It was determined that tapetum cell disruption occurs at the early to mid uninucleate microspore stage in apg-barnase transformants. The data presented show that after this point in microspore development the tapetum is no longer essential for the production of viable pollen in tobacco.     

Multiple enzyme purifications from muscle extracts by using affinity-elution-chromatographic procedures.

1. Starting with (NH4)2SO4 fractions of muscle extracts, procedures for purifying four to six separate enzymes from each fraction by using affinity-elution-chromatographic techniques are described. 2. Schemes for purifying 12 separate enzymes from rabbit muscle, and eight from chicken muscle extracts, are included. In nearly all cases the overall procedure involves three steps: the initial (NH4)2SO4 fractionation, the ion-exchange chromatography with affinity elution of the enzyme, and gel filtration. The specific activities of the enzymes so purified are comparable with the highest values in the literature. 3. The five schemes described include illustrations of affinity elution of the separate enzymes at different pH values, at different ionic strengths and in combination with conventional gradient elution. They also include stepwise adsorption on columns at different pH values. 4. Separation of two electrophoretically differing forms of phosphoglycerate kinase was achieved by gradient affinity elution from CM-cellulose. The lower-pI form was eluted by a lower concentration of substrate than the higher-pI form.     

Sphingosine 1-phosphate lyase enzyme assay using a BODIPY-labeled substrate

Sphingosine 1-phosphate lyase (SPL) is responsible for the irreversible catabolism of sphingosine 1-phosphate, which signals through five membrane receptors to mediate cell stress responses, angiogenesis, and lymphocyte trafficking. The standard assay for SPL activity utilizes a radioactive dihydrosphingosine 1-phosphate substrate and is expensive and cumbersome. In this study, we describe an SPL assay that employs an ω-labeled BODIPY-sphingosine 1-phosphate substrate, allowing fluorescent product detection by HPLC and incorporating advantages of the BODIPY fluorophore. The major aldehyde product is confirmed by reaction with 2,4-dinitrophenylhydrazine. The SPL-catalyzed reaction is linear over a 30 min time period and yields a K_m of 35 μM for BODIPY-sphingosine 1-phosphate.     

Measurement of Pi dissociation from actin filaments following ATP hydrolysis using a linked enzyme assay

Using glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase as a linked enzyme assay for determination of free inorganic phosphate, as described by Trentham et al. (1972, Biochem. J. 126, 635-644) we have been able to monitor the time course of Pi release from F-actin following ATP hydrolysis that accompanies ATP-actin polymerization. The rate constant for Pi dissociation from Mg-F-actin is 0.006 s-1 at 25 degrees C and pH 7.8, both in the presence of 1 mM Mg and 0.1 M KCl + 1 mM Mg. This result confirms the existence of ADP-Pi-F-actin as a major intermediate in the polymerization of ATP-actin (Carlier and Pantaloni, 1986, Biochemistry 25, 7789-7792). The method is potentially useful for other enzymes hydrolyzing triphosphate nucleotides, provided that the rate of Pi release is appreciably lower than 0.1 s-1.     

Determining acetylcholinesterase inhibitory activity in plant extracts using a fluorimetric flow assay

A fluorometric assay for acetylcholinesterase inhibitory activity was developed in a flow system using the fluorogenic substrate 7-acetoxy-1-methyl quinolinium iodide which is hydrolysed to the highly fluorescent 7-hydroxy-1-methyl quinolinium iodide. The detection limit of galanthamine is 0.5 microM, which is about 20 times more sensitive than in the colorimetric flow assay. In the presence of 30% methanol or of 5% acetonitrile, about 70% of the enzyme activity could still be detected. Various plant extracts have been screened using the described system including bulbs of Galanthus nivalis, Eucharis amazonica (E. x grandiflora), Crinum powelli and Nerine bowdenii (all members of the Amaryllidaceae), which showed strong AchE inhibitory activity.     

