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Abstract

Hypoxia-ischemia (H-I) at the time of birth may cause neonatal death or lead to persistent brain damage. The search for an effective treatment of asphyxiated infants has not resulted in an effective therapy, and hypothermia remains the only available therapeutic strategy. Among possible experimental therapies, the induction of ischemic tolerance is promising. Recent investigations have shown that activation of group II metabotropic glutamate receptors (mGluR2/3) can provide neuroprotection against H-I, but the mechanism of this effect is not clear. The aim of this study was to investigate whether an mGluR2/3 agonist applied before H-I reduces brain damage in an experimental model of birth asphyxia and whether a decrease in oxidative stress plays a role in neuroprotection. Neonatal H-I on seven-day-old rats was used as an experimental model of birth asphyxia. Rats were injected intraperitoneally with the mGluR2/3 agonist 24 or 1 h before H-I (5 mg/kg). reduced the infarct area in the ischemic hemisphere. Application of the agonist at both times also reduced the elevated levels of reactive oxygen species (ROS) in the ipsilateral hemisphere observed after H-I and prevented the increase in antioxidant enzyme activity in the injured hemisphere. The decrease in glutathione (GSH) level was also restored after agonist application. The results suggest that the neuroprotective mechanisms triggered by the activation of mGluR2/3 before H-I act through the decrease of glutamate release and its extracellular concentration resulting in the inhibition of ROS production and reduction of oxidative stress. This, rather than induction of ischemic tolerance, is probably the main mechanism involved in the observed neuroprotection.

2.1. Induction of Cerebral Hypoxia–Ischemia

Neonatal cerebral H-I was induced according to Rice et al. (1981). Briefly, seven-day-old Wistar rat pups (weight 12–18 g) were anaesthetized with isoflurane (4% for induction, and 1.5–2.0% for maintenance) in a mixture of nitrous oxide and oxygen (0.6:1). The left common carotid artery was exposed and cut between double ligatures of silk sutures or only exposed (sham control). Animals were placed in home cages, allowed to recover for 60 min, placed in a humidified chamber (35 °C), and exposed to hypoxic conditions (8% oxygen in nitrogen) for 75 min. This duration of hypoxia-ischemia is typically associated with infarction predominantly of the cerebral hemisphere ipsilateral to the carotid artery occlusion [,]. After hypoxic treatment the animals were returned to the cages and housed at room temperature (22 °C) with a 12:12 h light–dark cycle and with ample food and water.

2.2. Drug Application

Animals were injected intraperitoneally (i.p.) with , a highly selective agonist of group II mGluRs, for which its affinity to mGluR2 is two times greater than that to mGluR3. The animals received a single injection made 24 or 1 h before H-I in a dose of 5 mg/kg of body weight. The dose was based on previous findings [,]. Sham-operated and H-I control rats were injected with saline.

3.1. LY379268 Reduces H-I Evoked Brain Damage

H-I resulted in brain tissue damage in the ipsilateral brain hemisphere. In the present study the evaluation of the infarct area using TTC staining showed that H-I produced 42% infarction of the ipsilateral hemisphere (). applied 24 or 1 h before H-I significantly decreased the area of infarction to 18.8% and 8.5%, respectively (p < 0.001). However, the infarct area measured after application of 1 h before H-I was significantly smaller than when the agonist was applied 24 h before the insult (p < 0.001).

3.2. LY379268 Reduces the H-I Evoked Increase in ROS Level

H-I resulted in an increase in the ROS level in the left ischemic hemisphere by as much as a factor of three (p < 0.001; ). The application of at both times significantly reduced the ROS level (p < 0.005). applied 24 h before H-I decreased the ROS level by 30% (p < 0.001), whereas when applied 1 h before H-I, it decreased the ROS level by 45%. However, the difference between the ROS level after agonist application at 24 or 1 h before H-I was not statistically significant. Neither of the treatments changed the ROS level in the right hemisphere.

