Isolation and characterization of an s-ethyl-N,N-dipropylthiocarbamate-degrading Arthrobacter strain and evidence for plasmid-associated s-ethyl-N,N-dipropylthiocarbamate degradation.

Arthrobacter sp. strain TE1 isolated from s-ethyl-N,N-dipropylthiocarbamate (EPTC)-exposed soil degraded this herbicide effectively and could grow on EPTC as the sole carbon source. TE1 harboured four plasmids of 65.5, 60, 50.5, and 2.5 megadaltons. Spontaneous mutants unable to degrade EPTC arose at a high frequency, and this was further increased by treatment of the culture with acridine orange or incubation at high temperature. All EPTC degradation-deficient (E-) mutants lacked the 50.5-megadalton plasmid. This plasmid could be transferred from TE1 to E- mutants by conjugation, resulting in the restoration of EPTC-degrading ability to the mutants.     

Cloning of the genes for degradation of the herbicides EPTC (S-ethyl dipropylthiocarbamate) and atrazine from Rhodococcus sp. strain TE1.

The degradation of the herbicides EPTC (S-ethyl dipropylthiocarbamate) and atrazine (2-chloro-4-ethyl-amino-6-isopropylamino-1,3,5-triazine) is associated with an indigenous plasmid in Rhodococcus sp. strain TE1. Plasmid DNA libraries of Rhodococcus sp. strain TE1 were constructed in a Rhodococcus-Escherichia coli shuttle vector, pBS305, and transferred into Rhodococcus sp. strain TE3, a derivative of Rhodococcus sp. strain TE1 lacking herbicide degradation activity, to select transformants capable of growing on EPTC as the sole source of carbon (EPTC+). Analysis of plasmids from the EPTC+ transformants indicated that the eptA gene, which codes for the enzyme required for EPTC degradation, residues on a 6.2-kb KpnI fragment. The cloned fragment also harbored the gene required for atrazine N dealkylation (atrA). The plasmid carrying the cloned fragment could be electroporated into a number of other Rhodococcus strains in which both eptA and atrA were fully expressed. No expression of the cloned genes was evident in E. coli strains. Subcloning of the 6.2-kb fragment to distinguish between EPTC- and atrazine-degrading genes was not successful.     

Metabolism of the herbicide atrazine by Rhodococcus strains.

Rhodococcus strains were screened for their ability to degrade the herbicide atrazine. Only rhodococci that degrade the herbicide EPTC (s-ethyl-dipropylthiocarbamate) metabolized atrazine. Rhodococcus strain TE1 metabolized atrazine under aerobic conditions to produce deethyl- and deisopropylatrazine, which were not degraded further and which accumulated in the incubation medium. The bacterium also metabolized the other s-triazine herbicides propazine, simazine, and cyanazine. The N dealkylation of triazine herbicides by Rhodococcus strain TE1 was associated with a 77-kb plasmid previously shown to be required for EPTC degradation.     

The interaction of protectants with EPTC on field bean, and tri-allate on wheat

Protectants (antidotes) were tested for their potential to protect field beans (Vicia faba L.) from EPTC damage, or wheat (Triticum aestivum L.) from triallate damage. For both crops there was considerable variation in the degree of protection shown from similar treatments in different experiments. For field bean, a seed treatment of 1,8-naphthalic anhydride (NA) at 5 mg/g seed gave some protection from EPTC applied pre-planting at 4–8 kg a.i./ha but not in all experiments. NA also caused marked chlorosis of the foliage. N, N-diallyl-2, 2-dichloroacetamide (R25788) at 20 mg/g seed severely damaged field bean in the absence of herbicide but 5 mg/g gave comparable protection from EPTC to that given by NA and did not cause chlorosis. Mixing R25788 with EPTC in the spray tank gave reduced protection. In a single experiment R4115 (chemistry undisclosed) gave some protection against EPTC damage. For wheat, a seed treatment of 5–20 mg/g NA sometimes countered damage from tri-allate applied pre-planting at 1 kg a.i./ha but not generally from higher doses. R25788 sometimes protected from weight loss due to tri-allate at 1 kg a.i./ha but not from damage symptoms, whereas R4115 at 20 mg/g seed alleviated these symptoms but did not prevent weight loss. R25788 at 4 kg a.i./ha mixed in the spray tank with the herbicide partially reduced weight loss and damage symptoms from a dose of 2 kg a.i./ha. Some treatments of R29148 gave complete protection from tri-allate at 1 kg a.i./ha. The results are discussed in the context of the full data from the two series of experiments.     

