RESUMEN
Glutathione S-transferases (GSTs) are multi-functional enzymes, known to conjugate xenobiotics and degrade peroxides. Herein, we report on the potential of four Zea mays GST isoforms (Zm GST I-I, Zm GST I-II, Zm GST II-II and Zm GST III-III) to act as binding and protection proteins. These isoforms bind protoporphyrin IX (PPIX), mesoporphyrin, coproporphyrin, uroporphyrin and Mg-protoporpyhrin, but do not form a glutathione conjugate. The binding is non-covalent and inhibits GSTs enzymatic activity, dependent on the type of the porphyrin and GST isoform tested. I(50) values are in the range of 1 to 10 microM for PPIX, the inhibition by mesoporphyrin and Mg-protoporphyrin (Mg-PPIX) is two to five times less. The mode of binding is non-competitive for the hydrophobic substrate and competitive for glutathione. Binding affinities (K(D) values) of the GST isoforms are between 0.3 and 0.8 microM for coproporphyrin and about 2 microM for mesoporphyrin.Zm GST III-III prevents the nonenzymatic autoxidation of protoporphyrinogen to the phytotoxic PPIX. Zm GST II-II can reduce the oxidative degradation of hemin. This points to a specific ligand role of distinct GST isoforms to protect tetrapyrroles in the plant cell.
Asunto(s)
Glutatión Transferasa/metabolismo , Proteínas de Plantas/metabolismo , Porfirinas/metabolismo , Zea mays/enzimología , Fluorescencia , Glutatión Transferasa/antagonistas & inhibidores , Hemina/metabolismo , Oxidación-Reducción , Protoporfirinas/metabolismoRESUMEN
Complementary chromatic adaptation was studied in two unicellular diazotrophic Synechocystis-type cyanobacteria, strains BO 8402 and BO 9201. Strain BO 8402 was isolated from Lake Constance as a mutant lacking phycobilisomes due to an insertion sequence element in the gene apcA, encoding alpha-allophycocyanin. Strain BO 9201 recovered the ability to assemble functional phycobilisomes after a spontaneous excision of the insertion sequence element in apcA. Simultaneously, the strain became able to perform group II complementary chromatic adaptation by regulating the synthesis of phycoerythrin. The two strains had identical phycoerythrin operons, cpeBA, and similar-sized transcripts were formed upon induction by green light. However, in strain BO 8402 the cpeBA transcript level was approx. 20-fold lower than in strain BO 9201. Because strain BO 8402 cannot synthesize allophycocyanin and phycocyanin is sequestered in paracrystalline inclusion bodies, non-assembled phycoerythrin may accumulate inside the cells. It was examined whether non-assembled phycoerythrin or other effects caused by the absence of phycobilisomes, such as a permanently oxidized redox status of the photosynthetic electron transport chain or a distorted ratio of C and N assimilation mediated the repression of cpeBA transcription in strain BO 8402. No such links could be established. We therefore concluded that in these diazotrophic Synechocystis-type cyanobacteria the green light-induced transcription of the cpe operon directly required a functional apc operon.