3.3. LY379268 Pretreatment Reduces the H-I Increased Activity of SOD and Catalase

SOD activity in the brain tissue measured 3 h after H-I increased four times compared to the SOD activity in the control sham-operated animals (). Application of 24 h before H-I decreased SOD activity by 25% compared to the H-I group and by 42% when application of the agonist was performed 1 h before H-I.

Abstract

Domoic acid (DA) is a potent toxin, marine biotoxin, and primarily produced by Pseudo-nitzschia. The DA hapten was coupled with bovine serum albumin (BSA), and ovalbumin (OVA) as carrier proteins. DA-BSA conjugate was used as immunogen and DA-OVA as coating antigen. Cell fusion between spleen cells and sp2/0 myeloma cells developed 1C3 hybridoma clone producing 1C3 monoclonal antibody (mAb). Hybridoma was injected into the mice to produce ascites, and further purified by caprylic acid/ammonium sulfate method. The mAb was of IgG3 subclass, and was specific to DA with high affinity (2.5 × 108 L/mol). Moreover, western blot exhibited significant specificity to the DA antigens. Indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) showed DA working range of 0.006–0.2 ng/mL. The IC50 was 0.03 ng/mL with low limit of detection (LOD) of 0.006 ng/mL. Average DA recovery from spiked shellfish extract was 100.56 ± 2.8% with the coefficient variation of 0.01–0.1%. Hence, mAb producing 1C3 hybridoma was successfully developed and could be used to detect DA in contaminated samples.

2.4. Subclass and Chromosome Count of 1C3 Hybridoma Cell Line

The hybridoma cell line 1C3 against DA was analyzed for subclass using a commercial isotyping kit (IgM, IgG1, IgG2a, IgG2b, IgA, and IgG3). The results of the subclass analysis showed that the mAb producing 1C3 hybridoma cell had the IgG3 subclass (A). The chromosome count analysis by Geimsa staining exhibited that the chromosome number of 1C3 hybridoma cell line was 102 ± 4 (B) among the experiment replicates, and was produced by cell fusion between splenocytes with chromosome number 66 ± 4 and Sp2/0 myeloma cells with chromosome number 39 ± 1, respectively []. Therefore, the results indicated that the 1C3 hybridoma clone was produced by successfully fusing splenocyte and the Sp2/0 cell.

2.8. Preparation of the Standard Curve

The standard curve and recovery test of the 1C3 mAb was analyzed by competitive inhibition ELISA. The standard curve was prepared and the data was analyzed by Microcal OriginPro 9.1 to test the relationship of the concentration of DA with its inhibition. The standard curve was analyzed by the logistic curve and the equation was y = 0.447/(1 + (x/0.0349)0.795), and the correlation coefficient (R2) was about 0.98 (A). The linear equation was y = 0.2507 − 0.0452x with the correlation coefficient (R2) 0.99 (B). In the present study, the half inhibitory concentration (IC50) was 0.03 ng/mL, and the linear range of detection was 0.006–0.2 ng/mL with a lower LOD of 0.006 ng/mL.

2.9. Analysis of Recovery Test and Sample Detection by ic-ELISA Using 1C3 mAb

Recovery test was carried out to assess the quality of the established ic-ELISA, Non-contaminated shellfish extract was diluted properly to minimize the matrix effect and was spiked with DA at various concentrations (0.5, 5, 100, 1000 ng/mL) (). The results demonstrated that the recovery range was from (95.03 ± 5.4)% to (106.18 ± 1.7)% with an average recovery of (100.56 ± 2.8)%, and its coefficient of variation (CV) was ranged from 0.01 to 0.1% with an average CV of 0.03% (). Real shellfish samples (abalone, sea mussel, fresh oyster, giant clam, king scallop, razor clam, fresh water mussel and triangular clam) were randomly purchased from the seafood market for sample analysis. After extraction, the samples were assayed independently in duplicates to check the working of the established ic-ELISA for DA detection in complex matrix. The results showed that four samples from all shellfish samples were DA positive with levels under 30 ng/mL (120 ng/g), and the extracts of razor clam and giant clam showed the highest levels of DA at 6.70 ng/mL (26.80 ng/g) and 2.89 ng/mL (11.56 ng/g), respectively. Moreover, extracts of abalone (0.76 ng/mL) and fresh oyster (0.02 ng/mL) exhibited least detection, demonstrated DA levels lower than 1.0 ng/mL (4 ng/g). Similarly, all other shellfish samples were DA free with no contamination compared with the control in ic-ELISA at OD 450 nm using 1C3 mAb in the study ().

