Production of dyestuffs from indole derivatives by naphthalene dioxygenase and toluene dioxygenase

AIMS: To isolate and characterize the phorate [O,O-diethyl-S-(ethylthio)methyl phosphoradiothioate] degrading bacteria from agricultural soil, and their assessment for multifarious biological activities of environmental and agronomic significance. METHODS AND RESULTS: Based on their morphological and biochemical characteristics, the selected isolates PS-1, PS-2 and PS-3 were presumptively identified as Rhizobium, Pseudomonas and Proteus species, respectively. The HPLC analysis of phorate in bioaugmented soil revealed its complete disappearance within 40 days. The degradation isotherms of the isolates PS-1, PS-2 and PS-3 suggested time-dependent disappearance of phorate following the first-order rate kinetics at the corresponding rate constants of 0.04, 0.05 and 0.04 d-1. Besides, the isolates concurrently exhibited substantial phosphate solubilization, indole acetic acid (IAA) and siderophore production, as well as limited biocontrol activity against fungal phytopathogens. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The data revealed the potential of isolates for collateral plant growth promotion, biocontrol and bioremediation. The selected strains may serve as an important bioresource for development of effective super-bioinoculants.     

Studies on the persistence and effects on soil fauna of some soil-applied systemic insecticides

Granular formulations of menazon, phorate and thionazin were applied to a sandy loam soil (pH = 6·1)in April and May as in-row treatments at commercial rates. They were also broadcast and mixed into the top 4 in of soil at concentrations (10 and 250 ppm of dry soil) which simulated the local in-row concentrations in areas large enough for sampling. Bioassays showed that 50 % of phorate, thionazin and menazon equivalents had disappeared in about 68, 57 and 23 days respectively from soil treated with 10 ppm of the insecticides. Small residues of the 250 ppm treatments still remained 2 years later. The initial rate of loss of activity of thionazin applied at 250 ppm was much slower than from a 10 ppm application whereas the activities of 250 and 10 ppm of menazon disappeared at similar rates. Phorate at 250 ppm killed almost all earthworms, Collembola, Acarina, free-living saprophytic and parasitic nematodes and Protozoa; 10 ppm phorate and 250 ppm of menazon also killed almost all Collembola and Acarina but the menazon was relatively harmless to earthworms. Collembola and mite populations began to increase when residues of phorate and menazon equivalents had decreased to about 2 and 20 ppm respectively, and after 15 months they were similar to those in untreated plots. The broadcast treatments initially decreased the rate of breakdown of leaf discs put in the soil but, after about 4 weeks, breakdown increased, sometimes above that in untreated plots. This was associated with a large increase in numbers of Enchytraeidae which were apparently unaffected by the insecticides. Four months after application, 1 lb/acre of phorate and 2 lb of menazon applied in seed drills 2 ft apart were not affecting Collembola in soil between the rows but were still decreasing the numbers within them. In -row phorate distributed along a 1·5 in diameter band of soil, 3 in deep, killed Collembola 3 in on either side but not 6 in away. It did not spread upwards in toxic quantities, but after rainfall, sufficient to kill Collembola leached at least 3 in downwards. We conclude that commercial in-row applications of chemicals like phorate are most unlikely to harm soil fertility, especially as the leaf-litter-destroying function of Collembola and other animals killed by phorate may be taken over by Enchytraeidae.     

Integrated management of root-knot nematode, Meloidogyne incognita infestation in tomato and grapevine

An integrated approach with the obligate bacterial parasite, Pasteuria penetrans and nematicides was assessed for the management of the root-knot nematode, Meloidogyne incognita infestation in tomato and grapevine. Seedlings of tomato cv. Co3 were transplanted into pots filled with sterilized soil and inoculated with nematodes (5000 juveniles/pot). The root powder of P. penetrans at 10 mg/pot was applied alone and in combination with carbofuran at 6 mg/pot. Application of P. penetrans along with carbofuran recorded lowest nematode infestation (107 nematodes/200 g soil) compared to control (325 nematodes/200 g soil). The rate of parasitization was 83.1% in the carbofuran and P. penetrans combination treatment as against 61.0% in the P. penetrans treatment only. The plant growth was also higher in the combination treatment compared to all other treatments. A field trial was carried out to assess the efficacy of P. penetrans and nematicides viz., carbofuran and phorate in the management of root-knot nematode, M. incognita infestation of grapevine cv. Muscat Hamburg. A nematode and P. penetrans infested grapevine field was selected and treatments either with carbofuran or phorate at 1 g a.i/vine was given. The observations were recorded at monthly interval. The results showed that the soil nematode population was reduced in nematicide treated plots. Suppression of nematodes was higher under phorate (117 nematodes/200 g soil) than under carbofuran (126.7 nematodes/200 g soil) treatment. The number of juveniles parasitized was also influenced by nematicides and spore load carried/juvenile with phorate being superior and the increase being 17.0 and 29.0% respectively over the control. The results of these experiment confirmed the compatibility of P. penetrans with nematicides and its biological control potential against the root-knot nematode.     