Asunto(s)
Cianobacterias/genética , Genes Bacterianos , Luz , Ficocianina/genética , Proteínas Bacterianas/aislamiento & purificación , Northern Blotting , Western Blotting , Cianobacterias/química , Cianobacterias/crecimiento & desarrollo , Elementos Transponibles de ADN , Diurona/farmacología , Complejos de Proteína Captadores de Luz , Datos de Secuencia Molecular , Operón , Fotosíntesis/efectos de los fármacos , Ficobilisomas , Ficoeritrina/biosíntesis , Ficoeritrina/química , Ficoeritrina/genética , Proteínas/análisis , Proteínas/genética , ARN Mensajero/biosíntesis , Mapeo Restrictivo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido Nucleico , Espectrometría de Fluorescencia , Transcripción Genética/efectos de los fármacosRESUMEN
Glutathione S-transferases (GSTs) are ubiquitous enzymes and abundant in plants. They are intimately involved in plant metabolism and stress defense related to reactive oxygen species. Our project assigned particular reactions including novel ones to certain GST-isoforms. Transformed E. coli was used to express recombinant GST-isoforms from maize. An N-terminal His tag allowed their purification by affinity chromatography. Three GST-monomers had a molecular weight of 26, 27, 29 kDa, and aggregated to dimers when assayed for their enzymic properties. Four dimeric isoforms were used to study how they interact with tetrapyrroles (of the chlorophyll biosynthesis pathway). It was found that protoporphyrin IX (Proto IX), Mg-protoporphyrin and other tetrapyrroles are bound non-covalently ("liganded") to GSTs but not conjugated with reduced glutathione. This binding is non-covalent, and results in inhibition of conjugation activity, the degree depends on type of the porphyrin and GST-isoform. I50-values between 1-10 microM were measured for Proto IX, the inhibition by mesoporphyrin and Mg-protoporphyrin was 2- to 5-fold less. The ligand binding is noncompetitive for the substrate 1-chloro-2,4-dinitrobenzene and competitive for glutathione. The dimer GST 26/26 prevents the (non-enzymic) autoxidation of protoporphyrinogen to Proto IX, which produces phytotoxic reactive oxygen species in the light. GST 27/27 protects hemin against degradation. Protoporphyrinogen is formed in the plastid and then exported into the cytosol. Apparently binding by a suitable GST-isoform ensures that the highly autoxidizable protoporphyrinogen can safely reach the mitochondrium where it is processed to cytochrome.
Asunto(s)
Glutatión Transferasa/metabolismo , Cloroplastos/enzimología , Dimerización , Escherichia coli/enzimología , Escherichia coli/genética , Glutatión Transferasa/química , Hemina/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Ligandos , Mitocondrias/enzimología , Protoporfirinas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Zea mays/enzimologíaRESUMEN
Inhibitors of p-hydroxyphenylpyruvate dioxygenase (HPPD) are bleaching compounds impairing the formation of colored carotenoids. This activity makes them promising candidates for herbicides. Detailed studies on enzyme-inhibitor complexes or on the binding niche of the enzyme have still to be performed. Enzyme preparation from plants is time-consuming and the yield is poor. This paper describes in relevant detail the preparation of recombinant enzyme from Arabidopsis thaliana with good yield and high specific activity.
Asunto(s)
4-Hidroxifenilpiruvato Dioxigenasa/metabolismo , 4-Hidroxifenilpiruvato Dioxigenasa/genética , Secuencia de Aminoácidos , Arabidopsis/enzimología , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Clonación Molecular , ADN Complementario/genética , ADN de Plantas/genética , Cinética , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
A series of replacement experiments of [(14)C]-triazines, [(14)C]-atrazine and [7-(14)C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine, bound to thylakoids isolated from wild-type and atrazine-resistant Chenopodium album (lambsquarters) were conducted. Replacement experiments of [(14)C]-triazines bound to wild-type Chenopodium thylakoids with non-labeled atrazine and 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine were carried out, to elucidate whether benzylamino-1,3,5-triazines use the same binding niche as atrazine. [(14)C]-Atrazine and [7-(14)C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine bound to wild-type thylakoids were replaced by non-labeled 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine and non-labeled atrazine, respectively. The above two replacements showed mutual competition. To clarify further whether benzylamino-1,3,5-triazines bind at the D1-protein to amino acid residue(s) different from atrazine or not, experiments to replace [7-(14)C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazines bound to atrazine-resistant Chenopodium thylakoids by non-labeled atrazine, 2-(4-bromobenzylamino)-4-methyl-6-trifluoromethyl-1,3,5-triazine, DCMU and DNOC were carried out. Although the bound [7-(14)C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine was difficult to be replaced even with high concentrations of atrazine, [(14)C]-labeled 1,3,5-triazine was competitively replaced by non-labeled 2-(4-bromobenzylamino)-4-methyl-6-trifluoromethyl-1,3,5-triazine, DCMU or DNOC. Thus, 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine herbicides are considered to bind to the same niche at the D1 protein as atrazine, but use amino acid residue(s) different from those involved with atrazine binding.