Abstract

As primary p53 antagonists, Mdm2 and the closely related Mdm4 are relevant cancer therapeutic targets. We have previously described a series of cell-permeable stapled peptides that bind to Mdm2 with high affinity, resulting in activation of the p53 tumour suppressor. Within this series, highest affinity was obtained by modification of an obligate tryptophan residue to the non-natural L-6-chlorotryptophan. To understand the structural basis for improved affinity we have solved the crystal structure of this stapled peptide (M011) bound to Mdm2 (residues 6–125) at 1.66 Å resolution. Surprisingly, near identity to the structure of a related peptide (M06) without the 6-chloro modification is observed. Further analysis of linear and stapled peptides comprising 6-Me-tryptophan provides mechanistic insight into dual Mdm2/Mdm4 antagonism and confirms L98 of Mdm4 as a mutable steric gate. The results also highlight a possible role of the flexible hinge region in determining Mdm2/Mdm4 plasticity.

Structural determination of M011 bound to Mdm2-M62A

The sequence compositions of peptides used in this study are shown in . The crystal structure of M011 bound to Mdm2-M62A shows 2 unique complexes in the asymmetric unit (, ). Within each, a single molecule of M011 interacts with a pronounced hydrophobic cleft that binds endogenous p53 (residues 19 to 26). As with other peptide antagonists, an interfacing signature triad of F19, W23 (6-chloro) and L26 residues (numbering based on p53 sequence) project their side chains into discrete apolar sub-pockets (“Phe”, “Trp” and “Leu” pockets) (). Comparison with the structure of a similar stapled peptide (M06) [], comprising native tryptophan bound to the same Mdm2 construct shows remarkable similarity. Each M011 complex, from the asymmetric unit, shows high structural similarity to the corresponding complex bound to M06 (all-atom derived RMSDs = 0.167 and 0.126 Å). This is particularly evident in the binding site, where minimal variation is observed in the ligand conformations (all-atom RMSDs = 0.055 and 0.031 Å). The major difference arises from rotation around the CHI1 bond of the L57 side chain in Mdm2 to widen the Trp pocket, with other residues comprising this pocket (F86, F91, I99) remaining unchanged (). This optimal formation of Van der Waals contacts around the 6-chloro moiety, resulting from the change in conformation of L59, explains the ~8-fold increased binding affinity of M011 over the parental stapled peptide lacking the 6-chloro modification (PM2, ). Structural differences are also observed in the lid/hinge region between the two M011: Mdm2 complexes in the asymmetric unit (). These result in a small conformational change of the M011 L26 residue. The observed changes in the hinge regions are primarily the result of crystal packing effects. However, these differences highlight how capping of the peptide binding groove by the hinge region can potentially impact on the binding of ligands to Mdm2.

2. Results and Discussion

The toxicity of BMAA in comparison to l-4-fluorophenylalanine (FPA) and m-tyr was determined by evaluating metabolic activity and apoptosis/necrosis in three human cell lines, so as to determine whether BMAA produced the same type of toxicity at similar concentrations as the known misincorporating amino acid analogues. The rationale was that if BMAA did not produce any toxicity, or produced a different profile of apoptosis and necrosis with increasing concentrations in any of the cell lines, the mechanism of toxicity must differ. Amino acid ratios, including the ratio of the analogue to the replaced encoded amino acid, of SDS-PAGE purified proteins from exposed cell lines were then used to determine whether any toxicity observed was the result of misincorporation of the analogue.