Saposin C is required for normal resistance of acid beta-glucosidase to proteolytic degradation

Saposins (A, B, C, and D) are small sphingolipid activator proteins that are derived by proteolytic processing of a common precursor, prosaposin. In the lysosomal sphingolipid degradation pathway, acid beta-glucosidase (GCase) requires saposin C for optimal in vitro and in vivo hydrolysis of glucocerebroside. The deficiency of prosaposin/saposins (PS-/-) in humans and mice leads to a decrease of GCase activity in selected tissues. Concordant decreases (>50%) of GCase protein and in vitro activity were detected in extracts of cultured fibroblasts and hepatocytes from PS-/- mice and human prosaposin-deficient fibroblasts. GCase RNA in the PS-/- cells was at wild-type levels. Compared with that in wild-type cells (t(1/2) >24 h), the GCase protein in the PS-/- cells had a faster disappearance rate (t(1/2) approximately 1 h in mouse and approximately 8 h in human) as determined by metabolic labeling and immunoprecipitation with anti-GCase antibodies. Treatment of PS-/- cells with leupeptin, an inhibitor of cysteine proteases, led to significant increases (approximately 2-fold) in GCase protein and in vitro activity. Loading saposin C to human PS-/- fibroblasts resulted in an enhancement of GCase protein and in vitro activity. Saposin D loading had no effect. These data indicate that saposin C is required for GCase resistance to proteolytic degradation in the cell. Thus, diminished in vivo GCase activity would be greater than expected only from the lack of GCase activation by saposin C. These results indicate a new property for saposin C, an anti-proteolytic protective function toward GCase.     

Insecticidal effects on soil microorganisms and their biochemical processes related to soil fertility

Among the four insecticides under study, hexachlorocyclohexane (BHC) followed by phorate significantly stimulated the populations of (total) bacteria, actinomycetes, fungi, aerobic non-symbiotic N2-fixing bacteria and P-solubilizing microorganisms in soil. Carbofuran significantly stimulated total as well as N2-fixing bacteria. Fenvalerate had no effect on P-solubilizers. All the insecticides stimulated the proportion of Penicillium in soil. Similarly, Pseudomonas with BHC, Sarcina with phorate, Corynebacterium, Azotobacter and Streptomyces with fenvalerate were also stimulated. On the other hand, Erysipelothrix with BHC, Staphylococcus with phorate, Staphylococcus, Nocardia and Fusarium with fenvalerate were inhibited. Almost all the insecticides reduced the proportions of Micrococcus and Rhizopus in soil. Insecticides also augmented the non-symbiotic N2-fixing and P-solubilizing capacities of the soil and the augmentation was more pronounced with BHC followed by phorate.     

Isolation and characterization of a phorate degrading bacterium

Aims:  To study the degradation of phorate by a bacterium isolated from phorate-contaminated sites.
Methods and Results:  Ralstonia eutropha strain AAJ1 isolated from soil was found to degrade phorate (supplied as sole carbon source) upto 85% in 10 days in liquid medium. Half-life ( t _½) of phorate in the liquid medium in control (uninoculated) and in experimental (inoculated with R. eutropha , strain AAJ1) samples was recorded as 36·49 and 6·29 days, respectively. Kinetics revealed that phorate degradation depends on time and the reaction follows the first order kinetics. Diethyl dithiophosphate was one of the degradation products, which is markedly less toxic than the parent compound; other degradation products included phorate sulfoxide and phorate sulfone. Release of inorganic phosphates and sulfates indicated the potential of the isolate to further degrade the above-mentioned metabolites to simpler forms. The strain was also found to posses phosphomonoesterase and phosphodiesterase enzymatic activity, which are involved in biodegradation of organophosphorus compounds.
Conclusions:  Ralstonia eutropha AAJ1 could degrade and detoxify phorate upto 85% in 10 days in laboratory conditions.
Significance and Impact of the Study:  The isolate has the potential to be utilized for remediation of phorate-contaminated water and soil.     

