F. Bilka

Involvement of lipoxygenase in elicitor-stimulated sanguinarine accumulation in Papaver somniferum suspension cultures

The involvement of lipoxygenase (LOX, EC 1.13.11.12) in elicitor-induced opium poppy defense response was investigated. Papaver somniferum L. suspension cultures were treated with abiotic elicitor methyl jasmonate (MJ), fungal elicitor (Botrytis cinerea homogenate) and phenidone (specific inhibitor of LOX) to determine the involvement of this enzyme in production of sanguinarine, the major secondary metabolite of opium poppy cultures. P. somniferum suspension cultures responded to elicitor treatment with strong and transient increase of LOX activity followed by sanguinarine accumulation. LOX activity increased in elicited cultures, reaching 9.8 times of the initial value at 10 h after MJ application and 2.9 times after B. cinerea application. Sanguinarine accumulated to maximal levels of 169.5 ± 12.5 μg g⁻¹ dry cell weight in MJ-elicited cultures and 288.0 ± 10.0 μg g⁻¹ dry cell weight in B. cinerea-elicited cultures. The treatment of cells with phenidone before elicitor addition, significantly reduced sanguinarine production. The relative molecular weight of P. somniferum LOX (83 kDa) was estimated by using immunobloting and its pH optimum was shown to be pH 6.5.

An amine oxidase in seedlings of Papaver somniferum L.

Amine oxidase (AO) from 4-d-old seedlings of Papaver somniferum L. (Papaveraceae) was purified (58-fold) by using ammonium sulphate precipitation and chromatography on Sephadex G-150 and HA-Ultrogel columns. The most readily oxidized substrate was tyramine and other aromatic amines, while aliphatic amines cadaverine and putrescine were oxidized more slowly. Cu chelating and carbonyl reagents are the most effective inhibitors of poppy amine oxidase. Immunoblotting analysis showed cross reactivity of AO protein from poppy seedlings with polyclonal antisera against AO from pea. Obtained Mr value for AO from poppy (83 kDa) corresponds to that of copper AOs (75 – 90 kDa). These results suggest that the amine oxidase from poppy seedlings is a copper containing and tyramine specific AO.

Biochemical characterization of antigens detected with anti-platelet monoclonal antibodies

A panel of 18 monoclonal antibodies (mAbs) defined by the third workshop as specific for platelets, clustered in three preliminary groups: PC7, PC13 and PC27. These mAbs were further analysed by immunoprecipitation using extracts of iodinated and biotinylated peripheral blood mononucleated cells (PBMC) and platelets. We could confirm the existence of mAbs with specificities to WC9 (in PC7) and CD41/61 (in PC13). Two mAbs formed a new cluster, WC13, which may be homologous to human CD31 (in PC27). The influence of EDTA and thrombin on the expression of the different antigens on the platelet membrane was assessed by flow cytometry (FCM) analysis, as well as cross-reactivity with platelets from different species.

Characterization of Polyphenol Oxidase from the Latex of Opium Poppy

Polyphenol oxidase from the latex of opium poppy was purified to the electrophoretic homogeneity by affinity chromatography using p-aminobenzoic acid as a ligand coupled to Sepharose CL-4B by divinyl sulphone activation method. The purified enzyme was used to prepare the polyclonal antibodies. The purified latex PPO exhibited high diphenolase activity in comparison with almost unmeassurable monophenolase activity. Both of these activities were sensitive to the activation with sodium dodecyl sulphate. Two isoforms (65 and 40 kDa) of latex PPO were separated by the gel filtration. There were no differences in substrate specifity (weak monophenolase and high diphenolase activity) and sensitivity to inhibitors between these isoforms, but they showed differences in electrophoretic mobility.

Effect of N-Feruloylserotonin and Methotrexate on Severity of Experimental Arthritis and on Messenger RNA Expression of Key Proinflammatory Markers in Liver.

Rheumatoid arthritis (RA) is a chronic inflammatory disease, leading to progressive destruction of joints and extra-articular tissues, including organs such as liver and spleen. The purpose of this study was to compare the effects of a potential immunomodulator, natural polyphenol N-feruloylserotonin (N-f-5HT), with methotrexate (MTX), the standard in RA therapy, in the chronic phase of adjuvant-induced arthritis (AA) in male Lewis rats. The experiment included healthy controls (CO), arthritic animals (AA), AA given N-f-5HT (AA-N-f-5HT), and AA given MTX (AA-MTX). N-f-5HT did not affect the body weight change and clinical parameters until the 14th experimental day. Its positive effect was rising during the 28-day experiment, indicating a delayed onset of N-f-5HT action. Administration of either N-f-5HT or MTX caused reduction of inflammation measured as the level of CRP in plasma and the activity of LOX in the liver. mRNA transcription of TNF-α and iNOS in the liver was significantly attenuated in both MTX and N-f-5HT treated groups of arthritic rats. Interestingly, in contrast to MTX, N-f-5HT significantly lowered the level of IL-1β in plasma and IL-1β mRNA expression in the liver and spleen of arthritic rats. This speaks for future investigations of N-f-5HT as an agent in the treatment of RA in combination therapy with MTX.

