Field applications of zein as a precise nanoscale delivery system for methoxyfenozide.

When insecticides are applied in the environment, much of the product does not reach the target pest. Biopolymeric nanoparticles as nanocarriers have the potential to improve insecticide efficacy by improving absorption, coverage, and permeability while protecting the insecticide active ingredient from abiotic conditions and extending efficacy through controlled release. We conducted a series of experiments using a biopolymeric nanoparticle synthesized from zein, a biodegradable maize protein, to compare efficacy of a nanodelivered hydrophobic insect growth regulator (methoxyfenozide) against a commercial formulation. Positively charged zein nanoparticles (empty and loaded with methoxyfenozide) were compared to the formulated product, Intrepid 2F, as a foliar spray in-field on soybean. Chrysodeixis includens (Walker) was used as a model and was fed sprayed soybean leaves to evaluate efficacy of the tested foliar products over time. A separate set of leaves was sampled to measure residue levels of methoxyfenozide (MFZ) over time following foliar application using QuEChERS extraction and high-resolution liquid chromatography-mass spectrometry. Regression analysis found no differences in mortality slopes between positively charged zein nanoparticles loaded with methoxyfenozide [(+)ZNP(MFZ)] and Intrepid 2F, suggesting comparable efficacy of the synthesized nanoparticles to a commercial product. Higher concentrations of MFZ were present in (+)ZNP(MFZ)-treated in leaf tissue at 3 d following spray when compared to Intrepid 2F. The multiyear study results demonstrate that nanoparticles loaded with MFZ are comparable to Intrepid 2F under field conditions, with potential short-term benefits.

Endophyte-enhanced phytoremediation of DDE-contaminated using Cucurbita pepo: A field trial.

Although the use of the pesticide 2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane (DDT) was banned from the mid-1970s, its most abundant and recalcitrant degradation product, 2,2-bis(p-chlorophenyl)-1,1-dichloro-ethylene (DDE), is still present in terrestrial and aquatic ecosystems worldwide. Zucchini (Cucurbita pepo ssp. pepo) has been shown to accumulate high concentrations of DDE and was proposed for phytoremediation of contaminated soils. We performed a field trial covering a full plant life cycle. C. pepo plants inoculated with the plant growth-promoting endophytic strains Sphingomonas taxi UH1, Methylobacterium radiotolerans UH1, Enterobacter aerogenes UH1, or a consortium combining these 3 strains were grown on a DDE-contaminated field for 100 days. The effects of these inoculations were examined at both the plant level, by evaluating plant weight and plant DDE-content, and at the level of the cultivable and total endophytic communities. Inoculating plants with S. taxi UH1, M. radiotolerans UH1, and the consortium increased plant weight. No significant effects of the inoculations were observed on DDE-concentrations in plant tissues. However, the amount of DDE accumulated by C. pepo plants per growing season was significantly higher for plants that were inoculated with the consortium of the 3 strains. Therefore, inoculation of C. pepo with DDE-degrading endophytes might be promising for phytoremediation applications.

Macroinvertebrate Taxonomic and Functional Trait Compositions within Lotic Habitats Affected By River Restoration Practices.

The widespread degradation of lotic ecosystems has prompted extensive river restoration efforts globally, but many studies have reported modest ecological responses to rehabilitation practices. The functional properties of biotic communities are rarely examined within post-project appraisals, which would provide more ecological information underpinning ecosystem responses to restoration practices and potentially pinpoint project limitations. This study examines macroinvertebrate community responses to three projects which aimed to physically restore channel morphologies. Taxonomic and functional trait compositions supported by widely occurring lotic habitats (biotopes) were examined across paired restored and non-restored (control) reaches. The multivariate location (average community composition) of taxonomic and functional trait compositions differed marginally between control and restored reaches. However, changes in the amount of multivariate dispersion were more robust and indicated greater ecological heterogeneity within restored reaches, particularly when considering functional trait compositions. Organic biotopes (macrophyte stands and macroalgae) occurred widely across all study sites and supported a high alpha (within-habitat) taxonomic diversity compared to mineralogical biotopes (sand and gravel patches), which were characteristic of restored reaches. However, mineralogical biotopes possessed a higher beta (between-habitat) functional diversity, although this was less pronounced for taxonomic compositions. This study demonstrates that examining the functional and structural properties of taxa across distinct biotopes can provide a greater understanding of biotic responses to river restoration works. Such information could be used to better understand the ecological implications of rehabilitation practices and guide more effective management strategies.