Isolation and characterization of a novel acidic polysaccharide containing tartrate and glyoxylate residues from the mineralized scales of a unicellular coccolithophorid alga Pleurochrysis carterae.

The characterization of mineral-associated polyanions from the unicellular alga Pleurochrysis carterae is described. This species is useful for the study of mineralization, because it produces calcified scales known as coccoliths in homogeneous cell culture. Three acidic polysaccharides (PS-1, PS-2, and PS-3) were extracted from the coccoliths with EDTA and were separated and purified by differential precipitation with magnesium ions and chromatography on DEAE-cellulose. PS-1 and PS-3 are predominantly polymers of galacturonic acid containing lesser amounts of other monosaccharides. PS-2 has an unusual structure. Chemical, enzymatic, and two-dimensional NMR analyses demonstrate that the repeating unit of PS-2 is [----4)D-glucuronate(beta 1----2)meso-tartrate(3----1)glyoxylate(1-]n. Thus PS-2 has a high density of negatively charged groups available for calcium ion binding, similar to the phosphoprotein polyanions of other species. Polysaccharides containing tartrate and/or glyoxylate have not been previously described; these residues may be introduced into PS-2 by a postpolymerization process involving oxidative cleavage of glucuronate or mannuronate residues.     

Genetic and cytological analyses of a partial-female-sterile mutant (PS-1) in soybean (Glycine max; Leguminosae)

Soybean partial-female-sterile mutant 1 (PS-1) was recovered from a gene-tagging study. The objectives were to study the inheritance, linkage, allelism, and certain aspects of the reproductive biology of the PS-1 mutant. For inheritance and linkage tests, PS-1 was crossed to flower color mutant Harosoy-w4 and to chlorophyll-deficient mutant CD-1, also recovered from the gene-tagging study. For allelism tests, reciprocal crosses were made with PS-1 and three other partial-sterile mutants (PS-2, PS-3, and PS-4) recovered from the same gene-tagging study. The PS-1 mutant is inherited in a 3:1 ratio and is a single recessive gene. Linkage results indicated that the gene for partial sterility in PS-1 is not linked either to the w4 locus or to the CD-1 locus. Allelism tests showed that the gene in PS-1 is nonallelic to the gene in PS-2, PS-3, and PS-4. Investigations of developing and mature pollen indicated no differences in morphology, stainability, or fluorescence between normal and partial-sterile genotypes. The PS-1 mutant is completely male fertile. Confocal scanning laser microscopy was used to determine that early embryo abortion in PS-1 is due indirectly to abnormal migration of the fused polar nucleus, which prevented it from being fertilized. Subsequent absence of endosperm development leads directly to abortion of the proembryo.     

The isolation, partial characterization, and biosynthesis of the paragonial substances, PS-1 and PS-2 of Drosophila funebris

In methanolic extracts of accessory glands (paragonia) from Drosophila funebris, two specific, ninhydrin-positive substances, PS-1 and PS-2, were found. PS-1 and PS-2 were isolated by column chromatography. PS-1 consists of 27 amino acid residues. Two forms of PS-1 are present in the ratio of 7:3 which differ only in the content of valine and leucine. Fractions containing partially purified valine-PS-1 and leucine-PS-1, respectively, have the same biological activity. All males in the population synthesize both forms of PS-1. PS-2 is a low molecular weight substance containing glycine and ammonia as ninhydrin-positive components and carbohydrate as indicated by several sugar tests. In vitro studies showed that copulation provides the stimulus for enhanced synthesis of paragonial substances.     