RESUMEN
In various water depths of the littoral zone of Lake Constance (Bodensee) cyanobacteria of the Synechococcus-type were isolated from biofilms (periphyton) on three natural substrates and an artificial one (unglazed tiles). From one tile three strains of phycoerythrin (PE)-rich Synechococcus spp. were isolated, the first examples of these organisms in the epibenthos. Phylogenetic inference based on the 16S-23S rRNA intergenic spacer (ITS-1) assigned all periphytic isolates to two clusters of the picophytoplankton clade (evolutionary lineage VI of cyanobacteria). The sequence divergence in the ITS-1 was used to design specific PCR primers to allow direct, culture-independent detection and quantification of isolated Synechococcus strains in natural periphytic and pelagic samples. Denaturing gradient gel electrophoresis (DGGE) analysis revealed depth-related differences of Synechococcus spp. distribution on tiles placed in the littoral zone. Synechococcus genotypes were observed which occurred in both the periphyton (on tiles) and in the pelagic picoplankton. A strain with one of these genotypes, Synechococcus sp. BO 8805, was isolated from the pelagic zone in 1988. Its genotype was found on tiles that had been exposed at different water depths in the littoral zone in spring and autumn of the year 2000. Quantitative analysis with a genotype-specific TaqMan probe and real-time Taq nuclease assays (TNA) confirmed its presence in the pelagic zone, although appearance of this and related genotypes was highly irregular and exhibited strong differences between consecutive years. Our results show that the ability to form significant subpopulations in pelagic and periphytic communities exists in three out of four phylogenetic clusters of Synechococcus spp. in Lake Constance. This versatility may be a key feature in the ubiquity of the evolutionary lineage VI of cyanobacteria.
RESUMEN
Chloroacetamide herbicides inhibit very-long-chain fatty acid elongase, and it has been suggested that covalent binding to the active site cysteine of the condensing enzyme is responsible [Pest Manage Sci 56 (2000), 497], but direct evidence was not available. The proposal implied that other condensing enzymes might also be targets, and therefore we have investigated four purified recombinant type III plant polyketide synthases. Chalcone synthase (CHS) revealed a high sensitivity to the chloroacetamide metazachlor, with 50% inhibition after a 10 min pre-incubation with 1-2 molecules per enzyme subunit, and the inactivation was irreversible. Stilbene synthase (STS) inactivation required 20-fold higher amounts, and 4-coumaroyltriacetic acid synthase and pyrone synthase revealed no response at the highest metazachlor concentrations tested. A similar spectrum of differential responses was detected with other herbicides that also inhibit fatty acid elongase (metolachlor and cafenstrole). The data indicate that type III polyketide synthases are potential targets of these herbicides, but each combination has to be investigated individually. The interaction of metazachlor with CHS was investigated by mass spectrometric peptide mapping, after incubation of the enzymes with the herbicides followed by tryptic digestion. A characteristic mass shift and MS/MS sequencing of the respective peptide showed that metazachlor was covalently bound to the cysteine of the active site, and the same was found with STS. This is the first direct evidence that the active site cysteine in condensing enzymes is the primary common target of these herbicides.
Asunto(s)
Acetamidas/metabolismo , Acetamidas/farmacología , Cisteína/metabolismo , Herbicidas/metabolismo , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/metabolismo , Acetamidas/química , Acetiltransferasas/metabolismo , Aciltransferasas/antagonistas & inhibidores , Aciltransferasas/genética , Aciltransferasas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Radioisótopos de Carbono , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Elongasas de Ácidos Grasos , Herbicidas/química , Concentración 50 Inhibidora , Isomerismo , Malonil Coenzima A/análogos & derivados , Malonil Coenzima A/metabolismo , Datos de Secuencia Molecular , Complejos Multienzimáticos/genética , Fragmentos de Péptidos/análisis , Proteínas de Plantas/antagonistas & inhibidores , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
The response of Nostoc commune and Nostoc flagelliforme (terrestrial blue-green algae), grown in their natural habitat, towards rewetting after prolonged drought periods (2 weeds up to five years) has been investigated. In Nostoc flagelliforme, the energy charge (EC) about 0.18 in dry condition increases rapidly (EC=0.7 after 1 h) and more slowly in a second phase (EC=0.8 after 6 h). The total content of AXP (=ATP+ADP+AMP) apparently increases due to de novo synthesis of adenine nucleotides. ATP-build-up after a drought period is probably provided by oxidative phosphorylation. It has been found to be about the same, regardless of whether the foregoing drought period had been extended over 6 months or 5 years.Dry samples of colony mats of N. commune exhibit very low ATP-, but high ADP-contents. Within 6 h after rewetting, the final level of extractable ATP (60-100 nmol/mg chlorophyll) is recovered.