Long-term river invertebrate community responses to groundwater and surface water management operations.

River flow regimes have been transformed by groundwater and surface water management operations globally, prompting widespread ecological responses. Yet, empirical evidence quantifying the simultaneous effects of groundwater and surface water management operations on freshwater ecosystems remains limited. This study combines a multi-decadal freshwater invertebrate dataset (1995-2016) with groundwater model outputs simulating the effects of different anthropogenic flow alterations (e.g. groundwater abstraction, effluent water returns) and river discharges. A suite of flow alteration- and flow-ecology relationships were modelled that tested different invertebrate community responses (taxonomic, functional, flow response guilds, individual taxa). Most flow alteration-ecology relationships were not statistically significant, highlighting the absence of consistent, detectable ecological responses to long-term water management operations. A small number of significant statistical models provided insights into how flow alterations transformed specific ecological assets; including Ephemeroptera, Plecoptera and Trichoptera taxa which are rheophilic in nature being positively associated with groundwater abstraction effects reducing river discharges by 0-15%. This represents a key finding from a water resource management operation perspective given that such flow alteration conditions were observed on average in over two-thirds of the study sites examined. In a small number of instances, specific invertebrate responses displayed relative declines associated with the most severe groundwater abstraction effects and artificial hydrological inputs (predominantly effluent water returns). The strongest flow-ecology relationships were recorded during spring months, when invertebrate communities were most responsive to antecedent minimum and maximum discharges, and average flow conditions in the preceding summer months. Results from this study provide new evidence indicating how groundwater and surface water resources can be managed to conserve riverine ecological assets. Moreover, the ensemble of flow alteration- and flow-ecology relationships established in this study could be used to guide environmental flow strategies. Such findings are of global importance given that future climatic change and rising societal water demands are likely to further transform river flow regimes and threaten freshwater ecosystems.

Particle-size dependent bactericidal activity of magnesium oxide against Xanthomonas perforans and bacterial spot of tomato.

Bacterial spot, caused by Xanthomonas spp., is a highly destructive disease of tomatoes worldwide. Copper (Cu) bactericides are often ineffective due to the presence of Cu-tolerant strains. Magnesium oxide (MgO) is an effective alternative to Cu bactericides against Xanthomonas spp. However, the effects of particle size on bactericidal activity and fruit elemental levels are unknown. In this study, nano (20 nm) and micron (0.3 and 0.6 µm) size MgO particles were compared for efficacy. Nano MgO had significantly greater in vitro bactericidal activity against Cu-tolerant X. perforans than micron MgO at 25-50 µg/ml. In field experiments nano and micron MgO applied at 200 and 1,000 µg/ml were evaluated for disease control. Nano MgO at 200 µg/ml was the only treatment that consistently reduced disease severity compared to the untreated control. Inductively Coupled Plasma Optical Emission Spectroscopy revealed that nano MgO applications did not significantly alter Mg, Cu, Ca, K, Mn, P and S accumulation compared to fruits from the untreated plots. We demonstrated that although both nano MgO and micron MgO had bactericidal activity against Cu-tolerant strains in vitro, only nano MgO was effective in bacterial spot disease management under field conditions.

Membrane transport influences the rate of accumulation of cytosine arabinoside in human leukemia cells.