Managing lepidopteran pests in cabbage with herbicide-induced resistance, in combination with a pyrethroid insecticide

S-ethyldipropylthiocarbamate (EPTC) applied as a soil treatment or over-the-top spray on cabbage plants (Brassica oleracea L.) caused the leaves to turn ‘glossy’ for as long as 30 days. EPTC-induced glossy plants were damaged significantly less than untreated plants by diamondback moth,Plutella xylostella (L.), imported cabbage worm,Pieris rapae (L.), and cabbage looper,Trichoplusia ni (Hbn.). Reductions in damage were equivalent to those obtained from treatment with permethrin. When used in combination with permethrin, EPTC provided additive control of damage by these pests. Our calculations show EPTC-induced resistance to be cost-effective. This use of EPTC has several limitations, however. Younger plants (<9 leaves) were killed or injured by the herbicide. The growth of older plants was not affected, but plants did not become glossy for ca. 10 days after they were treated with EPTC. The crop must be protected with insecticides until the plants are mature enough to treat with EPTC, and until treated plants become glossy. In addition, since the glossy trait is only effective against first instar larvae, populations of later instars on glossy plants must be reduced with an application of insecticide. Finally, EPTC formulations are water-soluble and can be washed away from the plants by heavy rains and irrigation, which may make this use of EPTC impractical in some situations. Where its use is practical, and the indicated precautions are taken, EPTC-induced resistance could reduce dependence on chemical insecticides and reduce selection for insecticide resistance in diamondback moth.     

The Arabidopsis her1 mutant implicates GABA in E-2-hexenal responsiveness

When wounded or attacked by herbivores or pathogens, plants produce a blend of six-carbon alcohols, aldehydes and esters, known as C6-volatiles. Undamaged plants, when exposed to C6-volatiles, respond by inducing defense-related genes and secondary metabolites, suggesting that C6-volatiles can act as signaling molecules regulating plant defense responses. However, to date, the molecular mechanisms by which plants perceive and respond to these volatiles are unknown. To elucidate such mechanisms, we decided to isolate Arabidopsis thaliana mutants in which responses to C6-volatiles were altered. We observed that treatment of Arabidopsis seedlings with the C6-volatile E-2-hexenal inhibits root elongation. Among C6-volatiles this response is specific to E-2-hexenal, and is not dependent on ethylene, jasmonic and salicylic acid. Using this bioassay, we isolated 18 E-2-hexenal-response (her) mutants that showed sustained root growth after E-2-hexenal treatment. Here, we focused on the molecular characterization of one of these mutants, her1. Microarray and map-based cloning revealed that her1 encodes a gamma-amino butyric acid transaminase (GABA-TP), an enzyme that degrades GABA. As a consequence of the mutation, her1 plants accumulate high GABA levels in all their organs. Based on the observation that E-2-hexenal treatment induces GABA accumulation, and that high GABA levels confer resistance to E-2-hexenal, we propose a role for GABA in mediating E-2-hexenal responses.     

Protein kinase casein kinase 2-mediated upregulation of N-cadherin confers anoikis resistance on esophageal carcinoma cells

Previously, we reported that high PKCK2 activity could protect cancer cells from death receptor-mediated apoptosis through phosphorylation of procaspase-2. Because anoikis is another form of apoptosis, we asked whether PKCK2 could similarly confer resistance to anoikis on cancer cells. Human esophageal squamous cancer cell lines with high PKCK2 activity (HCE4 and HCE7) were anoikis-resistant, whereas cell lines with low PKCK2 activity (TE2 and TE3) were anoikis-sensitive. Because the cells showed different sensitivity to anoikis, we compared the expression of cell adhesion molecules between anoikis-sensitive TE2 and anoikis-resistant HCE4 cells using cDNA microarray. We found that E-cadherin is expressed only in TE2 cells; whereas N-cadherin is expressed instead of E-cadherin in HCE4 cells. To examine whether PKCK2 activity could determine the type of cadherin expressed, we first increased intracellular PKCK2 activity in TE2 cells by overexpressing the PKCK2α catalytic subunit using lentivirus and found that high PKCK2 activity could switch cadherin expression from type E to N and confer anoikis resistance. Conversely, a decrease in PKCK2 activity in HCE4 cells by knockdown of PKCK2α catalytic subunit using shRNA induced N- to E-cadherin switching and the anoikis-resistant cells became sensitive. In addition, N-cadherin expression correlated with PKB/Akt activation and increased invasiveness. We conclude that high intracellular PKCK2 activity confers anoikis resistance on esophageal cancer cells by inducing E- to N-cadherin switching. Mol Cancer Res; 10(8); 1032-8. ?2012 AACR.     