2.1. Metabolic Activity

shows the activity, as measured by the metabolic reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), of FPA, m-tyr, and BMAA-exposed HepG2 (A), Caco-2 (B) and HeLa (C) cells at analogue concentrations ranging from 0.25 mM to 2 mM. FPA decreased metabolic activity at all the concentrations tested in all cell lines. m-Tyr decreased metabolic activity in both Caco-2 and HeLa cell lines at all tested concentrations, but only at the highest tested concentration in HepG2 cells. BMAA had no significant effect on HepG2 or HeLa cells at any of the tested concentrations. Interestingly, in Caco-2 cells, BMAA at 1 mM and 2 mM significantly enhanced the metabolic activity. These data indicate an absence of BMAA toxicity in these non-neuronal cell lines.

2.2. Apoptosis/Necrosis

A concentration-dependent decrease in total cell numbers was observed for both HepG2 (A) and Caco-2 (A) cells exposed to m-tyr and FPA. A slight but significant decrease in cell number was observed for HepG2 cells exposed to BMAA at the highest tested concentrations (2 mM) but BMAA did not affect cell growth and division at any of the tested concentrations in Caco-2 cells (). The different response of cells to BMAA, compared to known misincorporating amino acid analogues, suggests that BMAA, where toxic at the highest concentration for the one cell line, might have a different mechanism of toxicity. This is supported by the absence of an effect by BMAA on MTT reduction.

2.3.2. l-4-Fluorophenylalanine Analysis

As for m-tyr, there is a clear and steady increase in the FPA/l-phenylalanine ratios with increasing concentration of FPA as shown in . Again, there is also a trend of increasing FPA/l-serine and FPA/l-glycine concentrations with increasing FPA exposure concentrations, as well as decreasing l-phenylalanine/l-serine and l-phenylalanine/l-glycine ratios as more l-phenylalanine is replaced with higher concentrations of FPA.

Results

Among the investigated compounds 1–9, the PSMA inhibitors 5 and 6 showed the highest PSMA affinity (lowest IC50 values) after the introduction of a naphthylalanine modification. The affinity was up to three times higher compared to the reference PSMA I&T. Extended aromatic systems such as the biphenylalanine residue in 4 impaired the interaction with the lipophilic binding pocket of PSMA, resulting in a tenfold lower affinity. The IC50 of DOTAGA-conjugated 10 was slightly increased compared to the acetylated analog; however, efficient PSMA-mediated internalization and 80% plasma protein binding of 68Ga-10 resulted in effective tumor targeting and low uptake in non-target tissues of LNCaP tumor-bearing CD-1 nu/nu mice at 1 h p.i., as determined by small-animal PET imaging and biodistribution studies. For prolonged tumor retention, the plasma protein binding was increased by insertion of 4-iodo-d-phenylalanine resulting in 97% plasma protein binding and 16.1 ± 2.5% ID/g tumor uptake of 177Lu-11 at 24 h p.i.

Conclusions

Higher lipophilicity of the novel PSMA ligands 10 and 11 proved to be beneficial in terms of affinity and internalization and resulted in higher tumor uptake compared to the parent compound. Additional combination with para-iodo-phenylalanine in the spacer of ligand 11 elevated the plasma protein binding and enabled sustained tumor accumulation over 24 h, increasing the tumor uptake almost fourfold compared to 177Lu-PSMA I&T. However, high renal uptake remains a drawback and further studies are necessary to elucidate the responsible mechanism behind it.