A novel lactone-forming carboxylesterase: molecular identification of a tuliposide A-converting enzyme in tulip

Tuliposides, the glucose esters of 4-hydroxy-2-methylenebutanoate and 3,4-dihydroxy-2-methylenebutanoate, are major secondary metabolites in tulip (Tulipa gesneriana). Their lactonized aglycons, tulipalins, function as defensive chemicals due to their biological activities. We recently found that tuliposide-converting enzyme (TCE) purified from tulip bulbs catalyzed the conversion of tuliposides to tulipalins, but the possibility of the presence of several TCE isozymes was raised: TCE in tissues other than bulbs is different from bulb TCE. Here, to prove this hypothesis, TCE was purified from petals, which have the second highest TCE activity after bulbs. The purified enzyme, like the bulb enzyme, preferentially accepted tuliposides as substrates, with 6-tuliposide A the best substrate, which allowed naming the enzyme tuliposide A-converting enzyme (TCEA), but specific activity and molecular mass differed between the petal and bulb enzymes. After peptide sequencing, a novel cDNA (TgTCEA) encoding petal TCEA was isolated, and the functional characterization of the recombinant enzyme verified that TgTCEA catalyzes the conversion of 6-tuliposide A to tulipalin A. TgTCEA was transcribed in all tulip tissues but not in bulbs, indicating the presence of a bulb-specific TgTCEA, as suggested by the distinct enzymatic characters between the petal and bulb enzymes. Plastidial localization of TgTCEA enzyme was revealed, which allowed proposing a cytological mechanism of TgTCE-mediated tulipalin formation in the tulip defensive strategy. Site-directed mutagenesis of TgTCEA suggested that the oxyanion hole and catalytic triad characteristic of typical carboxylesterases are essential for the catalytic process of TgTCEA enzyme. To our knowledge, TgTCEA is the first identified member of the lactone-forming carboxylesterases, specifically catalyzing intramolecular transesterification.     

Time relationships of sporopollenin synthesis associated with tapetum and microspores in Lilium

Summary The development of the sporopollenin orbicules (Ubisch bodies) on the tapetal cells of Lilium begins while the spores are still enclosed in the meiotic tetrads. Spherosome-like structures, the pro-orbicular bodies, accumulate in the vicinity of the plasmalemma early in the tetrad period, and are extruded into the space within the degenerating inner walls of the tapetal cells. There they acquire a coating of sporopollenin, the accretion continuing until after the release of the spores from the tetrads. Some orbicules remain attached to the plasmalemma by stalks. Synthesis of a material of the general class of sporopollenin begins in the primexine of the young spore in the mid-tetrad period, again outside of the cell membrane, but within the callose tetrad wall.A general scheme for sporopollenin formation in the anther is given. According to this, (a) precursors are synthesised both in the young spores and in the tapetum, and released into the extracellular space; and (b) polymerisation occurs on initiating sites outside of the cell membranes, these sites being the surface of the proorbicular bodies and of the special lamellae concerned in exine growth.Synthesis of sporopollenin in the anther is virtually complete before the main synthesis of the pigmented pollen coat substances (Pollenkitt) begins in the tapetum. It is therefore improbable that the carotenoids produced in the final phase of metabolic activity in the tapetum can be sporopollenin precursors.

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Zusammenfassung Die Entwicklung der Sporopollenin-Orbicula (Ubisch-Körper) in den Tapetenzellen von Lilium beginnt wenn die Sporen noch in den meiotischen Tetraden zusammen geschlossen sind. Sphärosomenähnliche Gebilde, die Pro-Orbicularkörper, werden im frühen Tetradenstadium in der Nähe des Plasmalemmas angehäuft und dann in den Raum innerhalb der degenerierenden Innenwände der Tapetenzellen ausgestoßen. Dort werden sie mit einer Hülle aus Sporopollenin versehen; dieser Vorgang dauert noch an, wenn die Sporen aus dem Tetradenverband freigesetzt worden sind. Einige der Orbicular bleiben mit dem Plasmalemma mittels Stielchen verbunden. Im mittleren Tetradenstadium setzt in den Primexinen der jungen Sporen die Synthese eines Materials ein, welches der allgemeinen Klasse der Sporopollenine angehört; auch dieser Vorgang verläuft außerhalb der Zellmembran, aber innerhalb der aus Callose bestehenden Tetradenwand.Ein allgemeines Schema für Sporenpollenbildung in der Anthere wird vorgeschlagen. Danach werden a) Vorstufen sowohl in der jungen Spore als auch im Tapetum synthetisiert und in den extracellulären Raum ausgeschieden, und b) findet Polymerisation an spezifischen Stellen außerhalb der Zellmembran, und zwar den Oberflächen der Pro-Orbicularkörper und der für das Wachstum der Exine maßgeblichen, speziellen Lamellen, statt.Die Synthese des Sporopollenins in der Anthere ist praktisch beendet bevor die Synthese der pigmentierten Substanzen der Pollenhaut (Pollenkitt) im Tapetum einsetzt. Es ist daher unwahrescheinlich, daß die Carotinoide, die in den Endstadien der Stoffwechseltätigkeit der Tapetenzellen gebildet werden, Vorstufen des Sporopollenins sein können.