Proteinase Activity during Tracheary Element Differentiation in Zinnia Mesophyll Cultures

The zinnia (Zinnia elegans) mesophyll cell culture tracheary element (TE) system was used to study proteinases active during developmentally programmed cell death. Substrate-impregnated gels and single-cell assays revealed high levels of proteinase activity in differentiating TEs compared with undifferentiated cultured cells and expanding leaves. Three proteinases (145, 28, and 24 kD) were exclusive to differentiating TEs. A fourth proteinase (59 kD), although detected in extracts from all tissues examined, was most active in differentiating TEs. The 28- and 24-kD proteinases were inhibited by thiol proteinase inhibitors, leupeptin, and N-[N-(L-3-trans-carboxirane-2-carbonyl)-L-leucyl]-agmatine (E-64). The 145- and 59-kD proteinases were inhibited by the serine proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF). Extracts from the TE cultures contained sodium dodecyl sulfate-stimulated proteolytic activity not detected in control cultures. Sodium dodecyl sulfate-stimulated proteolysis was inhibited by leupeptin or E-64, but not by PMSF. Other tissues, sucrose-starved cells and cotyledons, that contain high levels of proteolytic activity did not contain TE-specific proteinases, but did contain higher levels of E-64-sensitive activities migrating as 36- to 31-kD enzymes and as a PMSF-sensitive 66-kD proteinase.     

Impact of agricultural land use on aquatic insect assemblages in the Garonne river catchment (SW France)

The impact of agricultural land use on the composition and structure of aquatic insect assemblages (i.e., taxa of Ephemeroptera, Plecoptera, Trichoptera, and Coleoptera (EPTC)) was investigated in tributary streams of the Garonne river basin, southern France. The self-organizing map (SOM) method was applied to compare both instream environmental conditions and EPTC assemblages between forest and agricultural streams. According to the SOM model, the study sites were classified into three main clusters corresponding to distinct EPTC assemblages. The SOM cluster associated with most of the agricultural sites had lower EPTC species richness and diversity. This cluster was also characterized by high levels of total dissolved solids, nitrate (NO₃), and chemical oxygen demand. Overall, our study shows that agricultural streams when compared with forest streams had lower biological integrity. In accordance with the European Water Framework Directive, our results indicate that the sites most impacted by agricultural land use should be restored and that the least-impacted forest sites could serve as reference conditions.     

Ganglioside Composition of Normal and Mutant Mouse Embryos

The enrichment of gangliosides in neuronal membranes suggests that they play an important role in CNS development. We recently found a marked tetrasialoganglioside deficiency in twl/twl mutant mouse embryos at embryonic day (E)-11. The recessive twl/twl mutants die at embryonic ages E-9 to E-18 from failed neural differentiation in the ventral portion of the neural tube. In the present study, we examined the composition and distribution of gangliosides in twl/twl mutant mouse embryos at E-12. The total ganglioside sialic acid concentration was significantly lower in the mutants than in normal (+/-) embryos. The mutants also expressed significant deficiencies of gangliosides in the "b" metabolic pathway (GD3, GD1b, GT1b, and GQ1b) and elevations in levels of gangliosides in the "a" metabolic pathway (GM3, GM2, GM1, and GD1a). These findings suggest that the mutants have a partial deficiency in the activity of a specific sialyltransferase in the b pathway. Regional ganglioside distribution was also studied in E-12 normal mouse embryos. The ganglioside composition in heads and bodies was similar to each other and to whole embryos. Total ganglioside concentration and the distribution of b pathway gangliosides were significantly higher in neural tube regions than in nonneural tube regions. These findings suggest that b pathway gangliosides accumulate in differentiating neural cells and that the deficiency of these gangliosides in the twl/twl mutants is closely associated with failed neural differentiation.     

