The trapping fungus Dactylaria Brochopaga: induction of trap formation,attraction, trapping and the development in some phytonematodes

All the six species of the phytonematodes induced traps directly on the spores of all the five isolates of Dactylaria brochopaga. The maximum rings induction was observed in the presence of second-stage juveniles of Meloidogyne incognita followed by Meloidogyne graminicola (J₂), Xiphinema basiri, Helicotylenchus dihystera, Tylenchorynchus brassicae and Hoplolaimus indicus. Maximum number of induced traps was recorded at temperature 28°C (98.3%) and pH 7 (96.7%) in the presence of H. indicus. In attraction assay, irrespective of the isolates of the fungus, all the six phytonematodes were attracted towards the fungal discs of the different isolates of D. brochopaga. Among all the five isolates, Isolate D attracted maximum (34.7%) number of nematodes closely followed by Isolate C (31.0%), whereas, Isolate A, B and E did not differ significantly. Maximum in vitro trapping was recorded in the case of M. incognita (J₂) followed by M. graminicola (J₂), H. dihystera and T. brassicae. The maximum ringing (pre-capturing), trapping and killing was recorded for the second-stage juveniles of M. incognita followed by M. graminicola, T. brassicae and H. dihystera. The inflation of ring cells did not occur immediately after ringing; however, the time between ringing and inflation varied with different nematodes species. Clear hyphae were observed in the invaded nematodes body after the content of nematode body was consumed. External growth of hyphae occurred only from the inflated cells of the constricting rings, which further developed several rings. Higher number of rings was observed in mycelia developed from bigger nematodes.     

Modulation of nitric oxide formation by endothelial nitric oxide synthase gene haplotypes

Nitric oxide (NO) is a major regulator of the cardiovascular system. However, the effects of endothelial nitric oxide synthase (eNOS) gene polymorphisms or haplotypes on the circulating concentrations of nitrite (a sensitive marker of NO formation) and cGMP are unknown. Here we examined the effects of eNOS polymorphisms in the promoter region (T-786C), in exon 7 (Glu298Asp), and in intron 4 (4b/4a) and eNOS haplotypes on the plasma levels of nitrite and cGMP. We hypothesized that eNOS haplotypes could have a major impact on NO formation. We genotyped 142 healthy subjects by PCR-RFLP. To assess NO formation, the plasma concentrations of nitrite and cGMP were determined using an ozone-based chemiluminescence assay and an enzyme immunoassay. Haplotypes were inferred using the PHASE 2.1 program. No significant differences were found in age, body mass index, systolic and diastolic arterial blood pressure, heart rate, total cholesterol, triglycerides, cGMP, or nitrite among the genotype groups for the three polymorphisms studied here (all p>0.05). Interestingly, the C-4b-Glu haplotype was associated with lower plasma nitrite concentrations than those found in the other haplotype groups (p<0.05), but not with different cGMP levels (p>0.05). These findings suggest that eNOS gene variants combined within a specific haplotype modulate NO formation, although individual eNOS polymorphisms probably do not have major effects.     

Cross-talk between calcium-calmodulin and nitric oxide in abscisic acid signaling in leaves of maize plants

Using pharmacological and biochemical approaches, the signaling pathways between hydrogen peroxide （H2O2）, calcium （Ca^2＋）-calmodulin （CAM）, and nitric oxide （NO） in abscisic acid （ABA）-induced antioxidant defense were investigated in leaves of maize （Zea mays L.） plants. Treatments with ABA, H2O2, and CaCl2 induced increases in the generation of NO in maize mesophyll cells and the activity of nitric oxide synthase （NOS） in the cytosolic and microsomal fractions of maize leaves. However, such increases were blocked by the pretreatments with Ca^2＋ inhibitors and CaM antagonists. Meanwhile, pretreatments with two NOS inhibitors also suppressed the Ca^2＋-induced increase in the production of NO. On the other hand, treatments with ABA and the NO donor sodium nitroprusside （SNP） also led to increases in the concentration of cytosolic Ca^2＋ in protoplasts of mesophyll cells and in the expression of calmodulin 1 （CaM1） gene and the contents of CaM in leaves of maize plants, and the increases induced by ABA were reduced by the pretreatments with a NO scavenger and a NOS inhibitor. Moreover, SNP-induced increases in the expression of the antioxidant genes superoxide dismutase 4 （SOD4）, cytosolic ascorbate peroxidase （cAPX）, and glutathione reductase 1 （GR1） and the activities of the chloroplastic and cytosolic antioxidant enzymes were arrested by the pretreatments with Ca^2＋ inhibitors and CaM antagonists. Our results suggest that Ca^2＋-CaM functions both upstream and downstream of NO production, which is mainly from NOS, in ABA- and H2O2-induced antioxidant defense in leaves of maize plants.     