RESUMEN
The response of the terrestrial blue-green algae Nostoc flagelliforme, Nostoc commune, and Nostoc spec. to water uptake has been investigated after a drought period of approximately 2 years. Rapid half-times of rewetting (0.6, 3.3, and 15.5 min, respectively) are found. The surfaceto-mass ratio of the three species is inversely correlated to the speed of water uptake and loss. The ecological relevance of these different time courses is discussed.Respiration starts immediately after a 30-min rewetting period, whereas photosynthetic oxygen evolution reaches its maximum activity after 6 and 8 h with N. commune and N. flagelliforme, respectively. In the dark, recovery of oxygen uptake by N. commune is somewhat impaired, while slightly stimulated with N. flagelliforme. With both species, recovery of photosynthesis is inhibited by darkness.Using colonies kept dry for two years, nitrogenase activity of N. commune attains its maximum 120 to 150 h after rewetting, while only 50 h were needed with algal mats kept dry for two days.Thus, after a 2-year drought period, the physiological sequence of reactivation is respiration-photosynthesis-nitrogen fixation. Respiration and photosynthesis precede growth and are exhibited by existing vegetative cells, whereas recovery of nitrogen fixation is dependent on newly differentiated heterocysts.
RESUMEN
O-[1-Ethyl-2-(3-trifluoromethylphenoxy)]ethyl-N-benzylcarbamate exhibits a marked inhibition of carotenoid biosynthesis. Forty-one analogues were synthesized and assayed for plant-type phytoene desaturase (PDS) and zeta-carotene desaturase (ZDS) inhibition in a cell-free system using recombinant enzymes obtained from Escherichia coli transformants. The target enzyme of all carbamates synthesized in this study is PDS and not ZDS; no inhibition of ZDS was observed using a 10(-4) M inhibitor concentration. Four compounds, O-[1-ethyl-2-(3-trifluoromethylphenoxy)]ethyl-N-(2-phenylethyl)carbamate (23), O-[1-ethyl-2-(3-trifluoromethylphenoxy)]ethyl-N-(2-chlorobenzyl)carbamate (25), O-[1-ethyl-2-(3-trifluoromethylphenoxy)]ethyl-N-(2-chlorobenzyl)carbamate (26), and O-[1-methyl-2-(3-trifluoromethylphenoxy)]ethyl-N-benzylcarbamate (30), were the most potent PDS inhibitors. Their pI(50) values, the negative logarithms of the molar concentration that produces a 50% inhibition, were 7.5, representing the same inhibitory activity as norflurazon. With respect to a structure-activity relationship the oxygen atom of the phenoxy group and a carbamate structure in O-(1-ethyl-2-phenoxy)ethyl-N-aralkylcarbamates studied were found to be essential for strong PDS inhibitors. Also, introduction of an ethyl group at the alpha-position of the ethylene bridge between the phenoxy group and the carbamate was important for a strong PDS inhibitor. Substituents at the 2- and/or 3-position of the phenoxybenzene ring were found to be favorable to a strong PDS inhibition of the analogues.
Asunto(s)
Carbamatos/análisis , Inhibidores Enzimáticos/farmacología , Oxidorreductasas/antagonistas & inhibidores , Carbamatos/farmacología , Carotenoides/biosíntesis , Inhibidores Enzimáticos/química , Piridazinas/farmacología , Proteínas Recombinantes , Relación Estructura-ActividadRESUMEN
At present the use-rate of modern herbicides is in the range of 100-300 g AI ha-1, with a tendency to decline. The low use-rate (ca 10 g AI ha-1) of the original sulfonylurea and cyclic imide herbicides prompted agrochemical scientists to look for even more active compounds which led to the successive discoveries of many new herbicidal acetolactate synthase inhibitors and no less than 18 cyclic imides in the class of protoporphyrinogen-IX oxidase inhibitors in the 1990s. In this paper, mechanisms of action related to function and biosynthesis of chlorophylls, carotenoids, plastoquinone, amino acids, fatty acids and photosynthetic electron transport and other metabolic processes are discussed as plant-specific herbicidal target domains.