The role of membrane transport in the cellular accumulation of 1-beta-D-arabinofuranosylcytosine (ara-C) was studied in freshly isolated human acute leukemia cells. Patient cells had low rates for ara-C transport as compared with human and murine experimental cells and correspondingly low binding capacities for the nucleoside transport inhibitor, nitrobenzylmercaptopurine riboside (NBMPR). At 1 microM ara-C, the rate of net cellular accumulation was close to the membrane transport rate, and NBMPR inhibited transport and accumulation to the same extent. The rate of ara-C accumulation was half maximal at only 3-5 microM, a level much lower than that required for murine cells (67-85 microM). At concentrations below 1 microM the rate of ara-C accumulation was determined primarily by the transport rate, but at higher concentrations above 10 microM, phosphorylation capacity was the principal determinant of the net uptake rate. This difference in the role of transport at high and low ara-C concentrations may explain, in part, the efficacy of high-dose ara-C in patients refractory to standard dose protocols.

Role of endocytosis and lysosomal pH in uptake of N-(phosphonacetyl)-L-aspartate and its inhibition of pyrimidine synthesis.

The mechanism of uptake and retention of N-(phosphonacetyl)-L-aspartate (PALA) was examined. Uptake of [3H]PALA by Ehrlich ascites tumor cells appeared to be biphasic. A small, variable quantity of PALA associated with cells within 5 min; the significance of this rapid uptake component is unclear. Between 15 min and 5 h, uptake was linear and consistent from experiment to experiment. The properties of the slow phase of PALA uptake are consistent with fluid-phase endocytosis. The intracellular PALA concentration approached the extracellular level very slowly, at a rate of approximately 1%/hr. The velocity of PALA uptake in these cells was proportional to the concentration in the media from 10(-6) to 10(-2) M. Uptake of PALA was identical to that of the extracellular marker inulin. Uptake of both PALA and inulin was inhibited by colchicine and stimulated by phorbol myristate acetate. The microtubule antagonist and the phorbol ester are known to, respectively, inhibit and stimulate endocytosis in other cell types. Phorbol myristate acetate enhanced the ability of PALA to inhibit incorporation of [14C]bicarbonate into pyrimidine nucleotides, presumably through an increase in PALA uptake. This inhibitory action of PALA was almost completely blocked by two agents known to neutralize lysosomal pH, NH4Cl and methylamine. These results suggest that intracellular PALA is initially compartmentalized in a pinosomal vesicle which may later fuse with cellular lysosomes. Neutralization of lysosomal pH prevents the protonation of some or all of the four negatively charged groups found in the structure of PALA which may be necessary for its diffusion across the lysosomal membrane and eventual inhibition of aspartate transcarbamylase in the cytoplasm. Since partitioning of the fully charged molecule into the lipid phase of the plasma membrane for diffusion out of the cell should be minimal, the effects of PALA on cellular metabolism are expected to be prolonged.

Role of uridine triphosphate in the phosphorylation of 1-beta-D-arabinofuranosylcytosine by Ehrlich ascites tumor cells.

Pyrimidine nucleotide pools were investigated as determinants of the rate of phosphorylation of 1-beta-D-arabinofuranosylcytosine (ara-C) by Ehrlich ascites cells and cell extracts. Cells were preincubated for 2 h with 10 microM pyrazofurin, 10 mM glucosamine, 50 microM 3-deazauridine, or 1 mM uridine in order to alter the concentrations of pyrimidine nucleotides. Samples of the cell suspensions were taken for assay of adenosine 5'-triphosphate (ATP), uridine 5'-triphosphate (UTP), cytidine 5'-triphosphate, guanosine 5'-triphosphate, deoxycytidine 5'-triphosphate (dCTP), and deoxythymidine 5'-triphosphate; then 1 microM [3H]ara-C was added and its rate of intracellular uptake was measured for 30 min. 3-Deazauridine lowered dCTP and stimulated ara-C uptake; however, pyrazofurin and glucosamine were potent inhibitors of ara-C uptake although they also decreased dCTP levels. Uridine stimulated ara-C uptake despite an increase in dCTP. A crude cytoplasmic extract was prepared by a procedure which permitted results of ara-C kinase assays to be expressed as pmol per min per 10(6) cells as in the cellular uptake studies. When assayed in the presence of mixtures of ribo- and deoxyribonucleoside triphosphates at concentrations close to their cellular levels, ara-C kinase activity closely approximated the cellular uptake rate for the five incubation conditions. Deletion of cytidine 5'-, guanosine 5'-, or deoxythymidine 5'-triphosphate from the assay mixture had little effect, while deletion of dCTP increased kinase activity 9-fold. Elimination of ATP also did not alter kinase activity in the presence of the remaining five ribo- and deoxyribonucleoside triphosphates; however, deletion of UTP reduced activity to 22% of the rate with the control mixture. When ara-C kinase was assayed with only 3 mM ATP, dCTP was a very potent inhibitor (50% inhibition concentration = 0.4 microM). Inhibition was complete at 10 microM dCTP, a concentration below the intracellular dCTP level in control cells (25 microM). With 0.9 mM UTP, enzyme activity was 2-fold greater in the absence of dCTP and the dCTP was 15-fold less potent as an inhibitor (50% inhibition concentration = 6 microM). We conclude that the actual phosphate donor for the phosphorylation of 1 microM ara-C in Ehrlich cells is UTP and not ATP. These observations suggest that successful combination protocols aimed at stimulating ara-C uptake by means of a decrease in dCTP levels must simultaneously preserve or increase UTP pools.