Background

In the literature, para-halogenated aromatic systems conjugated to the Nɛ-amino group of KuE seem favorable in terms of PSMA affinity [] and PSMA inhibitors containing a para-iodo-benzoic acid derivative as albumin binder demonstrated an increased tumor uptake in vivo [, ]. In addition, para-halogenated aromatic systems are reported to increase plasma protein binding [–], and a reduced tracer uptake in the kidneys was reported for radiopharmaceuticals modified with an albumin-binding entity []. Therefore, we designed several derivatives of the known PSMA inhibitor PSMA I&T in terms of structural modification of the spacer unit with lipophilic moieties, and additionally, we introduced a para-halogenated aromatic residue, para-iodo-phenylalanine (I-f), into the linker between the binding motif and the spacer unit. Subsequently, all PSMA ligands were evaluated regarding PSMA affinity, and the most promising structure 6 was further used to develop the PSMA inhibitors 10 and 11 (Fig. ). These two ligands were radiolabeled with 68GaIII and 177LuIII and comparatively evaluated with particular respect to internalization in LNCaP cells, lipophilicity, plasma protein binding, and albumin binding. In order to assess the influence of the structural modifications in vivo, both ligands were evaluated in micro-PET imaging and biodistribution studies and compared to PSMA I&T.Fig. 2Chemical structures of the DOTAGA-conjugated PSMA inhibitors PSMA I&T, 10, and 11

Synthesis

Hydrolytic stability was especially important for the synthesis of PSMA inhibitor 11. To address plasma proteins, a PSMA inhibitor with a halogenated aromatic moiety (4-iodo-d-phenylalanine; I-f) was developed on the basis of 10. Substitution of the suberic acid spacer with glutaric acid-(I-f) resulted in DOTAGA-y-nal-k(Glut-(I-f)-KuE) (11). For the synthesis of 11, PfpO-Glut-(I-f)-(OtBu)KuE(OtBu)2 was synthesized in solution from (OtBu)KuE(OtBu)2, Fmoc-d-iodo-phenylalanine, and the bis-pentafluorophenylester of glutaric acid (Glut(OPfp)2) and was purified using RP-HPLC. Glut(OPfp)2 was prepared from glutaric acid using pyridine and pentafluorophenol in 87% yield after flash chromatography. Synthesis yields of PSMA inhibitors 1–11 are summarized in Additional file : Table S1.

Abstract

The development of radiation countermeasures for acute radiation syndrome (ARS) has been underway for the past six decades, leading to the identification of multiple classes of radiation countermeasures. However, to date, only two growth factors (Neupogen and Neulasta) have been approved by the United States Food and Drug Administration (US FDA) for the mitigation of hematopoietic acute radiation syndrome (H-ARS). No radioprotector for ARS has been approved by the FDA yet. Gamma-tocotrienol (GT3) has been demonstrated to have radioprotective efficacy in murine as well as nonhuman primate (NHP) models. Currently, GT3 is under advanced development as a radioprotector that can be administered prior to radiation exposure. We are studying this agent for its safety profile and efficacy using the NHP model. In this study, we analyzed global metabolomic and lipidomic changes using ultra-performance liquid chromatography (UPLC) quadrupole time-of-flight mass spectrometry (QTOF-MS) in serum samples of NHPs administered GT3. Our study, using 12 NHPs, demonstrates that alterations in metabolites manifest only 24 h after GT3 administration. Furthermore, metabolic changes are associated with transient increase in the bioavailability of antioxidants, including lactic acid and cholic acid and anti-inflammatory metabolites 3 deoxyvitamin D3, and docosahexaenoic acid. Taken together, our results show that the administration of GT3 to NHPs causes metabolic shifts that would provide an overall advantage to combat radiation injury. This initial assessment also highlights the utility of metabolomics and lipidomics to determine the underlying physiological mechanisms involved in the radioprotective efficacy of GT3.

1. Introduction

Here, we have studied GT3 induced changes in metabolomic and lipidomic profiles as part of its safety and toxicity testing in the NHP model. We report GT3-induced longitudinal changes in metabolic and lipidomic serum profiles of NHPs using a global metabolomic and lipidomic approach with an ultra-performance liquid chromatography (UPLC) quadrupole time-of-flight mass spectrometry (QTOF-MS) platform. We found few changes in metabolite profiles during the first 24 h following GT3 administration. There were significant changes in profiles of several metabolites over time (1–3 days) that returned to baseline levels over the next 20-day follow-up period. Furthermore, there was a significant overlap of metabolite profiles for the three doses of GT3 that were tested in this study. The findings from metabolic profiles were consistent with complete blood count (CBC) and blood chemistry analyses of the same set of animals post GT3 administration. Taken together, these results suggest that GT3 administration does not have adverse consequences on overall metabolism and is safe for use as a potential radiation countermeasure.