Isolation and characterization of 2,4-dichlorophenoxyacetic acid-catabolizing bacteria and their biodegradation efficiency in soil

Bacterial isolates (NJ 10 and NJ 15) capable of degrading the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) were isolated from agricultural soil by enrichment culture technique. The isolates exhibited substantial growth in mineral salt medium supplemented with 0.1–0.5% of 2,4-D as a sole source of carbon and energy. Based on their morphological, cultural and biochemical characteristics, the isolates NJ 10 and NJ 15 have been identified as Pseudomonas species and Pseudomonas aeruginosa, respectively. Biodegradation studies in a soil microcosm enriched with pure cultures of the isolates demonstrated a time-dependent disappearance of 2,4-D from the 100 mg/kg herbicide-amended soil. The HPLC data analysis revealed 96.6 and 99.8% degradation in the soil inoculated with the pure cultures of isolates NJ 10 and NJ 15, respectively with in 20 days of incubation at 30 °C. Both the isolates showed significant solubilization of inorganic phosphate [Ca₃(PO₄)₂] on the specific Pikovskaya's medium.     

Genetic and cytological analyses of three lethal ovule mutants in soybean (Glycine max; Leguminosae).

Soybean partially sterile mutants 2, 3, and 4 (PS-2, PS-3, and PS-4), recovered from a gene-tagging experiment, were studied to clarify their inheritance, linkage, allelism, and reproductive biology. The PS-2, PS-3, and PS-4 mutants were maintained as heterozygotes and upon self-pollination segregated l fertile: l partially-sterile. For inheritance and linkage tests, all three PS mutants were crossed to flower color mutant Harosoy-w4 and to chlorophyll-deficient (CD) mutants CD-1 and CD-5, also recovered from the tagging study. For allelism tests, reciprocal crosses were made among the three partially sterile mutants. Linkage results indicated that the gene for partial sterility in the PS-2, PS-3, and PS-4 mutants was not linked either to the w4 locus or to the genes for chlorophyll deficiency. Studies of pollen development, pollen viability, and pollen-tube germination indicated no difference between normal and partially sterile genotypes. Linkage and allelism tests indicated that the gene in the three partially sterile mutants was not transmitted through the female when they were used as a female parent. A study of megagametogenesis indicated that the ovules from partially sterile plants had normal embryo sac development. Ovule abortion was due to failure of fertilization.     

Cytotoxic and necrotic responses in human amniotic epithelial (WISH) cells exposed to organophosphate insecticide phorate

The in vitro interaction of the organophosphorous insecticide (OPs) phorate with calf thymus DNA (ctDNA), and its potential to cause changes in cell cycle, membrane damage, and cytotoxicity leading to cell death (necrosis) was investigated in human amnion epithelial (WISH) cells. Fluorescence quenching revealed high binding affinity (K(a)=5.62×10(4)M(-1)) of phorate to ctDNA. Molecular modeling of the phorate-ctDNA interaction suggested the binding of phorate at AT rich regions on minor groove of DNA. The interaction ensued alkylation of the N-6, N-7 of adenine and C-4 carbonyl oxygen of thymine. Binding of phorate was stronger in the presence of the transition metal ion copper II (Cu(2+)), and has accentuated the destabilization of the DNA secondary structure. A discernable change in the voltammetric E(1/2) (E(0')) with lesser cathodic (i(pc)) and anodic (i(pa)) peak currents confirmed the formation of phorate-DNA and phorate-DNA-Cu (II) association complexes. Furthermore, the MTT and NRU assays demonstrated substantial phorate cytotoxicity due to loss of mitochondrial and lysosomal membrane integrity, and reduction in mitochondrial membrane potential (ΔΨm) of treated WISH cells. Cell cycle analysis of WISH cells treated with 1000μM phorate exhibited 13.7-fold (p<0.01) augmentation in the sub-G(1) peak. Annexin V-PE and 7-ADD staining of phorate treated cells reaffirmed the development of late apoptotic or necrotic cell population in a concentration dependent manner. Thus, this study demonstrated the phorate induced DNA structural alterations and cellular damage in cultured human cells.     