Spin trapping of nitric oxide by aci anions of nitroalkanes.

In alkaline solutions, nitroalkanes (RCH2NO2) undergo deprotonation and rearrange to an aci anion (RHC=NO2-), which may function as a spin trap. Using electron paramagnetic resonance (EPR) spectroscopy, we have investigated suitability of aci anions of a series of nitroalkanes (CH3NO2, CH3CH2NO2, CH3(CH2)2NO2, and CH3(CH2)3NO2) to spin trap nitric oxide (*NO). Based on the observed EPR spectra, the general structure of the adducts, formed by addition of *NO to RHC=NO2-, was identified as nitronitroso dianion radicals of general formula [RC(NO)NO2]*2- in strong base (0.5 M NaOH), and as a mono-anion radical [RCH(NO)NO2]*- in alkaline buffers, pH 10-13. The hyperfine splitting on 14N in the -NO2 moiety (11.2-12.48 G) is distinctly different from the splitting on 14N in the -NO moiety of the adducts (5.23-6.5 G). The structure of the adducts was verified using 15N-labeled *NO, which produced radicals, in which triplet due to splitting on 14N (I = 1) in 14NO/aci nitro adducts was replaced by a doublet due to 15N (I = 1/2) in 15NO/aci nitro adducts. EPR spectra of aci nitromethane/NO adduct recorded in NaOH and NaOD (0.5 M) showed that the hydrogen at alpha-carbon can be exchanged for deuterium, consistent with structures of the adducts being [CH(NO)NO2]*2- and [CD(NO)NO2]*2-, respectively. These results indicate that nitroalkanes could potentially be used as prototypes for development of *NO-specific spin traps suitable for EPR analysis.     

Phytoglobins in the nuclei,cytoplasm and chloroplasts modulate nitric oxide signaling and interact with abscisic acid

Symbiotic hemoglobins provide O₂ to N₂‐fixing bacteria within legume nodules, but the functions of non‐symbiotic hemoglobins or phytoglobins (Glbs) are much less defined. Immunolabeling combined with confocal microscopy of the Glbs tagged at the C‐terminus with green fluorescent protein was used to determine their subcellular localizations in Arabidopsis and Lotus japonicus. Recombinant proteins were used to examine nitric oxide (NO) scavenging in vitro and transgenic plants to show S‐nitrosylation and other in vivo interactions with NO and abscisic acid (ABA) responses. We found that Glbs occur in the nuclei, chloroplasts and amyloplasts of both model plants, and also in the cytoplasm of Arabidopsis cells. The proteins show similar NO dioxygenase activities in vitro, are nitrosylated in Cys residues in vivo, and scavenge NO in the stomatal cells. The Cys/Ser mutation does not affect NO dioxygenase activity, and S‐nitrosylation does not significantly consume NO. We demonstrate an interaction between Glbs and ABA on several grounds: Glb1 and Glb2 scavenge NO produced in stomatal guard cells following ABA supply; plants overexpressing Glb1 show higher constitutive expression of the ABA responsive genes Responsive to ABA (RAB18), Responsive to Dehydration (RD29A) and Highly ABA‐Induced 2 (HAI2), and are more tolerant to dehydration; and ABA strongly upregulates class 1 Glbs. We conclude that Glbs modulate NO and interact with ABA in crucial physiological processes such as the plant's response to dessication.     

Downregulation of nitric oxide formation by cytosolic phospholipase A2-released arachidonic acid

Exposure of PC12 cells to A23187 or thapsigargin caused a concentration-dependent release of arachidonic acid (AA) mediated by cytosolic phospholipase A2 (PLA2). Under the same conditions, however, analysis of nitric oxide (NO) formation revealed that activation of NO synthase (NOS) is best described by a bell-shaped curve. Reduced detection of NO observed at increasing A23187 or thapsigargin concentrations was not due to formation of peroxynitrite or to activation of NO-consuming processes, but rather to AA-dependent inhibition of NOS activity. Furthermore, NO formation observed under optimal conditions for NOS activity was suppressed by AA as well as by the PLA2 activator melittin. Finally, the effects of AA were not the consequence of direct enzyme inhibition, because this lipid messenger failed to inhibit formation of NO by purified neuronal NOS, but were mediated by an AA-dependent signaling and not by downstream products of the cyclooxygenase and lipoxygenase pathways. In conclusion, the present study underscores a novel mechanism whereby endogenous, or exogenous, AA promotes inhibition of NOS activity. Because AA is generated in response to various agonists acting on membrane receptors and extensively released in inflammatory conditions, these findings have important physiopathological implications.     