Asunto(s)
Herbicidas/farmacología , Proteínas del Complejo del Centro de Reacción Fotosintética/antagonistas & inhibidores , Pigmentos Biológicos/antagonistas & inhibidores , Acetolactato Sintasa/antagonistas & inhibidores , Aminoácidos/antagonistas & inhibidores , Aminoácidos/biosíntesis , Amoníaco/antagonistas & inhibidores , Amoníaco/metabolismo , Carotenoides/antagonistas & inhibidores , Carotenoides/biosíntesis , Pared Celular/efectos de los fármacos , Pared Celular/metabolismo , Celulosa/antagonistas & inhibidores , Celulosa/biosíntesis , Clorofila/antagonistas & inhibidores , Clorofila/biosíntesis , Transporte de Electrón/efectos de los fármacos , Transporte de Electrón/fisiología , Complejos de Proteína Captadores de Luz , Lípidos/antagonistas & inhibidores , Lípidos/biosíntesis , Fotosíntesis/efectos de los fármacos , Fotosíntesis/fisiología , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Pigmentos Biológicos/biosíntesis , Plastoquinona/antagonistas & inhibidores , Plastoquinona/metabolismo , Especificidad de la EspecieRESUMEN
In the present study membrane fatty acids were analyzed to find a link between the biosynthesis inhibition of very-long-chain fatty acids and the phytotoxic effects of herbicidal chloroacetamides. Accordingly, we have isolated membranes of cucumber seedlings (Cucumis sativus) by two-phase partitioning and analyzed their fatty acid content. Saturated VLCFAs ranging from C20 to C26 were found in high amounts (22%) in the plasma membrane fraction. Non-modified VLCFAs were predominantly present in phospholipids, while saturated 2-hydroxylated VLCFAs were identified in cerebrosides. Treatment of intact seedlings with chloroacetamides markedly reduced the VLCFA content in the plasma membrane. This result could be specified by fatty-acid labeling using [14C]malonate as a substrate for fatty acid elongation. De novo incorporation of VLCFAs into the plasma membrane and into microsomal membranes, respectively, was severely impaired by chloroacetamides with I50 values between 10 to 100 nM. These results confirm the previous finding that chloroacetamides inhibit VLCFA biosynthesis localized in the microsomes (Böger et al., Pest Manage. Sci. 56, 497-508, 2000). The direct consequence of this inhibition is a strong decrease of VLCFAs required as constituents of the plasma membrane and the substitution by shorter acyl chains. Apparently, physical properties and function of the plasma membrane are affected eventually leading to death of the plant.
Asunto(s)
Acetamidas/farmacología , Membrana Celular/efectos de los fármacos , Lípidos de la Membrana/metabolismo , Membrana Celular/metabolismo , Cucumis sativus , Ácidos Grasos/metabolismo , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/metabolismo , Fosfolípidos/metabolismo , Plastidios/metabolismoRESUMEN
Reports on stress response of tobacco expressing a bacterial glutathione reductase (GR) do not agree. To clarify this situation we investigated several parameters using the tobacco BelW3 line and its transformant BelW3gor expressing an E. coli GR. This alteration in the activity of GR led to an ambiguous modification of the antioxidative system. In contrast to the wild type, the transgenic tobacco suffered lipid peroxidation under moderate light intensities, while it was found to be more resistant towards oxidative stress induced by paraquat or hydrogen peroxide. Transcript levels for violaxanthin deepoxidase and cytosolic Cu-Zn-superoxide dismutase were strongly reduced in BelW3gor plants as compared to BelW3.