     

Simultaneous detection of three arboviruses using a triplex RT-PCR enzyme hybridization assay

Arboviruses represent a serious problem to public health and agriculture worldwide.Fast,accurate identification of the viral agents of arbovirus-associated disease is essential for epidemiological surv...     

Programmed cell death progressively models the development of anther sporophytic tissues from the tapetum and is triggered in pollen grains during maturation

To characterize the spatial and temporal occurrence of programmed cell death (PCD) in Lilium anther tissues, we used both microscopical and molecular markers of apoptosis for developmental stages from meiosis to pollen release. The first hallmarks of PCD include cell condensation and shrinkage of the cytoplasm, separation of chromatin into delineated masses, and DNA fragmentation in the tapetum as early as the premeiosis stage. PCD then extended to other anther sporophytic tissues, leading to anther dehiscence. Although the PCD clearly affected the endothecium and the epidermis, these two cell layers remained alive until anther dehiscence. In pollen, no sign of PCD was found until pollen mitosis I, after what apoptotic features developed progressively in the vegetative cell. In addition, DNA ladders were detected in all sporophytic tissues and cell types throughout pollen development, whereas in the male gametophyte DNA ladders were only detected during pollen maturation. Our data suggest that PCD is a progressive and active process affecting all the anther tissues, first being triggered in the tapetum.     

Validation of a colorimetric assay for the in vitro screening of inhibitors of angiotensin-converting enzyme (ACE) from plant extracts.

A new method for the in vitro screening of plant extracts with potential angiotensin-converting enzyme (ACE) inhibitory activity is proposed. The method is based on the cleavage of the substrate hippuryl-glycyl-glycine by ACE and subsequent reaction with trinitrobenzenesulfonic acid to form 2,4,6-trinitrophenyl-glycyl-glycine, whose absorbance is determined at 415 nm in a microtitre plate reader. Rabbit lung dehydrated by acetone was employed as an enzyme source. Validation of the method showed satisfactory intra-day (CV = 7.63%) and inter-day precision (CV = 13.61%), recovery (97-102.1%), sensitivity (IC50 = 14.1 nmol/l) and linearity in the range 7.5-120 mmol/l of glycyl-glycine (r2 = 0.9921). Besides, the method showed good correlation with a HPLC assay already established for the screening of ACE inhibitors (r = 0.9935 and 0.9034, respectively, for captopril solutions and for plant extracts). The method involves only inexpensive reagents and apparatus.     

An assay for angiotensin-converting enzyme using capillary zone electrophoresis

A sensitive and rapid method was developed for angiotensin-converting enzyme (ACE) activity determination by capillary zone electrophoresis. Hippuryl-l-histidyl-l-leucine, a synthetic tripeptide, was used as the ACE-specific substrate. Capillary zone electrophoresis was employed to separate the products of the enzymatic reaction and the ACE activity was determined by quantification of hippuric acid, a result of the enzymatic reaction on the tripeptide. The capillary electrophoresis was performed in a 27 cm x 75 micrometer i.d. fused-silica capillary using 200 mM boric acid-borate buffer (pH 9.0) as a run buffer with an applied voltage of 8.1 kV at a capillary temperature of 23 degrees C. The electrophoresis was monitored at 228 nm. Each electrophoretic run requires only a nanoliter of the enzymatic reactant solution, at only 6 min, rendering a powerful tool for the ACE assay.     

An immunoaffinity immobilized enzyme assay for neomycin phosphotransferase II in crude cell extracts.