A critical role for uridine nucleotides in the regulation of deoxycytidine kinase and the concentration dependence of 1-beta-D-arabinofuranosylcytosine phosphorylation in human leukemia cells.

The intracellular concentration of 1-beta-D-arabinofuranosylcytosine (ara-C) for half-maximal phosphorylation by leukemic blasts obtained directly from patients was 2.1 +/- 2.5 microM (median, 1.3 microM, N = 25), and the rate of ara-C accumulation actually declined at concentrations above 20 microM in 35% of these cell populations. These apparent Km values for cellular phosphorylation were an order of magnitude lower than the Km of deoxycytidine (dCyd) kinase for ara-C with ATP as phosphate donor. dCyd kinase was purified from human leukemia cells and assayed for [3H]ara-C kinase activity with a mixture of 7 nucleotides at their approximate cellular concentrations or with a single nucleotide deleted. At low or high ara-C concentrations, ATP, GTP, CTP, or dTTP could be eliminated without significantly altering the rate. The only potential phosphate donor that was clearly important was UTP, since its deletion reduced the rate to only 25% of that with the complete mix. As anticipated, eliminating dCTP, the end product of this salvage pathway, moderately increased the rate by 50% at 0.4 microM ara-C or by 26% at 40 microM ara-C. At 40 microM ara-C, deleting UDP from the mix increased the rate more than deleting dCTP. dCTP was less inhibitory against 1 mM UTP (50% inhibitory concentration, 26 microM) than against 4 mM ATP (50% inhibitory concentration, 2.2 microM). In kinetic assays with 4 mM ATP and variable ara-C, UDP was a potent uncompetitive inhibitor with a Ki of 4 microM; the Ki for ADP was 1000-fold higher. Direct fit of kinetic data to the Michaelis equation yielded a Km for ara-C of 49 microM with 4 mM ATP as the phosphate donor; however, there was evidence of negative cooperativity with a Hill coefficient of 0.7. High ara-C Km values were also obtained with GTP and CTP, but with no evidence of cooperativity. With 1 mM UTP, the Km was 1.5 microM with moderate substrate inhibition; thus the kinetic data with UTP were similar to those for ara-C phosphorylation by intact cells. UDP was less potent versus UTP than versus ATP. It lowered the Vmax and enhanced the ara-C substrate inhibition without altering the Km. When 1 mM UTP and 4 mM ATP were mixed, the kinetic pattern was similar to that for UTP alone. The Km for UTP with [3H]dCyd as the phosphate acceptor of 0.8 microM was 25-fold lower than the Km for ATP of 20 microM.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of 1-beta-D-arabinofuranosylcytosine transport and net accumulation by teniposide and etoposide in Ehrlich ascites cells and human leukemic blasts.