2.1. Identification of Metabolite Signatures of GT3 Administration in NHPs

Untargeted metabolomic and lipidomic profiling was performed for a total of 208 serum samples obtained from 12 NHPs (four per dosage group) in this pilot study. All NHPs were included in the analysis since there were no outliers. A total of 3667 features in the Electrospray Ionization (ESI) positive mode and 4914 features in the ESI negative mode were detected for metabolomics (Acquity BEH C18 column) while a similar analysis of lipidomics (Acquity CSH C18 column) yielded 1573 features in the ESI positive and 622 in ESI negative mode. Scores plots () were analyzed to evaluate group separation resulting from inherent differences in metabolite profiles of NHPs that received 9.375, 18.75, and 37.5 mg/kg of GT3 as compared to the pre-administration samples. Partial least squares-discriminant analysis (PLS-DA) plots of the negative ionization compounds and lipids are presented in . The coefficient of variation (CV) values of internal standards for quality control samples (QCS) as well as the total ion chromatogram (TIC) overlays have been presented as . We asked if GT3 administration induced changes in profiles in the short-term (first 24 h) (a) and/or if overall metabolite profiles changed over the long-term (2 days to 20 days) (b). Each time point following GT3 administration was defined as an independent class for multivariate analysis. Principal component analysis did not yield separation for any of the models. PLS-DA multi-class models including two components yielded R2 = 0.52 and Q2 = 0.19 for the short-term group and R2 = 0.43 and Q2 = 0.07 for the long-term group. There was a significant overlap between the treated groups with the pre-injection samples for time points monitored within the first 24 h, demonstrating that GT3 administration induced minimal changes in overall metabolite profiles. There was a modest separation of treated groups for later time points (1–3 days). Additionally, there was a significant overlap between groups for different doses of GT3. This suggests that a sample size of four may not be large enough to observe dose-dependent metabolic changes when the difference in GT3 concentration is only two-fold. Thus, we determined the mean for results obtained with three different doses of GT3 at each time point to compare with pre-drug injection samples.

2.1. Identification of Metabolite Signatures of GT3 Administration in NHPs

Next, chemical formula calculations for 2323 significantly dysregulated metabolites (using ANOVA, adjusted p < 0.05) were performed via accurate mass based search and the identity of 61 metabolites was confirmed using tandem mass spectrometry (). We used hierarchical clustering to visualize time and/or dose dependence of these validated metabolites, as a heat map (). While there were no appreciable changes in relative abundance within 24 h, we observed transient changes in abundance 1–3 days post-administration of GT3, and the endogenous levels returned to normal over the extended time course.

Abstract

The development of a biotechnological method for the production of new biologically active phosphatidylcholine containing monoterpene citronellic acid (CA) was the aim of this work. Incorporation of citronellic acid (CA) into egg-yolk phosphatidylcholine (PC) in the lipase-catalyzed acidolysis process was studied. Isoprenoid acid CA was used as an acyl donor and five commercially available immobilized lipases were examined as biocatalysts. The effects of organic solvent, enzyme load, reaction time and molar ratio of substrates on the incorporation of citronellic acid (CA) into the phospholipids were evaluated. Modified phospholipid fraction enriched with CA in the sn-1 position (39% of incorporation) was obtained in high 33% yield using Novozym 435 as biocatalyst. In this study a biotechnological method for production of new phospholipid biopreparation enriched with citronellic acid, which can play an important role as a nutraceutical, was applied.