Studies on gramicidin S synthetase. Purification and properties of the light enzyme obtained from some mutants of Bacillus brevis.

The phenylalanine-activating and/or-racemizing enzyme, i.e., the light enzyme, of gramicidin S synthetase was purified to a homogenous state by D-phenylalanine-Sepharose 4B chromatography from a wild and some gramicidin S-lacking mutant strains of Bacillus brevis. The light enzyme obtained from a mutant strain E-1 could activate phenylalanine but not racemize it, and had no phenylalanine-dependent ATP-[14C]AMP exchange activity, whereas the same enzyme obtained from other mutants and the wild strain had all three activities. Furthermore, the light enzyme of the mutant E-1 could form only acid-labile enzyme-bound phenylalanine, while the same fraction of the wild strain carried half of the enzyme-bound phenylalanine as acid-labile adenylate and half as a acid-stable thioester. These results suggest that the thiol site of the light enzyme of mutant E-1 might be damaged.     

In vitro regeneration of loblolly pine and random amplified polymorphic DNA analyses of regenerated plantlets

 Adventitious buds were induced from organogenic callus derived from mature zygotic embryos of three lines (E-311, E-440, and E-822) of loblolly pine (Pinus taeda L.) within 27 weeks of culture. The influence of cytokinins, silver nitrate, and low-temperature treatment on the differentiation of adventitious buds was analyzed. Elongation of adventitious buds was achieved on TE medium supplemented with 0.5 mg/l indole-3-butyric acid (IBA) and 1 mg/l 6-benzyladenine (BA). After adventitious shoots had rooted on TE medium supplemented with 0.5 mg/l IBA, 2 mg/l BA, and 0.5 mg/l gibberellic acid, 498 regenerated plantlets were transferred to a perlite:peatmoss:vermiculite (1 :>: 1) soil mixture; 351 of these survived in the field. Total DNA was extracted from 21 regenerated plantlets randomly chosen from the 151 regenerated plantlets of line E-822. Random amplified polymorphic DNA (RAPD) analysis using 80 arbitrary oligonucleotide 10-mers showed that 21 primers gave 107 clear reproducible bands, with the amplification products being monomorphic for all of the plantlets of line E-822 tested. A total of 2,247 bands obtained from these studies exhibited no aberration in RAPD banding patterns among the tested plantlets. These results suggest that somatic organogenesis can be used for clonal micropropagation of some lines of loblolly pine without the fear of the appearance of unwanted somaclonal variants. Received: 5 August 2000 / Revision received: 5 September 2000 / Accepted: 10 October 2000     

The hepatic glutathione transferases of the male little skate, Raja erinacea

Five cytosolic glutathione transferases were isolated from the liver of the male little skate, Raja erinacea, a marine elasmobranch. They were designated E-1 through E-5 in order of their elution from a DEAE-cellulose column with a 0 to 100 mM KCl gradient in 0.01 M Tris (pH 8.0). Each eluted peak of glutathione transferase activity, after concentration, was applied to an affinity column prepared by reaction of epoxy-activated Sepharose 6B with glutathione (GSH). Elution of the various glutathione transferases from this column with GSH resulted in the further purification of each enzyme; the major glutathione transferase, E-4 and E-1, were purified to apparent homogeneity by this procedure. Skate glutathione transferase E-4 is dimeric and the subunits are either very similar or identical in molecular weight (about 26 000 daltons). Enzymes E-2 through E-5 were acidic proteins (pI less than 7.0) and had high specific glutathione transferase activity (0.3--12 mumol/min/mg protein) with benzo[a]pyrene 4,5-oxide (BPO) as substrate, whereas the other enzyme (E-1) had low activity (0.01 mumol/min/mg) with BPO and a basic pI (greater than 9.5). Bilirubin and hematin, non-substrate ligands, bound tightly to homogeneous E-4, with dissociation constants in the micromolar range.     