The interactions of the epipodophyllotoxins, teniposide (VM-26) and etoposide (VP-16), with the nucleoside carrier were examined with emphasis on their effects on 1-beta-D-arabinofuranosylcytosine (ara-C) transport and net accumulation. VM-26 inhibited ara-C transport by Ehrlich ascites cells within 1 min of exposure, and inhibition was only partially reversed after 45 min in VM-26-free medium. ara-C transport was slowed by 50% by 7 microM VM-26 or by 35 microM VP-16. Since epipodophyllotoxins were noncompetitive inhibitors, fractional inhibition was independent of the ara-C concentration. Analysis of ara-C transport kinetics revealed only a single saturable transport route, and there was no indication of VM-26-insensitive transport. VM-26, VP-16, and ara-C were competitive inhibitors of the specific binding of nitrobenzylthioinosine to the nucleoside carrier with Ki values of 7.4 microM, 23 microM, and 2.2 microM, respectively. The rate of dissociation of nitrobenzylthioinosine (t 1/2 = 20.6 min) was accelerated by 5 microM ara-C (t 1/2 = 18.5 min) but slowed by 100 microM VM-26 (t 1/2 = 34.6). By these criteria, the interaction of VM-26 with the nucleoside carrier was qualitatively similar to that of dipyridamole. Although VM-26 inhibited ara-C transport, it did not significantly slow the rate of net intracellular accumulation of ara-C by Ehrlich cells, presumably because transport capacity far exceeds the capacity for phosphorylation in these cells. In freshly isolated human leukemic blasts, which have far less nucleoside transport activity, inhibition of ara-C accumulation by VM-26 was dependent on the ara-C concentration. At 1 microM ara-C, a concentration where transport was rate limiting for net uptake, VM-26 inhibited accumulation of ara-C over a 60-min time course. At 50 microM ara-C, transport was in excess, and VM-26 did not slow ara-C metabolism.

A basis for fluoropyrimidine-induced antagonism to methotrexate in Ehrlich ascites tumor cells in vitro.

The inhibitory effect of methotrexate on [3H]deoxyuridine incorporation into DNA is reduced as the basal rate of this reaction is inhibited by pretreatment of Ehrlich ascites tumor cells with fluoropyrimidines. This observation is a basis for fluoropyrimidine-methotrexate antagonism in anticancer regimens and supports the concept that the sensitivity of thymidylate synthesis in tumor cells to methotrexate is related, in part, to the basal rate of thymidylate synthesis from deoxyuridylate.

Formation of methotrexate polyglutamates in rat hepatocytes.

Polyglutamate derivatives of [3H]methotrexate (MTX) were detected in freshly isolated rat hepatocytes in suspension within 15 min after exposure to the folate analog. The rate of polyglutamate synthesis remained constant for at least one hr, and the polyglutamate derivatives accounted for an increasing proportion of the intracellular radiolabel with time. After initial exposure to 1 micron [3H]MTX, polyglutamate derivatives of Mtx continued to be synthesized even after the extracellular [3H]-MTX concentration had been reduced 20-fold. Prolonged exposure of hepatocytes in primary culture to 1 micron [3H]MTX resulted in the formation of longer-chain polyglutamate derivatives of MTX. The present studies demonstrate another important biosynthetic capacity of the freshly isolated hepatocyte and suggest the usefulness of this system for studying the mechanism of, and controlling factors in, the synthesis of polyglutamate derivatives of MTX. The ramifications of the formation of MTX polyglutamates on drug cytotoxicity in general and hepatotoxicity in particular are considered.

Transport, binding, and polyglutamation of methotrexate in freshly isolated rat hepatocytes.