2.1. Screening of Lipases

The progress of the reactions was monitored by collecting samples of the product mixtures after allowing the reactions to proceed for chosen length of time (12, 24, 48 and 72 h). The products mixtures were subsequently subjected to the solid phase extraction (SPE) to aid separation of PLs from the acids. In the next step, the fatty acid composition in the PL fraction was analyzed by gas chromatography (GC), whereas the information about the proportion between the modified and native PC was provided by high-performance liquid chromatography (HPLC) analysis. In these initial studies the PL were not fractionated into individual PC and lysophosphatidylcholine (LPC), which is formed in this process due to partial hydrolysis of phosphatidylcholine during the acidolysis reaction. The time course of the incorporation of CA into PC by lipases is presented in . It can be observed that only lipase B from C. antarctica immobilized on a macroporus acrylic resin (Novozym 435) was able to produce the desired PC with high incorporation level among all tested biocatalysts. This enzyme exhibits the highest activity in PC modification giving 19% incorporation of CA into phospholipid fraction after 48 h of reaction. Preparations of Lipozyme®, Lipozyme TL IM and CALB, showed significantly lower activity and the CA incorporation into phospholipid fraction did not exceed 9%, whereas lipase A from C. antarctica was inactive in acidolysis process. Novozym 435 was therefore used as the biocatalyst on the synthesis of phospholipid biopreparation enriched with citronellic acid for the following experiments.

2.6. Identification of Acidolysis Products

The best method for identification of products of enzymatic modifications and the composition of modified phospholipid fractions in our experiments was HPLC equipped with a Corona charged aerosol detector (CAD). This method was chosen because we had the standards of products that can be formed during the acidolysis reaction: 1-citroneloyl-2-palmitoyl-sn-glicero-3′-phosphatidylcholine (1-CA-2-PA-PC), as an analogue of modified PC and 1-citroneloyl-2-hydroxy-sn-glicero-3′-phosphatidylcholine (1-CA-2-OH-LPC). Those products we have obtained previously by the chemical methods []. We used also as the standards native PC (PC-egg yolk) and product of its enzymatic hydrolysis (LPC-egg yolk). Below we present the HPLC chromatogram of products mixture (phospholipid fraction) () and fatty acids (wt %) profile of modified phospholipid fraction () for the best variant of the reaction of acidolysis of PC with CA with optimized parameters: organic solvent (toluene), lipase (Novozym 435, enzyme load 30%), substrate molar ratio (PC:CA, 1:60) and reaction time (48 h).

Abstract

Systemic acquired resistance (SAR) is a defence mechanism that induces protection against a wide range of pathogens in distant, pathogen-free parts of plants after a primary inoculation. Multiple mobile compounds were identified as putative SAR signals or important factors for influencing movement of SAR signalling elements in Arabidopsis and tobacco. These include compounds with very different chemical structures like lipid transfer protein DIR1 (DEFECTIVE IN INDUCED RESISTANCE1), methyl salicylate (MeSA), dehydroabietinal (DA), azelaic acid (AzA), glycerol-3-phosphate dependent factor (G3P) and the lysine catabolite pipecolic acid (Pip). Genetic studies with different SAR-deficient mutants and silenced lines support the idea that some of these compounds (MeSA, DIR1 and G3P) are activated only when SAR is induced in darkness. In addition, although AzA doubled in phloem exudate of tobacco mosaic virus (TMV) infected tobacco leaves, external AzA treatment could not induce resistance neither to viral nor bacterial pathogens, independent of light conditions. Besides light intensity and timing of light exposition after primary inoculation, spectral distribution of light could also influence the SAR induction capacity. Recent data indicated that TMV and CMV (cucumber mosaic virus) infection in tobacco, like bacteria in Arabidopsis, caused massive accumulation of Pip. Treatment of tobacco leaves with Pip in the light, caused a drastic and significant local and systemic decrease in lesion size of TMV infection. Moreover, two very recent papers, added in proof, demonstrated the role of FMO1 (FLAVIN-DEPENDENT-MONOOXYGENASE1) in conversion of Pip to N-hydroxypipecolic acid (NHP). NHP systemically accumulates after microbial attack and acts as a potent inducer of plant immunity to bacterial and oomycete pathogens in Arabidopsis. These results argue for the pivotal role of Pip and NHP as an important signal compound of SAR response in different plants against different pathogens.