Specific substrate for histone kinase II: a synthetic nonapeptide

Based on the previously determined intrinsic substrate specificity of histone kinase II, a nonapeptide was synthesized which was a specific substrate for this enzyme. The Vmax value of phosphorylation of the peptide (Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide) was about the same as that for H1 histone and the apparent Km for the phosphorylation of the peptide was 0.2 mM, an order of magnitude higher than that for H1 histone. H1 histone inhibited the phosphorylation of the peptide, while the peptide did not inhibit the phosphorylation of H1 histone. In the crude extracts of calf thymus, spleen and liver, histone kinase II was the only enzyme which phosphorylated the synthetic peptide. The rate of phosphorylation of this peptide was used to determine the activity of histone kinase II in the crude extracts of several tissues obtained from different species.     

The 38 kDa Ca2+/membrane-binding protein of pig granulocytes needs a high Ca2+ concentration to be phosphorylated by protein kinase C

The 38 kDa Ca2+/membrane-binding protein reported to be the dominant substrate of protein kinase C in the extracts of pig neutrophil granulocytes was purified partially and its phosphorylation was investigated. In pig granulocytes type II protein kinase C was the major isoform, while type III isoenzyme was present only as a minor activity. Phosphorylation of the 38 kDa protein was performed with rat brain protein kinase C. Each of the three isoenzymes purified from rat brain was able to phosphorylate this protein, though on the conditions used in our experiments it was phosphorylated most intensively by type II protein kinase C. A phospholipid-dependent, but Ca2(+)-independent, form of protein kinase C was demonstrated with the aid of a synthetic oligopeptide substrate. Phosphorylation of the 38 kDa protein by the Ca2(+)-independent enzyme proceeded exclusively in the presence of Ca2+. The Ca2+ concentration necessary for the phosphorylation of the 38 kDa by either form of protein kinase C was by orders of magnitude higher than that required for the activation of protein kinase C.     

Salicylic acid and its derivatives elicit the production of diterpenes and sterols in corals and their algal symbionts: a metabolomics approach to elicitor SAR

Introduction

The production of marine drugs in its normal habitats is often low and depends greatly on ecological conditions. Chemical synthesis of marine drugs is not economically feasible owing to their complex structures. Biotechnology application via elicitation represents a promising tool to enhance metabolites yield that has yet to be explored in soft corals.

Objectives

Study the elicitation impact of salicylic acid (SA) and six analogues in addition to a systemic acquired resistance inducer on secondary metabolites accumulation in the soft coral Sarcophyton ehrenbergi along with the symbiont zooxanthellae and if SA elicitation effect is extended to other coral species S. glaucum and Lobophyton pauciliforum.

Methods

Post elicitation in the three corals and zooxanthella, metabolites were extracted and analyzed via UHPLC-MS coupled with chemometric tools.

Results

Multivariate data analysis of the UHPLC-MS data set revealed clear segregation of SA, amino-SA, and acetyl-SA elicited samples. An increased level ca. 6- and 8-fold of the diterpenes viz., sarcophytonolide I, sarcophine and a C₂₈-sterol, was observed in SA and amino-SA groups, respectively. Post elicitation, the level of diepoxy-cembratriene increased 1.5-fold and 2.4-fold in 1 mM SA, and acetyl-SA (aspirin) treatment groups, respectively. S. glaucum and Lobophyton pauciliforum showed a 2-fold increase of diepoxy-cembratriene levels.

Conclusion

These results suggest that SA could function as a general and somewhat selective diterpene inducing signaling molecule in soft corals. Structural consideration reveals initial structure–activity relationship (SAR) in SA derivatives that seem important for efficient diterpene and sterol elicitation.

     

Identification of four components from the female sex pheromone of the lima-bean pod borer, Etiella zinckenella

Four compounds, tetradecyl acetate, (Z)-9-tetradecenyl acetate, (E)-11-tetradecenyl acetate and (Z)-11-tetradecenyl acetate were identified from female sex pheromone extracts of Hungarian and Egyptian lima-bean pod borers (Etiella zinckenella Tr., Lepidoptera: Phycitidae) by gas chromatography with flame ionization (FID) and electroantennographic (EAD) detection. In EAG studies these monoun-saturated acetates gave the best responses in a series of other tetradecenyl acetates and tetradecenols. The four component blend of the identified components in similar ratios as in the pheromone extract attracted significant numbers of male lima-bean pod borers in both Hungary and Egypt. In a preliminary subtraction test best capture was achieved by the ternary mixture of the monounsaturated acetates.

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Résumé A partir de femelles d'E. zinckenella d'origines hongroise et égyptienne, nous avons isolé quatre composés par chromatographie en phase gazeuse avec ionisation de flamme et électroantennographie (EAD): l'acétate de tétradécanyl, l'acétate (Z)-11-tétradécényl, l'acétate (E)-11-tétradécényl et l'acétate (Z)-11-tétradécényl. Les acétates monoinsaturés donnent les meilleures réponses en EAG parmi une série d'acétates tétradécényls et de tétradécénols. Les quatre composés mélangés dans les mêmes proportions que dans l'extrait de la phéromone ont attiré un nombre significatif de mâles tant en Egypte qu'en Hongrie. Dans un test préliminaire de soustraction, la meilleure capture a été réalisée par le mélange ternaire d'acétates monoinsaturés.