Prevalence, development, and molecular mechanisms of bacteriocin resistance in Campylobacter

Bacteriocins (BCNs) are antimicrobial peptides produced by bacteria with narrow or broad spectra of antimicrobial activity. Recently, several unique anti-Campylobacter BCNs have been identified from commensal bacteria isolated from chicken intestines. These BCNs dramatically reduced C. jejuni colonization in poultry and are being directed toward on-farm control of Campylobacter. However, no information concerning prevalence, development, and mechanisms of BCN resistance in Campylobacter exists. In this study, susceptibilities of 137 C. jejuni isolates and 20 C. coli isolates to the anti-Campylobacter BCNs OR-7 and E-760 were examined. Only one C. coli strain displayed resistance to the BCNs (MIC, 64 μg/ml), while others were susceptible, with MICs ranging from 0.25 to 4 μg/ml. The C. coli mutants resistant to BCN OR-7 also were obtained by in vitro selection, but all displayed only low-level resistance to OR-7 (MIC, 8 to 16 μg/ml). The acquired BCN resistance in C. coli could be transferred at intra- and interspecies levels among Campylobacter strains by biphasic natural transformation. Genomic examination of the OR-7-resistant mutants by using DNA microarray and random transposon mutagenesis revealed that the multidrug efflux pump CmeABC contributes to both intrinsic resistance and acquired resistance to the BCNs. Altogether, this study represents the first report of and a major step forward in understanding BCN resistance in Campylobacter, which will facilitate the development of effective BCN-based strategies to reduce the Campylobacter loads in poultry.     

Ganglioside abnormalities associated with failed neural differentiation in a T-locus mutant mouse embryo

The T-locus on mouse chromosome 17 contains a number of mutations that disrupt cellular differentiation and embryonic development. Because of their purported role in neuronal differentiation and brain development, gangliosides were studied in mouse embryos homozygous for two T-locus mutations: T and twl. Mice homozygous for the dominant T mutation die from failed mesodermal differentiation in the notochord, whereas mice homozygous for the recessive twl mutation die from failed neural differentiation in the ventral portion of the neural tube. No major ganglioside abnormalities were found in T/T mutant embryos at Embryonic Day 10 (E-10). In contrast, E-11 twl/twl mutants expressed a marked deficiency of the tetrasialoganglioside GQ1. Since this ganglioside migrates with GQ1b in three different thin-layer solvent systems, it may have the same structure as GQ1b. To gain insight into regional distribution, gangliosides were examined in head regions and body regions of normal (+/+) E-11 embryos. The ganglioside composition of these regions was the same as that of the whole embryo, with GM3 and GD3 comprising about 75% of the total ganglioside distribution. Moreover, N-acetylneuraminic acid was the only sialic acid species detectable in the E-10 and the E-11 embryos. These findings indicate that N-acetylneuraminic acid-containing gangliosides are synthesized actively in E-10 and E-11 mouse embryos and also suggest that the GQ1 deficiency in the twl/twl mutants is closely associated with failed neural differentiation.     

Type II Thioesterase ScoT, Associated with Streptomyces coelicolor A3(2) Modular Polyketide Synthase Cpk, Hydrolyzes Acyl Residues and Has a Preference for Propionate

Type II thioesterases (TE IIs) were shown to maintain the efficiency of polyketide synthases (PKSs) by removing acyl residues blocking extension modules. However, the substrate specificity and kinetic parameters of these enzymes differ, which may have significant consequences when they are included in engineered hybrid systems for the production of novel compounds. Here we show that thioesterase ScoT associated with polyketide synthase Cpk from Streptomyces coelicolor A3(2) is able to hydrolyze acetyl, propionyl, and butyryl residues, which is consistent with its editing function. This enzyme clearly prefers propionate, in contrast to the TE IIs tested previously, and this indicates that it may have a role in control of the starter unit. We also determined activities of ScoT mutants and concluded that this enzyme is an α/β hydrolase with Ser90 and His224 in its active site.     