Differential effect of buffer on the spin trapping of nitric oxide by iron chelates.

Nitric oxide synthase (NOS) generates nitric oxide (NO*) by the oxidation of l-arginine. Spin trapping in combination with electron paramagnetic resonance (EPR) spectroscopy using ferro-chelates is considered one of the best methods to detect NO* in real time and at its site of generation. The spin trapping of NO* from isolated NOS I oxidation of L-arginine by ferro-N-dithiocarboxysarcosine (Fe(DTCS)2) and ferro-N-methyl-d-glucamide dithiocarbamate (Fe(MGD)2) in different buffers was investigated. We detected NO-Fe(DTCS)2, a nitrosyl complex, resulting from the reaction of NO* and Fe(DTCS)2, in phosphate buffer. However, Hepes and Tris buffers did not allow formation of NO-Fe(DTCS)2. Instead, both of these buffers reacted with Fe2+, generating sparingly soluble complexes in the absence of molecular oxygen. Fe(DTCS)2 and Fe(MGD)2 were found to inhibit, to a small degree, NOS I activity with a greater effect observed with Fe(MGD)2. In contrast, Fe(MGD)2 was more efficient at spin trapping NO* from the lipopolysaccharide-activated macrophage cell line RAW264.7 than was Fe(DTCS)2. Data suggested that Fe(DTCS)2 and Fe(MGD)2 are efficient at spin trapping NO* but their maximal efficiency may be affected by experimental conditions.     

The rational design of inhibitors of nitric oxide formation by inducible nitric oxide synthase

A series of compounds was rationally designed as inhibitors of dimer formation of the inducible isoform of nitric oxide synthase, and subsequent nitric oxide production. The conformation of two fragments obtained from a crystal structure was utilized to design a tether connecting those same two fragments. The resulting compounds were potent dimerization inhibitors that bound to the enzyme in a similar conformation as the fragments.     

Inactivation of nitric oxide by uric acid

The 1980 identification of nitric oxide (NO) as an endothelial cell-derived relaxing factor resulted in an unprecedented biomedical research of NO and established NO as one of the most important cardiovascular, nervous and immune system regulatory molecule. A reduction in endothelial cell NO levels leading to "endothelial dysfunction" has been identified as a key pathogenic event preceding the development of hypertension, metabolic syndrome, and cardiovascular disease. The reduction in endothelial NO in cardiovascular disease has been attributed to the action of oxidants that either directly react with NO or uncouple its substrate enzyme. In this report, we demonstrate that uric acid (UA), the most abundant antioxidant in plasma, reacts directly with NO in a rapid irreversible reaction resulting in the formation of 6-aminouracil and depletion of NO. We further show that this reaction occurs preferentially with NO even in the presence of oxidants peroxynitrite and hydrogen peroxide and that the reaction is at least partially blocked by glutathione. This study shows a potential mechanism by which UA may deplete NO and cause endothelial dysfunction, particularly under conditions of oxidative stress in which UA is elevated and intracellular glutathione is depleted.     

Nitric oxide counteracts the senescence of rice leaves induced by abscisic acid

In the present study, we evaluate the protective effect of nitric oxide (NO) against senescence of rice leaves promoted by ABA. Senescence of rice leaves was determined by the decrease of protein content. ABA treatment resulted in (1) induction of leaf senescence, (2) increase in H2O2 and malondialdehyde (MDA) contents, (3) decrease in reduced form glutathione (GSH) and ascorbic acid (AsA) contents, and (4) increase in antioxidative enzyme activities (superoxide dismutase, ascorbate peroxidase, glutathione reductase, and catalase). All these ABA effects were reduced by free radical scavengers such as sodium benzoate and GSH. NO donors [N-tert-butyl-alpha-phenylnitrone (PBN), sodium nitroprusside, 3-morpholinosydonimine, and AsA + NaNO2] were effective in reducing ABA-induced leaf senescence. PBN prevented ABA-induced increase in the contents of H2O2 and MDA, decrease in the contents of GSH and AsA, and increase in the activities of antioxidative enzymes. The protective effect of PBN on ABA-promoted senescence, ABA-increased H2O2 content and lipid peroxidation, ABA-decreased GSH and AsA, and ABA-increased antioxidative enzyme activities was reversed by 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a NO-specific scavenger, suggesting that the protective effect of PBN is attributable to NO released. Reduction of ABA-induced senescence by NO in rice leaves is most likely mediated through its ability to scavenge active oxygen species including H2O2.     