Presenilin-2 in the cynomolgus monkey brain: investigation of age-related changes

Localization of presenilin-2 (PS-2), a transmembrane protein implicated in early onset familial Alzheimers disease, was examined in the brains of 30 cynomolgus monkeys aged 4 to 36 years. Anti-PS-2 antibody N20, which recognizes PS-2 amino acid residues 2–20, and anti-PS-2 antibody C20, which recognizes PS-2 amino acid residues 535–554, stained mainly the cytoplasm of large pyramidal neurons and large neurites. This finding was also confirmed by double immunohistochemical investigations using N20 or C20 and anti-NeuN antibody. In the brain of the oldest monkey, swollen neurites containing senile plaques were immunostained with C20, but not with N20. Western blot analyses of microsomal fractions isolated from the brains of three adult monkeys revealed that much less PS-2 was present compared to presenilin-1 (PS-1). Age-related assessment of PS-2 in brain homogenates from young and adult monkeys showed that PS-2 levels and PS-2 subcellular localization were unchanged with increasing age. Because PS-2 expression was much less robust than that of PS-1, we conclude that PS-2 mainly localizes to large neurons and does not show so drastic age-related changes as PS-1.     

Effect of cations on the quantum yield and the lifetime of the chloroplast fluorescence]

The interrelationship between the cation-induced fluorescence changes and the state of the photosystem 2 (PS-2) reaction centers for pea chloroplasts and their osmotic fragments was studied. The effects of K+ and Mg2+ on the fluorescence quantum yield (phi f1) under varying light intensities as well as on the fluorescence lifetime (tau f1) in the saturating light were demonstrated. K+ induces the decrease in tau f1; Mg2+ exerts an opposite effect. The effects were more pronounced when the reaction centers of PS-2 were converted into an inactive state by illuminating the sample with a saturating light or by adding DCMU. Under these conditions the cations' effect on tau f1 was accompanied by proportional changes in tau f1. It was concluded that in Mg-deficient chloroplasts an efficient channel of the excitation quenching appears in antenna chlorophyll of PS-2 with the rate constant of 7 . 10(8) s-1. The simultaneous measurements of tau f1 by phase and modulation type techniques allowed to reveal the emission heterogeneity within the nanosecond time interval and the DCMU-sensitive delayed fluorescence with the lifetime exceeding 10(-7) s and the overall quantum yield approximately equal to 2 . 10(-3).     

The breakdown of malathion in soil and soil components

The disappearance of the organophosphorus insecticide, malathion, from a silt loam soil and from its organic and inorganic components was examined. Half-lives and the time taken for 90% decomposition in nonsterile, sodium azide-treated, and 2.5 Mrad-irradiated soils were similar (3/4–1 1/2 days and 4–6 days, respectively) but breakdown in autoclaved soils was negligible. Decay in nonsterile sand, silt, and clay minus organic matter fractions was 3–6 times slower than that recorded in the original soil. Breakdown of malathion in the clay plus organic matter fraction (organo-mineral complex) was rapid (half-life, 1 day), as was the case in the separated organic matter (half-life, 1 3/4 days). Filter-sterilized organic matter was not as effective in catalyzing the breakdown of malathion (half-life, 4 days), and no loss occurred from any of the autoclaved components. Irradiation doses of 2.5 and 5.0 Mrad had little influence on the ability of soil to degrade malathion. Thereafter, increases up to 20 Mrad had a more drastic, though far from totally inhibitory, effect. Our results suggest that either the colloidal organic matter itself, or a fraction associated with it, is the most important single factor concerned with the rapid breakdown of malathion in the soil studied. Direct microbial metabolism is a slower process and may have a significant role in malathion disappearance in coarsetextured soils low in colloidal organic matter. The catalytic component of the organic matter is suggested to be a stable exoenzyme and is supportive of reports by other workers. The quantitative effect of organo-mineral complex (containing the active degradative ingredient) additions to sand and silt fractions on the rate of subsequent malathion decay is also described.     

Presenilin-1 inhibits delta-catenin-induced cellular branching and promotes delta-catenin processing and turnover

Although delta-catenin/neural plakophilin-related armadillo protein (NPRAP) was reported to interact with presenilin-1 (PS-1), the effects of PS-1 on delta-catenin have not been established. In this study, we report that overexpression of PS-1 inhibits the delta-catenin-induced dendrite-like morphological changes in NIH 3T3 cells and promotes delta-catenin processing and turnover. The effects of PS-1 on endogenous delta-catenin processing were confirmed in hippocampal neurons overexpressing PS-1, as well as in the transgenic mice expressing the disease-causing mutant PS-1 (M146V). In addition, disease-causing mutant PS-1 (M146V and L286V) enhanced delta-catenin processing, whereas PS-1/gamma-secretase inhibitors could block the formation of processed forms of delta-catenin. Together, our findings suggest that PS-1 can affect delta-catenin-induced morphogenesis possibly through the regulation of its processing and stability.     