An immunoaffinity immobilized enzyme assay for neomycin phosphotransferase II (NPT II) has been developed. This method combines affinity purification with an enzyme-catalyzed reaction. The assay is mechanically simple and can be semiautomatable since all steps are performed in a microtiter plate. An immunoaffinity step separates NPT II from endogenous kinases, which may produce false positive results, and from endogenous phosphatases and inhibitors, which decrease the apparent NPT activity. This method thus exploits two modes of specificity: antigen-antibody specificity and enzyme catalysis specificity. This gives a high degree of specificity and allows quantitation of 0.1 ppm NPT in crude plant protein extracts. The catalytic ability of the NPT is not significantly hampered by its attachment to the gel, in the Km values for ATP and neomycin and the catalytic number for immobilized NPT are comparable to those for the NPT in solution.     

The tapetum inSchizaea pectinata (Schizaeaceae) and a comparison with the tapetum inPsilotum nudum (Psilotaceae)

A combination tapetum consisting of a cellular, parietal component and a plasmodial component occurs inSchizaea pectinata. A single, tapetal initial layer divides to form an outer parietal layer which maintains its cellular integrity until late in spore wall development. The inner tapetal layer differentiates into a plasmodium which disappears after the outer exospore has developed. In the final stages of spore wall development, granular material occurs in large masses and is dispersed as small granules throughout the sporangial loculus. No tapetal membrane develops. Comparisons are drawn with the combination tapetum found inPsilotum nudum.     

Double-stranded RNA-stimulated enzyme activities isolated from human placental extracts

A (2′–5′)A_n synthetase activity was isolated from human placental extracts by affinity chromatography on poly(rI)·poly(rC)-agarose. The oligonucleotide (2′–5′)A_n was identified by (1) chromatography on PEI-cellulose and DEAE-cellulose, (2) inhibition of polypeptide synthesis in lysed rabbit reticulocytes (3) competition of the binding of pppA(pA)3,3′-[³²P]pCp to rabbit reticulocyte lysates, and (4) alkaline phosphatase digestion. The synthetase activity in most placental preparations is activated by natural or synthetic dsRNA. However, in a few placental synthetase preparations, dsRNA is only marginally stimulatory and only becomes effective by prior treatment of the enzyme preparations with the calcium-dependent micrococal nuclease. This suggeststhat there is an endogenous placental dsRNA contaminant in the enzyme preparations. In some synthetase preparations, a second dsRNA-stimulated product, tentatively identified as the nucleotide 5′-IMP, is also observed. Because the specific AMP deaminase inhibitor coformycin (10 μM) blocks the formation of IMP from ATP and causes a quantitative accumulation of AMP, and because the formation of IMp becomes independent of dsRNA when ADP or AMP is used in plase of ATP, the presence of a dsRNA-stimulated ATP phosphohydrolase (ATPase) activity in human placenta is suggested.     

Chemical synthesis of cellulose and cello-oligomers using a hydrolysis enzyme as a catalyst

Regio- and stereo-selective synthesis of polysaccharides and oligosaccharides has been achieved by using glycosyl fluorides as substrates for cellulases. This methodology has successfully been applied to the first synthesis of cellulose via a non-biosynthetic pathway as well as to a selective preparation of cello-oligosaccharides and unnatural oligosaccharides. Using the enzymatic polymerization, it is possible to control the relative direction (parallel or anti-parallel) of each glucan chain in the synthetic cellulose in vitro. Based on these results, a new concept of ‘allos-selectivity’ in polymer synthesis has been proposed.     

Analysis of nitrate in food extracts using a thermostable formate linked nitrate reductase enzyme system

Summary A method for reduction of nitrate to nitrite for determination of nitrate is described, using a thermostable formate linked nitrate reductase enzyme system (FLNR). The reduction of nitrate to nitrite was found to be quantitative in water and in various food samples containing nitrate. The method is suggested as an alternative for the cadmium reduction method.     

Measurement of immunoglobulin synthesis using the ELISPOT assay

We describe a method for the detection of specific antibody-producing cells from either in vitro or in vivo immunization. These techniques are especially useful for detecting antibodies from developing hybridomas. We have successfully used the system to detect isotype-specific antibodies to a variety of bacterial antigens which were produced by heterohybridomas.