Oxidative formation of lactic acid by a fungus

Experimental evidence has been presented for an aerobic biosynthesis of lactate from a C₂-precursor. The data indicate that the C₄-dicarboxylic acids function as intermediates; oxygen is essential. The aerobic process occurs concomitantly with glycolytic lactate synthesis and accounts for the extra lactate yield under vigorously aerobic conditions. A scheme is presented which accounts for the observed facts and it is evident that the oxidative synthesis of lactate in reality involves an oxidative synthesis of pyruvate. This latter synthesis is discussed from the point of view of its importance to autotrophic organisms, which must of necessity synthesize the important intermediate, pyruvate, from carbon fragments consisting of two carbons or less.     

Spin trapping of nitric oxide by ferro-chelates: kinetic and in vivo pharmacokinetic studies

Biologically generated nitric oxide appears to play a pivotal role in the control of a diverse series of physiologic functions. Iron-chelates and low-frequency EPR spectroscopy have been used to verify in vivo production of nitric oxide. The interpretation of in vivo identification of nitric oxide localized at the site of evolution in real time is complicated by the varied kinetics of secretion. The quantitative efficiency of the spectroscopic measurement, so important in understanding the physiology of nitric oxide, remains elusive. The development of a more stable iron-chelate will help better define nitric oxide physiology. In this report, we present data comparing the commonly used ferro-di(N-methyl-D-glucamine-dithiocarbamate) (Fe2+(MGD)2) and the novel chelate ferro-di(N-(dithiocarboxy)sarcosine) (Fe2+(DTCS)2) quantifying the in vitro and in vivo stability of the corresponding spin trapped adducts, NO-Fe(MGD)2 and NO-Fe(DTCS)2. Finally, very low frequency EPR spectroscopy has been used to evaluate the pharmacokinetics of NO-Fe(MGD)2 and NO-Fe(DTCS)2 in mice in real time.     

Effects of potnetial trap crops and planting date on soil infestation with potato cyst nematodes and root-knot nematodes

In 1997 and 1998 the stimulation of hatch of potato cyst nematodes (PCN) by a trap crop was studied at various times during the growing season in a container and a field experiment. Solanum nigrum‘90‐4750‐188’was used as the trap crop in both experiments and was sown on 1 May, 16 June or 1 August in two successive years on different plots. Neither experiment revealed much seasonal variation in hatchability of PCN juveniles under a trap crop. In the container experiment, the hatch of the Globodera pallida Pa3 population was equally and strongly stimulated (89%) at all sowing dates in both years, except for the 1 August sowing in 1998 (when the hatch was 77% under extremely wet soil conditions). In the control treatment with non‐hosts (flax followed by barley) the total spontaneous hatch was 50% over 2 yr. In the field experiment, the hatch of PCN, averaged over the four populations, was also equally stimulated (71%) at all sowing dates in both years. In the control treatment with non‐hosts (flax‐barley) the total spontaneous hatch was 36% over 2 yr. Total hatch under the trap crop over 2 yr varied between the four PCN populations from 63% to 80%. In 1998 and 1999, control of potato cyst nematodes (PCN) by the potential trap crops Solanum sisymbriifolium and S. nigrum‘90‐4750‐188’was studied in the field. Potato was also included as a trap crop. In the 1998 experiment, potato, S. sisymbriifolium and S. nigrum strongly stimulated the hatch of PCN compared with the non‐host white mustard (Sinapis alba). Roots of potato and white mustard were mainly found in the top 10 cm of soil, whereas roots of S. sisymbriifolium and S. nigrum were also abundant at depths of 10–20 cm and 20–30 cm. In the 1999 experiment, soil infestation with PCN decreased markedly with potato and S. sisymbriifolium as trap crops. In plots moderately to severely infested with 2‐yr old cysts (2–29 juveniles ml^?1 air dried soil), potato reduced soil infestation by 87% and S. sisymbriifolium by 77%. In plots moderately to severely infested with 1‐yr old cysts the reductions were 74% and 60%, respectively. The reduction was least on plots very severely infested with PCN (110–242 juveniles ml^?1 soil): 69% and 52% for potato and S. sisymbriifolium, respectively. Soil infestations of plots that were initially slightly to severely infested with the root‐knot nematode Meloidogyne hapla were greatly reduced under fallow and S. sisymbriifolium but increased under potato. From these and previous experiments it was concluded that, for several reasons, S. sisymbriifolium is a promising trap crop.     