Influx of [3H]methotrexate into freshly isolated hepatocytes in suspension is mediated by two routes, one with a high affinity (Km = 5.9 microM) and another with a low affinity for methotrexate. Both transport routes are equally sensitive to the sulfhydryl group inhibitor, p-chloromercuriphenylsulfonic acid, alterations in temperature, substitution of extracellular Na+ with choline, and inhibition by ouabain or azide. The high-affinity pathway for methotrexate shows specificity for the 4-amino group of the pteridine moiety as methotrexate and aminopterin similarly inhibit influx of the labeled drug. On the other hand, 100 microM concentrations of the naturally occurring folates, folic acid, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate, are not inhibitory to influx of 1 microM methotrexate. Once in the cell, methotrexate rapidly reaches molar equivalence with dihydrofolate reductase following which both exchangeable and nonexchangeable intracellular methotrexate accumulates. The exchangeable component reaches steady state within 0.5 hr while the nonexchangeable component increases for at least 1 hr. The nonexchangeable component represents both bound methotrexate and methotrexate polyglutamates. Polyglutamates of methotrexate are a trivial component of total 3H within the cell until about 15 min, but thereafter, their rate of accumulation is constant so that by 1 hr they represent approximately 30% of total intracellular 3H. At steady state, there is a transmembrane chemical gradient for exchangeable methotrexate of 2.4:1; this is 24 times greater than the chemical gradient predicted for equilibrium when the transcellular membrane potential is considered. These results indicate that there are multiple routes for methotrexate transport in the rat hepatocyte that appear to be, at least in part, distinct from the routes for folic acid and the tetrahydrofolate cofactors. The data suggest that transport is energy and Na+ dependent and that the transport carrier requires intact sulfhydryl groups. Net association of methotrexate with the cells is a complex process determined by transport and binding to multiple sites within the cell and metabolism to polyglutamate derivatives that are retained within the cell.

Relationship between membrane transport of methotrexate and endogenous cyclic adenosine 3':5'-monophosphate in the Ehrlich ascites tumor.

Transport of methotrexate in mammalian cells is a complex process. Although the drug is pumped uphill into cells, metabolic poisons enhance influx and transmembrane gradients for this agent. Structurally unrelated inorganic and organic anions nonspecifically depress these parameters in a variety of tumor cells. It has been suggested that cell cyclic adenosine 3':5'-Monophosphate (cyclic AMP) plays a regulatory role in the transport of this agent. To evaluate this further, a number of different experimental approaches were used to analyze the relationship between methotrexate transport and cell cyclic AMP in the Ehrlich ascites tumor. The following results indicate that for the Ehrlich ascites tumor, at least, there is no evidence for a regulatory role for cyclic AMP in the transport of methotrexate. (a) A marked increase in cell cyclic AMP by cholera-toxin or reduction in cyclic AMP by ascorbate was unaccompanied by changes in methotrexate influx. (b) Instantaneous and constant inhibition of methotrexate influx by isobutylmethylxanthine was temporally dissociated from the slower rise in cell cyclic AMP that was induced by this agent. (c) Although isobutylmethylxanthine and dibutyryl cyclic AMP augment cell cyclic AMP and decrease methotrexate influx, they have different effects on efflux and net transport of methotrexate. Isobutylmethylxanthine stimulates net methotrexate uptake and slows methotrexate efflux but dibutyryl cyclic AMP depresses net methotrexate uptake without an effect on methotrexate efflux. (d) Azide markedly stimulates methotrexate influx and net transport without a change in cell cyclic AMP. (d) Dinitrophenol inhibits methotrexate influx and augments net methotrexate accumulation but does not alter cell cyclic AMP.

Exchangeable intracellular methotrexate levels in the presence and absence of vincristine at extracellular drug concentrations relevant to those achieved in high-dose methotrexate-folinic acid "rescue" protocols.

