Ts. A. Egorov

Discovery of novel antimicrobial peptides with unusual cysteine motifs in dandelion Taraxacum officinale Wigg. flowers

Three novel antimicrobial peptides designated ToAMP1, ToAMP2 and ToAMP3 were purified from Taraxacum officinale flowers. Their amino acid sequences were determined. The peptides are cationic and cysteine-rich and consist of 38, 44 and 42 amino acid residues for ToAMP1, ToAMP2 and ToAMP3, respectively. Importantly, according to cysteine motifs, the peptides are representatives of two novel previously unknown families of plant antimicrobial peptides. ToAMP1 and ToAMP2 share high sequence identity and belong to 6-Cys-containing antimicrobial peptides, while ToAMP3 is a member of a distinct 8-Cys family. The peptides were shown to display high antimicrobial activity both against fungal and bacterial pathogens, and therefore represent new promising molecules for biotechnological and medicinal applications.

Defense peptides from barnyard grass (Echinochloa crusgalli L.) seeds.

A number of defense polypeptides from latent seeds of weed cereal barnyard grass (Echinochloa crusgalli L.) has been isolated and characterized using an acidic extraction and high performance liquid chromatography methods in combination with MALDI-TOF mass spectrometry and Edman sequencing. Members of three antimicrobial peptide families and two protease inhibitor families were found to be localized in barnyard grass seeds. Their biological activity concerning to Gram-Positive and Gram-Negative phytopathogenic bacteria, as well as oomycete Phytophthora infestans, has been investigated. Diversity of barnyard grass defense peptides is a significant factor that provides a resistance of E. crusgalli seeds to germination and latent phases.

Defense peptides of plant immunity

Antimicrobial peptides (AMPs) are natural antibiotics produced by all living organisms to fight pathogens. They are important effector molecules of the immune system both in animals and plants. AMPs are diverse in structure and mode of action. Based on the homology of amino acid sequences and 3D structures several AMP families have been distinguished. They are defensins, thionins, lipid transfer proteins, hevein- and knottin-like peptides, and cyclotides. AMPs display broad-spectrum antimicrobial activity and thus show promise for the development of disease-resistant crops by genetic engineering and for the production of new-generation drugs. In this paper, the properties of the main AMP families (defensins and hevein-like peptides) and of new 4-Cys plant AMP family are reviewed.

Heterologous expression of a synthetic gene encoding a novel hevein-type antimicrobial peptide of Leymus arenarius in Escherichia coli cells

A novel antifungal peptide, LAMP-1a, was isolated from sand-elymus (Leymus arenarius) seeds. Expression of a synthetic gene encoding this peptide in Escherichia coli cells was obtained. The target peptide was expressed as a fusion with thioredoxin. Identity of the recombinant peptide to native LAMP-1a was confirmed by chromatography, mass spectrometry, and amino acid sequencing. LAMP-1a displayed a high inhibitory activity in respect of a number of phytopathogenic fungi in in vitro assays, which opens up possibilities for the gene encoding it to be used for genetic transformation of plants and for engineering pathogen-resistant crops

The purification and characterization of a novel lipid transfer protein from caryopsis of barnyard grass (Echinochloa crusgalli)

A novel lipid transfer protein called Ec-LTP was isolated from resting caryopsis of weed barnyard grass Echinochloa crusgalli (L.) Beauv.; its molecular weight, amino acid content and N-terminal amino acid sequence were determined. Ec-LTP was a 9150 D protein, containing eight cysteine residues, which formed four disulfide bonds. The isolated protein could significantly inhibit the development of pathogenic fungi Phytophthora infestans and Helminthosporium sativum, causing the late blight of potato and tomato and the root rot of herbs, respectively.

Phenolic Glycoside Isolated from Seeds of the Greater Plantain (Plantago major L.)

To attain this goal, we developed a method of isolation of active components based on gel-filtration of extract with further purification of the active fraction D using the reverse-phase high-performance liquid chromatography (HPLC) (Fig. 1). As a result of the study, an active component capable of providing effective suppression of the growth of the fungus Helminthosporium sativum (syn. Bipolaris sorokiniana ) was isolated.

[Actinoporins from the Sea of Japan anemone Oulactis orientalis: isolation and partial characterization].