     

The assay of the activity of protein kinase C with the synthetic oligopeptide substrate designed for histone kinase II

The activity of histone kinase II was determined on the basis of its ability to phosphorylate the nonapeptide Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide designed previously as a specific substrate for this enzyme. Histone kinase II was purified from calf thymus extract by DEAE-cellulose chromatography followed by hydroxylapatite chromatography and high-performance liquid chromatography on a Protein Analysis column (I-125). The Mr value of histone kinase II estimated by the latter method was 50,000-55,000, but several observations indicated that histone kinase II was a product of a proteolytic process. Since the substrate specificity determinants for histone kinase II known from our previous investigations are very similar to those for protein kinase C, it was presumable that histone kinase II was the proteolytic fragment of protein kinase C. Therefore, the nonapeptide was tested as a substrate for protein kinase C prepared from rabbit brain extract by DEAE-cellulose chromatography. The activity of histone kinase II was also detected in brain extract. Histone kinase II was eluted from the DEAE-cellulose in the known position of the proteolytic fragment of protein kinase C. The nonapeptide Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide proved to be a better substrate than H1 histone for the detection of the activity of protein kinase C because it was not phosphorylated by the cAMP-dependent protein kinase and the Vmax of protein kinase C was about one order of magnitude higher with the peptide than with H1 histone. The apparent Km of protein kinase C for the peptide was identical with that of histone kinase II (0.2 mM).     

Flavonoid production in transgenic hop (Humulus lupulus L.) altered by PAP1/MYB75 from Arabidopsis thaliana L.

Hop is an important source of secondary metabolites, such as flavonoids. Some of these are pharmacologically active. Nevertheless, the concentration of some classes as flavonoids in wild-type plants is rather low. To enhance the production in hop, it would be interesting to modify the regulation of genes in the flavonoid biosynthetic pathway. For this purpose, the regulatory factor PAP1/AtMYB75 from Arabidopsis thaliana L. was introduced into hop plants cv. Tettnanger by Agrobacterium-mediated genetic transformation. Twenty kanamycin-resistant transgenic plants were obtained. It was shown that PAP1/AtMYB75 was stably incorporated and expressed in the hop genome. In comparison to the wild-type plants, the color of female flowers and cones of transgenic plants was reddish to pink. Chemical analysis revealed higher levels of anthocyanins, rutin, isoquercitin, kaempferol-glucoside, kaempferol-glucoside-malonate, desmethylxanthohumol, xanthohumol, α-acids and β-acids in transgenic plants compared to wild-type plants.     

Stilbene glucosides from the bulbs of Iris tingitana

Two new dimeric stilbene glucosides, tingitanol A (1) and tingitanol B (2) together with trans-resveratrol 3-O-glucopyranoside (3) in addition to three known isoflavones, 5-O-methylgenistein (4), 5-O-methylgenistein 7-O-β-d-glucopyranoside (5) and betavulgarin (6) have been isolated for the first time from the fresh bulbs of Iris tingitana Boiss. & Reut. Their structures were established on the basis of the spectral data and direct comparison with values from previously identified analogues. Additionally, the isolated compounds (1–6) were evaluated for the free radical scavenging activity.     

Agro-waste derived compounds (flax and black seed peels): Toxicological effect against the West Nile virus vector,Culex pipiens L. with special reference to GC–MS analysis

The development of different approaches to use agricultural residues as a source of high value-added products, become a must, especially after the problems emerged due to their accumulation. This contribution demonstrates the potential of agricultural residues, Linuim usitatissium (flax seed) and Nigella sativa (black seed) peels, as raw materials for the production of bioactive products, botanical insecticides, against Cx. pipiens, with deep analysis to their chemical constituents by gas chromatography-mass spectrometry, the larvicidal efficacies of the three crude extracts (methylene chloride, petroleum ether and methanol 70%) from the two plant waste peels were evaluated for the first time against the late third instar larvae of Cx. pipiens. Results indicated different lethal doses in larvae depending on the efficacy of organic solvent used. For both compounds methanol 70% extracts produced the highest dry yield. The most efficient solvent is petroleum ether in case of both flax and Black seed peels. Petroleum ether extract exhibited the highest toxicity against Cx. pipiens with an LC50 of 69.6383 ppm. The same results for black seed indicated that petroleum ether was the most efficient against Cx. pipiens with an LC50 of 40.7748 ppm. The study revealed for the first time the type of phytochemical constituents presents in peels of flax and black seeds using GC–MS analysis which revealed twenty-eight constituents among extracts of flax and black seed peels ranging from to 58.8711% to 99.99% of the total extracts. GC–MS profiling showed that a five constituents, 9-2-Methyl-Z, Z-3, 13 octadecadienol (terpenoid), 9,17-Octadecadienal, (Z)-, Nonanoic acid, 9-oxo-, methyl ester, 9,12-Octadecadienoic acid Z,Z and Octasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11,13,13,15,15-hexadecamethyl- have insecticidal activity beside many other biological activities as recorded from a variety of botanical extracts. While the constituents like Hexadecanoic acid, methyl ester and cis-9-Hexadecenal, both of them are larvicidal, cis-Vaccenic acid and 9-Oxononanoic acid showing only an insecticidal activity beside Undecanoic acid the mosquito repellent. The other six constituents Linoelaidic acid, Oleic Acid, Z-2-Octadecen-1-ol, 1-Methoxy-3-hydroxymethylheptane, Cis-11,14–Eicosadieonic acid-methyl ester and Heptasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11,13,13-tetradecamethyl- are constituents of other plant extracts which showed as a whole an insecticidal activity.