The uptake of phorate, a systemic insecticide, applied as a slurry to wheat and mustard seeds

The absorption by plants of wheat and mustard of a systemic organophosphorus insecticide (phorate), applied as a slurry seed dressing, was studied by caging the aphid Rhopalosiphum padi (L.) on the foliage of wheat, and the aphid Brevicoryne brassicae (L.) and the Chrysomelid beetle Phaedon cochleariae (F.) on white mustard, grown from phorate-treated seed.
Wheat and mustard plants quickly lost their toxicity to insects when they were transplanted, suggesting that most of the insecticide from a slurry seed treatment passes into the soil and is picked up by the roots. That phorate or its derivatives occur in the soil was shown by tests of anticholinesterase activity. Insecticide can also pass into the seed of wheat and move to the growing embryo. Phorate becomes closely bound to the testa of mustard, but does not penetrate it to reach the cotyledons or other parts of the embryo. Mustard cotyledons can become contaminated by insecticide as they emerge through the soil.
Young and old leaves of both wheat and mustard depend on continued absorption of insecticide from the soil to maintain their toxicity. No insecticide moves from old to young leaves. Old leaves lose their toxicity to insects more slowly than young ones. When treated seeds are sown close together, the overlapping zones of insecticide round each seed can increase strength and persistence of insecticidal effect. This happens more with dimethoate, which readily dissolves in water, than with phorate, which is almost insoluble. At normal sowing rates the zones of insecticide round each seed would rarely overlap.
Roots of wheat and mustard from treated seed did not excrete insecticide, and the roots did not carry insecticide through the soil.     

盐胁迫对VA菌根形成及接种VAM真菌对植物耐盐性的效应

用盆栽法研究了不同土壤含盐量条件下接种不同VAM真菌菌株对棉花、玉米、大豆和甜瓜耐盐性的影响。结果表明,随着土壤中NaCl水平提高,真菌对棉花、玉米、大豆和甜瓜的侵染率下降,其生长量亦均呈递减趋势但对VAM真菌的依赖性则呈明显递增趋势。接种VAM真菌促进了棉花、玉米和大豆的生长,如在NaCl施入量为0～3g·kg^-1时,接种M₁菌株使棉花干重提高4.6～80.9%;在NaCl施入量为0～2.5g·kg^-1时,接种M₁菌株的玉米干重比对照增加20～109.6%;NaCl施入量为1g·kg^-1时,接种M₁和M₂两菌株使大豆干重分别增加22.1%和10.2%.不同的VAM真菌菌株对同一植物的耐盐性以及同一种VAM真菌菌株对不同植物的耐盐性的影响程度不同。可以认为,VAM真菌与植物共生对植物在盐渍环境中的生存起着重要的作用;接种VAM真菌可以提高植物在盐渍土壤上的生产能力并减轻植物因盐害造成的产量损失。     

N141I mutant presenilin-2 gene enhances neuronal cell death and decreases bcl-2 expression

A missense mutation (N1411) in Presenilin-2 (PS-2) gene is associated with early-onset familial Alzheimer's disease. In this study, SK-N-SH human neuroblastoma cells were transfected with wild-type and mutant PS-2 gene to examine presenilin-2 effects on apoptosis. Serum deprivation resulted in enhanced apoptosis in mutant PS-2 comparing with wild-type PS-2. Similarly, mutant PS-2 induced lactate dehydrogenase release to greater extent than wild-type PS-2. Time course experiment demonstrated that the increase in caspase-3-like activity was more pronounced and accelerated in mutant PS-2, compared to wild-type PS-2. While a significant decrease in bcl-2, an anti-apoptotic molecule, occurred in the cells overexpressing mutant PS-2, no significant change was observed in bax, a pro-apoptotic molecule, as compared with the cells overexpressing wild-type PS-2. Our study demonstrated that mutant PS-2 induces apoptosis accompanied by increased caspase-3-like activity and decreased bcl-2 expression in neuronal cells after serum-deprivation.