Extending transfer entropy improves identification of effective connectivity in a spiking cortical network model

Transfer entropy (TE) is an information-theoretic measure which has received recent attention in neuroscience for its potential to identify effective connectivity between neurons. Calculating TE for large ensembles of spiking neurons is computationally intensive, and has caused most investigators to probe neural interactions at only a single time delay and at a message length of only a single time bin. This is problematic, as synaptic delays between cortical neurons, for example, range from one to tens of milliseconds. In addition, neurons produce bursts of spikes spanning multiple time bins. To address these issues, here we introduce a free software package that allows TE to be measured at multiple delays and message lengths. To assess performance, we applied these extensions of TE to a spiking cortical network model (Izhikevich, 2006) with known connectivity and a range of synaptic delays. For comparison, we also investigated single-delay TE, at a message length of one bin (D1TE), and cross-correlation (CC) methods. We found that D1TE could identify 36% of true connections when evaluated at a false positive rate of 1%. For extended versions of TE, this dramatically improved to 73% of true connections. In addition, the connections correctly identified by extended versions of TE accounted for 85% of the total synaptic weight in the network. Cross correlation methods generally performed more poorly than extended TE, but were useful when data length was short. A computational performance analysis demonstrated that the algorithm for extended TE, when used on currently available desktop computers, could extract effective connectivity from 1 hr recordings containing 200 neurons in ～5 min. We conclude that extending TE to multiple delays and message lengths improves its ability to assess effective connectivity between spiking neurons. These extensions to TE soon could become practical tools for experimentalists who record hundreds of spiking neurons.     

Are aquatic insect species sensitive to banana plant cultivation?

Ephemeroptera, Plecoptera, Trichoptera and Coleoptera (EPTC) insect fauna were collected in ten streams located in the southeastern state of São Paulo (five located in areas with banana cultivation and five located in preserved areas). Specimens were collected during October and November 2005, using a Surber sampler and network D (0.25 mm mesh size) in areas of rapids and backwaters. The organisms collected were identified to genus level, except for some small or damaged specimens that remained at the family level. In total, 1812 individuals of EPTC were identified, in which 1105 organisms were from streams in areas of banana cultivation and 706 from streams in preserved areas. Heterelmis (Elmidae) was dominant in both sets of streams. In preserved streams, Hexacylloepus (Elmidae), Tupiperla and Paragrypopteryx (Gripopterygidae) and Nectopsyche (Leptoceridae) also had high participation. Leptonema (Hydropsychidae) and Xenelmis (Elmidae) were dominant in streams located in areas of banana cultivation. The analyses of indicator species point out to five taxa on preserved streams and two taxa in the banana streams. The forested streams had higher richness and diversity of EPTC than banana plantation streams. Correspondence analysis point for two groups, one gathered in the streams near the banana plantation and the other in the preserved streams. The similarity test (ANOSIM) pointed to significant differences (P < 0.05) between these groups. This agricultural activity seems to influence the EPTC community structure in low order streams in the Atlantic Forest region. The identification of groups at the EPTC genus level was important to point out the differences between the fauna of streams impacted by banana cultivation and to establish indicator species.     

Insights into channel architecture and substrate specificity from crystal structures of two macrocycle-forming thioesterases of modular polyketide synthases

Modular polyketide synthases (PKSs) synthesize the polyketide cores of pharmacologically important natural products such as erythromycin and picromycin. Understanding PKSs at high resolution could present new opportunities for chemoenzymatic synthesis of complex molecules. The crystal structures of macrocycle-forming thioesterase (TE) domains from the picromycin synthase (PICS) and 6-deoxyerythronolide B synthase (DEBS) were determined to 1.8-3.0 A with an R(crys) of 19.2-24.4%, including three structures of PICS TE (crystallized at pH 7.6, 8.0, and 8.4) and a second crystal form of DEBS TE. As predicted by the previous work on DEBS TE [Tsai, S. C., et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 14808-14813], PICS TE contains an open substrate channel and a hydrophobic dimer interface. Notwithstanding their similarity, the dimer interfaces and substrate channels of DEBS TE and PICS TE reveal key differences. The structural basis for the divergent substrate specificities of DEBS TE and PICS TE is analyzed. The size of the substrate channel increases with increasing pH, presumably due to electrostatic repulsion in the channel at elevated pH. Together, these structures support previous predictions that macrocycle-forming thioesterases from PKSs share the same protein fold, an open substrate channel, a similar catalytic mechanism, and a hydrophobic dimer interface. They also provide a basis for the design of enzymes capable of catalyzing regioselective macrocyclization of natural or synthetic substrates. A series of high-resolution snapshots of a protein channel at different pHs is presented alongside analysis of channel residues, which could help in the redesign of the protein channel architecture.