Two cytolytic toxins (cytolysins Or-A and Or-G) were isolated from the Sea of Japan anemone Oulactis orientalis and characterized. Their purification scheme involved a hydrophobic chromatography on Polychrom-1, a gel filtration on Akrilex P-4, a cation-exchange chromatography on CM-32 cellulose, and a reverse-phase HPLC on a Nucleosil C18 column. The molecular masses of Or-A and Or-G were determined by SDS-PAGE in 14% PAG to be ca. 18 kDa. The absence of Cys residues and a high content of basic amino acid residues are characteristic of their amino acid compositions. The hemolytic activities of Or-A and Or-G were found to be 295.86 and 322.58 HU/mg, respectively; these are by three orders of magnitude lower than those of sphingomyelin-inhibitable cytolysins from the tropic sea anemones. The amino acid sequences of the N-terminal fragments of Or-A and Or-G were determined to be ATFRVLAK and GAIIAGAA, respectively. Action of the cytolysins on the erythrocyte membrane is inhibited by exogenous sphingomyelin. They form ion channels in bilayer lipid membranes with the conductivity of 16, 32, and 40 pSm in 0.1 M NaCl and 168, 240, and 320 pSm in 1 M NaCl at pH 7.2. Therefore, they were attributed to the group of actinoporins.

Isolation of the Lipid-transporting Protein Ns-LTP1 from Seeds of the Garden Fennel Flower ( Nigella sativa )

A novel lipid-transporting protein (Ns-LTP1) has been isolated from seeds of the garden fennel flower Nigella sativa. The molecular mass, N-terminal amino acid sequence, and amino acid composition of the protein have been determined. Ns-LTP1 has a molecular mass of 9602 Da and contains eight cysteine residues which form four disulfide bridges. The protein is capable of suppressing the development of some phytopathogenic fungi and oomycetes.

Increase of resistance of Arabidopsis thaliana plants to phytopathogenic fungi expressing hevein-like peptides from weed plant Stellaria media

It is shown that constitutive hyperexpression of new hevein-like peptides from the weed plant chickweed (Stellaria media)in mouse-ear cress (Arabidopsis thaliana) plants leads to a substantial increase of their resistance to phytopathogenic fungi Botrytis cinerea and Bipolaris sorokiniana. Thus, common chickweed peptides can play a definite role in protecting this weed plant and be useful as a new genetic tool for producing plants resistant to fungal diseases.

Comparative Analysis of Extracts of Nigella sativa Exhibiting Antifungal Activity against the Oomycete Phytophthora infestans

Phytophthorosis is one of the most economically significant potato diseases in the world and can led to total loss of a harvest. At present, the only effective means for limiting the spread of this disease in agricultural mass production is multiple applications of fungicides, residues of which persist in the final product. Antifungal properties, in particular, against the phytophthorosis vector, were obtained by using crude extracts of many plants [1–3]. Recent scientific research in this area is directed at answering the question of whether plant extracts can be used for battling diseases and pests of cultivated plants in the majority of developing countries [4]. We studied the plant Nigella sativa L. growing in Central and Southeast Asia [5], the seeds of which are traditionally used in folk medicine [6, 7]. We compared the antifungal activity of extracts of N. sativa seeds against the phytopathogenic oomycete Phytophthora infestans and characterized the active components. Total extract from seeds that were defatted beforehand of essential oils, lipids, and fatty acids was obtained by acidic extraction with subsequent precipitation by cold Me2CO. Preliminary fractionation of the extracted compounds was attained at this stage. The precipitate (extract No. 1) was dried in air and dissolved in distilled H2O to concentration 0.5 mg/mL. The extract had limited solubility in distilled H2O at higher (1.0 mg/mL and greater) concentrations. Therefore, this value was taken as the maximum. Test results of the obtained solution showed stable and pronounced suppression of the development of P. infestans on the surface of potato disks after incubation for 144 d (pathogen development was about 28% or “++”). The next purification stage of this extract consisted of its desalting primarily from low-molecular-weight compounds by RP-HPLC. The fraction bound to the column (extract No. 2) was eluted and analyzed by mass spectrometry (MS). Mass spectra were taken in linear mode in the range 1–17 kDa. According to MS, the total desalted extract of seeds contained principal masses in the peptide range (2.5–5.7 kDa) and in the range 9.6–11.3 kDa. The resulting total mass spectrum suggested that many compounds with molecular weights (MWs) in the peptide range were present in the desalted extract. This extract was separated further by affinity chromatography with a stepwise gradient of increasing NaCl concentration. As a result, three summed fractions called A, B, and C were collected. These were desalted and analyzed by MS (Table 1). A set of masses in the peptide range (<10 kDa) was identified in each of the obtained fractions. MS analysis of the fraction not bound to the column (extract No. 3) showed the presence of a predominant number of components with MWs primarily in the range 0.4–1.5 kDa (data not presented). Qualitative analysis of them was difficult because of overlap of part of the obtained spectrum on that of the matrix (2,5-dihydroxybenzoic acid). Thus, extract No. 2 that was obtained by desalting of the acidic (AcOH) Me2CO precipitate by RP-HPLC was dominated primarily by components of protein-peptide nature [8] that had basic properties whereas extract No. 3 consisted presumably of low-molecular-weight compounds (polyphenolic compounds, pigments, flavonoids, etc.).