Asunto(s)
Antioxidantes/metabolismo , Regulación de la Expresión Génica de las Plantas , Glutatión Reductasa/genética , Nicotiana/enzimología , Plantas Modificadas Genéticamente/química , Catalasa/genética , Escherichia coli/enzimología , Escherichia coli/genética , Glutatión Reductasa/metabolismo , Luz , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/crecimiento & desarrollo , ARN Mensajero/genética , ARN de Planta/genética , ARN de Planta/aislamiento & purificación , Nicotiana/genéticaRESUMEN
The first elongation step to form very-long-chain fatty acids (VLCFAs) is catalyzed by the VLCFA-synthase. CoA-activated fatty acids react with malonyl-CoA to condense a C2-unit. As shown with recombinant enzyme this reaction is specifically inhibited by chloroacetamide herbicides. The inhibition is alleviated when the inhibitor (e.g. metazachlor) is incubated together with adequate concentrations of the substrate (e.g. oleoyl-CoA). Malonyl-CoA has no influence. However, once a chloroacetamide has been tightly bound to the synthase after an appropriate time it cannot be displaced anymore by the substrate. In contrast, oleoyl-CoA, is easily removed from the synthase by metazachlor. The irreversible binding of the chloroacetamides and their competition with the substrate explains the very low half-inhibition values of 10(-8) M and below. Chiral chloroacetamides like metolachlor or dimethenamid give identical results. However, only the (S)-enantiomers are active.
Asunto(s)
Acetamidas/farmacología , Arabidopsis/enzimología , Coenzima A Ligasas/antagonistas & inhibidores , Proteínas de Arabidopsis/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Herbicidas/farmacología , Cinética , Malonil Coenzima A/metabolismo , Plásmidos , Saccharomyces cerevisiae/enzimologíaRESUMEN
The effect of 2-benzylamino-1,3,5-triazines on photosynthetic electron transport (PET) was measured with thylakoids isolated from atrazine-resistant, wild-type Chenopodium album, and spinach to find novel 1,3,5-triazine herbicides bearing a strong PET inhibition. The PET inhibition assay with Chenopodium (wild-type and resistant), yielded a resistance ratio (R/W = I50 (resistant)/I50 (wild-type)) of 324 for atrazine while for benzylamino-1,3,5-triazine derivatives of diamino-1,3,5-triazines a R/W of 11 to 160 was found. The compounds having a benzylamino group at one of the amino groups in the diamino-1,3,5-triazines have a resistant ratio down to one half to 1/30 of the atrazine value. The average resistance ratio of 21 benzylamino derivatives of monoamino-1,3,5-triazines was found to be about 4.0. The inhibition of 21 benzylamino-1,3,5-triazines assayed with atrazine-resistant Chenopodium thylakoids, indicated by pI50 (R)-values, correlated well with the PET inhibition pI50 (W) of wild-type thylakoids from Chenopodium.
Asunto(s)
Atrazina/farmacología , Chenopodium album/fisiología , Fotosíntesis/efectos de los fármacos , Tilacoides/efectos de los fármacos , Triazinas/farmacología , Chenopodium album/química , Chenopodium album/efectos de los fármacos , Resistencia a Medicamentos , Transporte de Electrón/efectos de los fármacos , Herbicidas/farmacología , Triazinas/química , Triazinas/aislamiento & purificaciónRESUMEN
Indanofan and its analogs inhibited the elongation of stearoyl- or arachidoyl-CoA by [2-14C]-malonyl-CoA in leek microsomes from Allium porrum. Although the precise mode of interaction of indanofan at the molecular level is not completely clarified by the present study, it is concluded that indanofan and analogs act as inhibitor of the elongase enzyme involved in de novo biosynthesis of fatty acids with an alkyl chain longer than C18, called very-long-chain fatty acids (VLCFAs). For a strong inhibition of VLCFA formation chloro substituents at the benzene ring and the oxirane group were necessary. Furthermore, the greenhouse test showed strong activity for indanofan and its analogs, and the scores coincided with cell-free elongation inhibition. The cell-free assay, however, failed to indicate any activity for an analog having a methylene instead of the oxirane group, while both Digitaria ciliaris and Echinochloa oryzicola were killed with 1 kg a.i./ha. This finding cannot be discussed because the applied use rate of 1 kg a.i./ha is too high to allow for a score differentiation. For high concentrations of this compound additional unknown inhibitory effects may be involved besides fatty acid elongation.