Detection of nitric oxide in tissue by spin trapping EPR spectroscopy and triacetylglycerol extraction

A modified method based on EPR spin trapping and triacetylglycerol extraction was used for tissue nitric oxide (NO) detection at room temperature. NO signal intensity was stable for about 1.5 h and the detection limit of this method was less than 200 pmol g^–1 tissue. Using this method, we report evidence that NO production in vivo can be inhibited by adriamycin in mice livers.     

Efficient formation of nitric oxide from selective oxidation of N-aryl N'-hydroxyguanidines by inducible nitric oxide synthase

Inducible nitric oxide synthase (NOS II) efficiently catalyzes the oxidation of N-(4-chlorophenyl)N'-hydroxyguanidine 1 by NADPH and O2, with concomitant formation of the corresponding urea and NO. The characteristics of this reaction are very similar to those of the NOS-dependent oxidation of endogenous Nomega-hydroxy-L-arginine (NOHA), i.e., (i) the formation of products resulting from an oxidation of the substrate C=N(OH) bond, the corresponding urea and NO, in a 1:1 molar ratio, (ii) the absolute requirement of the tetrahydrobiopterin (BH4) cofactor for NO formation, and (iii) the strong inhibitory effects of L-arginine (L-arg) and classical inhibitors of NOSs. N-Hydroxyguanidine 1 is not as good a substrate for NOS II as is NOHA (Km = 500 microM versus 15 microM for NOHA). However, it leads to relatively high rates of NO formation which are only 4-fold lower than those obtained with NOHA (Vm = 390 +/- 50 nmol NO min-1 mg protein-1, corresponding roughly to 100 turnovers min-1). Preliminary results indicate that some other N-aryl N'-hydroxyguanidines exhibit a similar behavior. These results show for the first time that simple exogenous compounds may act as NO donors after oxidative activation by NOSs. They also suggest a possible implication of NOSs in the oxidative metabolism of certain classes of xenobiotics.     

脱落酸对丛枝菌根真菌侵染和产孢的调控效应研究

[目的]揭示脱落酸(ABA)对丛枝菌根(AM)真菌侵染和产孢的影响,建立利用外源ABA促进孢子产量的高效菌剂扩繁方法.[方法]利用番茄毛状根和AM真菌Rhizophagus irregularis DAOM 197198建立双重培养体系,通过外源施用ABA、赤霉素(GA)或者使用ABA、GA的缺陷突变体,染色观察菌根侵...     

An endogenous rhythm of trap formation in the nematophagous fungus Arthrobotrys oligospora

In the predacious fungus Arthrobotrys oligospora Fres., the number and distribution of traps formed after the addition of living nematodes to the colonies were determined. At 21°C the traps were formed periodically; the mean period was 42.3±0.8 h. The periodicity was independent of light-dark (LD) cycles of 24 h (10:14). Temperature influenced the hyphal elongation but did not affect the periodic trap formation; at lower temperatures the peaks of trap formation were close together, showing partial overlapping. Induction of rhythmic mycelial growth and conidiation by chemical means was effective only in LD-cycles. The latter diurnal rhythm was weakly correlated with the trap formation and did not affect the endogenous period of approximately 42 h.Abbreviations LD light-dark - DD continuous darkness - LNM low-nutrient medium - CMA corn meal agar     

Interaction of intracellular hydrogen peroxide accumulation with nitric oxide production in abscisic acid signaling in guard cells

ABSTRACT

Reactive oxygen species and nitric oxide (NO?) concomitantly play essential roles in guard cell signaling. Studies using catalase mutants have revealed that the inducible and constitutive elevations of intracellular hydrogen peroxide (H₂O₂) have different roles: only the inducible H₂O₂ production transduces the abscisic acid (ABA) signal leading stomatal closure. However, the involvement of inducible or constitutive NO? productions, if exists, in this process remains unknown. We studied H₂O₂ and NO? mobilization in guard cells of catalase mutants. Constitutive H₂O₂ level was higher in the mutants than that in wild type, but constitutive NO? level was not different among lines. Induced NO? and H₂O₂ levels elicited by ABA showed a high correlation with each other in all lines. Furthermore, NO? levels increased by exogenous H₂O₂ also showed a high correlation with stomatal aperture size. Our results demonstrate that ABA-induced intracellular H₂O₂ accumulation triggers NO? production leading stomatal closure.