Studies were undertaken to (a) assess intracellular methotrexate (MTX) levels at extracellular drug concentrations comparable to those achieved in high-dose MTX-folinic acid rescue protocols and (b) establish whether there is a rationale for the use of vincristine in these regimens. The data indicate that only low levels of exchangeable MTX (intracellular MTX in excess of the tightly bound fraction) accumulated in Ehrlich ascites tumor cells at high extracellular MTX concentration. For instance, the exchangeable steady-state intracellular MTX level was approximately 6.5 muM when the extracellular drug concentration was 85 muM. Over the interval of these experiments, exchangeable intracellular MTX did not exceed approximately 10 muM even when extracellular MTX was raised to 250 muM. These exchangeable intracellular MTX concentrations are comparable to those levels required experimentally to suppress (a) tetrahydrofolate synthesis from dihydrofolate and (b) tetrahydrofolate-dependent purine, pyrimidine, and amino acid synthesis in these cells in vitro. Vincristine (10 muM) augmented net MTX accumulation when the extracellular MTX level was 10, 100, or 250 muM. The limited capacity of cells to accumulate exchangeable intracellular MTX and the apparent role for this intracellular MTX component in achieving the metabolic effects of this agent may account for the necessity for high MTX blood levels in the treatment of some tumors and may be the basis, in part, for the enhanced chemotherapeutic efficacy of high-dose MTX regimens. These studies provide a rationale for the combined use of vincristine and MTX in high-dose MTX protocols. The addition of vincristine may permit the achievement of the level of exchangeable intracellular MTX that is required to critically inhibit tetrahydrofolate synthesis without an increase in the extracellular MTX concentration. This may permit a reduced MTX dose, diminishing the excretory load on the kidney and minimizing nephrotoxicity due to deposition of MTX in the renal tubule and interstitium. While the data indicate that the ratio of the concentration of exchangeable intracellular MTX to the extracellular drug concentration may be very low under steady-state conditions at high extracellular drug levels, further studies are required to establish that these steady-state gradients for MTX represent nonequilibrium conditions.

Exposure of Cucurbita pepo to DDE-contamination alters the endophytic community: A cultivation dependent vs a cultivation independent approach.

2,2-bis(p-chlorophenyl)-1,1-dichloro-ethylene (DDE) is the most abundant and persistent degradation product of the pesticide 2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane (DDT) and is encountered in contaminated soils worldwide. Both DDE and DDT are classified as Persistent Organic Pollutants (POPs) due to their high hydrophobicity and potential for bioaccumulation and biomagnification in the food chain. Zucchini (Cucurbita pepo ssp. pepo) has been shown to accumulate high concentrations of DDE and other POPs and has been proposed as a phytoremediation tool for contaminated soils. The endophytic bacteria associated with this plant may play an important role in the remedial process. Therefore, this research focuses on changes in endophytic bacterial communities caused by the exposure of C. pepo to DDE. The total bacterial community was investigated using cultivation-independent 454 pyrosequencing, while the cultivable community was identified using cultivation-dependent isolation procedures. For both procedures, increasing numbers of endophytic bacteria, as well as higher diversities of genera were observed when plants were exposed to DDE. Several bacterial genera such as Stenotrophomonas sp. and Sphingomonas sp. showed higher abundance when DDE was present, while, for example Pseudomonas sp. showed a significantly lower abundance in the presence of DDE. These findings suggest tolerance of different bacterial strains to DDE, which might be incorporated in further investigations to optimize phytoremediation with the possible use of DDE-degrading endophytes.

Comparison between cultivated and total bacterial communities associated with Cucurbita pepo using cultivation-dependent techniques and 454 pyrosequencing.

Endophytic bacteria often have beneficial effects on their host plants that can be exploited for bioremediation applications but, according to the literature, only 0.001-1% of all endophytic microbes should be cultivable. This study compared the cultivated endophytic communities of the roots and shoots of Cucurbita pepo with the total endophytic communities as determined by cultivation-dependent techniques and 454 pyrosequencing. The ten most abundant taxa of the total communities aligned well with the cultivated taxa; however, the abundance of these taxa in the two communities differed greatly. Enterobacter showed very low presence in the total communities, whereas they were dominantly present in the cultivated communities. Although Rhizobium dominated in total root and shoot communities, it was poorly cultivable and even then only in growth media containing plant extract. Since endophytes likely contribute to plant-growth promotion, cultivated bacterial strains were tested for their plant-growth promoting capabilities, and the results were correlated with their abundance in the total community. Bacillus and Pseudomonas showed promising results when considering cultivability, abundance in the total community and plant-growth promoting capability. This study demonstrated that, although a limited number of bacterial genera were cultivable, current cultivation-dependent techniques may be sufficient for further isolation and inoculation experiments that aim to improve phytoremediation efficiency.

Membrane ordering effects of the anticancer agent VM-26.