Asunto(s)
Allium/metabolismo , Clorobencenos/farmacología , Ácidos Grasos/biosíntesis , Herbicidas/farmacología , Indanos/farmacología , Acilcoenzima A/metabolismo , S-Maloniltransferasa de la Proteína Transportadora de Grupos Acilo , Aciltransferasas/antagonistas & inhibidores , Radioisótopos de Carbono , Sistema Libre de Células , Ácidos Grasos/química , Malonil Coenzima A/metabolismo , Ácidos Esteáricos/metabolismo , Relación Estructura-ActividadRESUMEN
Quantitative Taq nuclease assays (TNAs) (TaqMan PCR), nested PCR in combination with denaturing gradient gel electrophoresis (DGGE), and epifluorescence microscopy were used to analyze the autotrophic picoplankton (APP) of Lake Constance. Microscopic analysis revealed dominance of phycoerythrin (PE)-rich Synechococcus spp. in the pelagic zone of this lake. Cells passing a 3- micro m-pore-size filter were collected during the growth period of the years 1999 and 2000. The diversity of PE-rich Synechococcus spp. was examined using DGGE to analyze GC-clamped amplicons of a noncoding section of the 16S-23S intergenic spacer in the ribosomal operon. In both years, genotypes represented by three closely related PE-rich Synechococcus strains of our culture collection dominated the population, while other isolates were traced sporadically or were not detected in their original habitat by this method. For TNAs, primer-probe combinations for two taxonomic levels were used, one to quantify genomes of all known Synechococcus-type cyanobacteria in the APP of Lake Constance and one to enumerate genomes of a single ecotype represented by the PE-rich isolate Synechococcus sp. strain BO 8807. During the growth period, genome numbers of known Synechococcus spp. varied by 2 orders of magnitude (2.9 x 10(3) to 3.1 x 10(5) genomes per ml). The ecotype Synechococcus sp. strain BO 8807 was detected in every sample at concentrations between 1.6 x 10(1) and 1.3 x 10(4) genomes per ml, contributing 0.02 to 5.7% of the quantified cyanobacterial picoplankton. Although the quantitative approach taken in this study has disclosed several shortcomings in the sampling and detection methods, this study demonstrated for the first time the extensive internal dynamics that lie beneath the seemingly arbitrary variations of a population of microbial photoautotrophs in the pelagic habitat.
Asunto(s)
Cianobacterias/fisiología , ADN Bacteriano/análisis , Calibración , Recuento de Colonia Microbiana , Cianobacterias/genética , Desoxirribonucleasas/metabolismo , Electroforesis , Filtración , Técnicas Genéticas , Variación Genética , Genoma Bacteriano , Ficoeritrina/metabolismo , Microbiología del AguaRESUMEN
Eight 5-arylimino-3,4-tetramethylene-1,3,4-thiadiazolidine-2-thiones and eight 4-aryl-1,2- tetramethylene-1,2,4-triazolidine-3,5-dithiones were synthesized and their phytotoxic activities were investigated using sawa millet (Echinochloa utilis), green microalgae (Scenedesmus acutus) and protoporphyrinogen-IX oxidase isolated from etiolated corn (Zea mays) seedlings. 5-Arylimino-3,4-tetramethylene-1,3,4-thiadiazolidine-2-thiones showed strong phytotoxic activities and the same herbicidal mode of action as known for peroxidizing herbicides. 5-Arylimino-3,4-tetramethylene-1,3,4-thiadiazolidine-2-thiones were not or very little converted into 4-aryl-1,2-tetram ethylene-1,2,4-triazolidine-3,5-dithiones either with E. utilis seedlings present for 7 days, with S. acutus cells, or using glutathione 5-transferase (GST) and glutathione (GSH). The phytotoxic activities of 4-aryl-1,2-tetram ethylene-1,2,4-triazolidine- 3,5-dithiones were stronger than those of 5-arylimino-3,4-tetram ethylene-1,3,4-thiadiazolidine- 2-thiones [cf. Sato, Y., et al., Z. Naturforsch. 49c, 49-56 (1994)].