Biochemical properties of lipoxygenase from opium poppy chloroplasts

Lipoxygenase (LOX) from opium poppy (Papaver somniferum L.) chloroplasts was isolated and 126.1-fold purified to electrophoretic homogeneity by combination of ion-exchange chromatography on HA-Ultragel column and affinity chromatography on a linoleyl-aminopropyl agarose column. The relative molecular mass of the LOX determined by SDS-PAGE was 92 kDa. Kinetic properties of purified LOX were determined in spectrophotometric assay by using of linoleic acid (KM = 1.78 mM and Vmax = 11.4 μmol mg−1 min−1) and linolenic acid (KM = 1.27 mM and Vmax = 10.2 μmol mg−1 min−1). The optimum pH was 6.0 for both linoleic and linolenic acid dioxygenation catalyzed by LOX. HPLC analysis of the products revealed a dual positional specificity of linoleic acid dioxygenation at pH 6.0 with ratio of 9- and 13-hydroperoxide products being about 1:1. The activity of purified LOX was stimulated by Mg2+ and Ca2+.

Evaluation of Manganese Chloride’s Effect on Biosynthetic Properties of In Vitro Cultures of Eschscholzia californica Cham.

The basal production of secondary metabolites in medicinal plants is limited. One of the effective approaches that encourages plants to produce a remarkable amount of precious compounds is an application of elicitors. Our work was focused on the elicitation of Eschscholzia californica Cham. suspension cultures using various concentrations of MnCl2 (5; 10; 15 mg/L) with the aim of evaluating its effect on sanguinarine, chelerythrine, and macarpine production and gene expression of enzymes involved in the biosynthesis of mentioned secondary metabolites (BBE, 4′-OMT, CYP80B1) or in defense processes (LOX). Suspension cultures were exposed to elicitor for 24, 48, and 72 h. The content of alkaloids in phytomass was determined on the basis of their fluorescence properties. The relative mRNA expression of selected genes was analyzed using the ΔΔCt value method. PCR products were evaluated by melting curve analysis to confirm the specific amplification. Our results demonstrated that Eschscholzia californica Cham. cell suspension cultures evince sensitivity to the presence of MnCl2 in growth media resulting in the increased production of benzophenanthridine alkaloids and gene expression of selected enzymes. Manganese chloride seems to be a potential elicitor supporting natural biosynthetic properties in plant cell cultures and can be applied for the sustained production of valuable secondary metabolites.

Ferulaldehyde Improves the Effect of Methotrexate in Experimental Arthritis

Methotrexate (MTX) is still the gold standard for treatment of rheumatoid arthritis (RA). The therapeutic efficacy of low-dose of MTX can be increased by its combination with a natural substance, ferulaldehyde (FRA). The aim of this study was to evaluate the effect FRA and MTX administered alone or in combination in adjuvant arthritis. The disease was induced to Lewis male rats by intradermal injection, which contains a suspension of heat-inactivated Mycobacterium butyricum in incomplete Freund’s adjuvant. The experiment of 28 days included: healthy animals, arthritic animals, arthritic animals with administration of FRA at the oral daily dose of 15 mg/kg, arthritic animals with administration of MTX at the oral dose of 0.3 mg/kg twice a week, and arthritic animals administered with FRA and MTX. FRA in monotherapy decreased significantly only the level of interleukin-1β (IL-1β) and matrix metalloproteinase-9 in plasma. Combination of FRA and low-dose MTX was more effective than MTX alone when comparing body weight, hind paw volume, arthritic score, plasmatic levels of IL-1β, activity of γ-glutamyl transferase, and relative mRNA expression of IL-1β in the spleen. Therefore, the combination treatment was the most effective. The obtained results are interesting for future possible innovative therapy of patients with RA.

Immunohistochemical reactivity of anti-platelet monoclonal antibodies

Ten mAbs of preliminary clusters PC13 and PC27 with specificity for bovine platelets were studied by immunohistochemistry. Cryostat sections of bovine lymph node, spleen, thymus, small intestine, liver, kidney and smears of bone marrow cells were used. Five mAbs (CAPP2, IVA30, IVA125, IL-A164 and IL-A166) assigned to cluster PC13 (CD41/CD61) stained platelets and non-lymphocytic cells of various tissues. Our data confirm the presence of two specificities in PC27: three mAbs (IVA120, IVA197 and IVA198) specific for fibrinogen strongly reacted with the endothelial and reticular tissues whereas the other two mAbs Co-3D1D4 and Buf13 (WC13) were negative.