The effect of the anticancer agent VM-26 on acyl chain order of cellular and model membranes was examined by electron spin resonance techniques. The order parameter for the paramagnetic probe 5-doxyl stearate was increased when VM-26 was incorporated into the bilayer of fluid-phase dimyristoylphosphatidylcholine (DMPC) or gel-phase dipalmitoylphosphatidylcholine (DPPC) liposomes at concentrations up to 4.8 mol%. The ordering effect of VM-26 in DMPC was greater than that of cholesterol on an equimolar basis. The less cytotoxic congener of VM-26, VP-16, was only one-third as active as VM-26 in its ordering effects on DMPC. Higher order parameters for 5-doxyl stearate were also noted in asolectin liposomes, Ehrlich ascites tumor cells, and CCRF-CEM cells treated with VM-26. We conclude that VM-26 has significant membrane associated activity in addition to its previously recognized nuclear effects.

Partitioning of teniposide into membranes and the role of lipid composition.

We have examined the partitioning behavior of the anticancer agent teniposide (VM-26) into multilamellar vesicles composed of various phospholipid species. Partitioning was found to be sensitive to the composition of the liposomal membrane since changes in the head group or acyl chain constituents could dramatically alter the affinity of the drug for the bilayer. [3H]VM-26 partitioned most readily into 1,2-monounsaturated species of phosphatidylcholine (PC) with a molar partition coefficient (Kp) of 4290 for dioleoyl-PC at 37 degrees C. Inclusion of additional phospholipids having a different head group reduced partitioning in the order cardiolipin greater than phosphatidylglycerol greater than phosphatidylserine greater than phosphatidylethanolamine. The Kp for dioleoyl-PC with 33 mol% cardiolipin was reduced to 1370. Partitioning into completely saturated species of PC was much less than that for unsaturated species and was inversely proportional to the hydrocarbon chain length at temperatures either above or below the chain melting temperature. The Kp for fluid phase dimyristoyl-PC was 2300. Partitioning into dimyristoyl-PC or dioleoyl-PC at 37 degrees C (fluid) or dipalmitoyl-PC at 25 degrees C (gel) was reduced by the addition of 5-30 mol% cholesterol in proportion to its bilayer concentration. Etoposide (VP-16) at concentrations up to 10 mol% did not compete with [3H]VM-26 for association with dioleoyl-PC. Addition of calf serum or serum albumin could significantly reduce the association of [3H]VM-26 with the liposomes.

Leukapheresis induced changes in cell cycle distribution and nucleoside transporters in patients with untreated acute myeloid leukemia.

Bone marrow leukemia cells from eight adults with untreated acute myeloid leukemia (AML) were evaluated before and after three daily leukaphereses to determine if mechanical cytoreduction can modulate the cell cycle distribution. The percentage of cells in S-phase and the proliferative fraction (PF = %S + %G2M) were determined by flow cytometry after dual labeling with bromodeoxyuridine and propidium iodide. Prior to pheresis the median %S and PF were 5.4 and 15.4%, respectively. The median change in %S was +2.5% (range -5.5 to +18.8) with increases greater than or equal to 3.7% in 4/8 patients. The median change in PF was +6.1% (range -13.8 to +25.3) with an increase of greater than or equal to 3.6% in 6/8 patients. The median absolute changes of 2.5 and 6.1% represent increases of 47% for %S and 40% for PF compared to the day 1 (pre-pheresis) median values. As the number of nucleoside transporters in the cell membrane [nitrobenzylmercaptopurine riboside (NBMPR) binding sites] has been related to the percentage of cells in S-phase and to cytosine arabinoside (ara-C) cellular pharmacology, these were also measured before and after leukapheresis. Changes in the number of NBMPR binding sites varied widely with a median increase of 365 sites per cell (range -26,061 to +10,396). The change in NBMPR sites was significantly and positively correlated with changes in %S (r = 0.829, p = 0.042). These data suggest that mechanical cytoreduction by leukapheresis can increase the fraction of leukemia cells in S-phase and the PF in some patients with AML. The increase in %S is accompanied by an increase in NBMPR binding sites per cell. These changes in leukemia cell characteristics would be expected to result in an increase in efficacy of ara-C or other S-phase specific agents.