Nowadays, there is growing interest in new compositions and drugs that can preserve and maintain human health. There are many studies on both known probiotics and new sources of biologically active substances which may potentially be new avenues for human longevity. Here, we describe the protective effect of cell lysates of the relict bacteria Bacillus sp. F (Bacilus Lyuba), isolated from the ancient permafrost of Central Yakutia, upon DNA damage, induced by hydrogen peroxide. The DNA damage was monitored by a comet assay in leukocytes isolated from the peripheral blood of experimental animals (BALB/c mice). Cell lysates were prepared using the French Press and ultrasonic treatment. The levels of DNA damage in the presence of bacterial lysates upon the stress test (20 μM hydrogen peroxide for 10 min at 37°C) were almost twice lower, compared with the buffer control. Of note, the maximal protective effect was observed in the temperature range of 50°C–60°C; with the increase of the temperature, this effect vanished. The obtained results do not give a clear answer on the mechanism of the protective properties of Bacillus sp. F. (Bacilus Lyuba) lysates. To answer this question, a detailed analysis of the protein composition of bacterial lysate at different temperatures should be performed.

The paper reports the study of the effects of N,N1-diphenylurea (DPU), which has cytokinin activity, on photosynthesis, photophosphorylation rate, and chloroplast structure in mesophyll cells in rapeseed plants in the flowering phase. DFM treatment affected photosystem 2 (PS-2) and changed the ratio between the rates of cyclic (CP) and noncyclic photophosphorylation (NCP) in thylakoid membranes isolated from flag leafs. CP rate in the chloroplasts increased by 25–30

The aim of our work was to study the colonization of potato, tomato, rapeseed and camelina by associative microorganisms Methylobacterium mesophilicum, Pseudomonas aureofaciens BS1393 and Pseudomonas putida BS3701; examine the resistance of colonized plants to biotic (phytopathogens Erwinia carotovora and Sclerotinia sclerotiorum) and abiotic (naphthalene, oil) stressors. Colonized plants were characterized by an increased growth rate (1.5–2.0 times higher) compared to non-colonized ones; flower-bud formation, flowering and fructification of the colonized plants also started earlier. An increased resistance of colonized plants to phytopathogens, naphthalene (100 mµ/ml) and oil (0.7 %) was noted, too. The level of superoxide dismutase (SOD) in control plants on a medium with naphthalene or oil increased by 160–150%; in colonized plants – by 20–18 %. Colonized plants were more viable because of the presence of P. putida BS3701 on the roots.

The biological activity of extracts from aloe and kalanchoe plants subjected to cold stress increases compared to extracts not subjected to cold stress.

The in vivo and in vitro interactions between tomato (Lycopersicon esculentum Mill.) and tobacco (Nicotiana tabacum L.) and the bacteria Pseudomonas fluorescens, Acinetobacter baumannii, Rhodococcus erythropolis,Pseudomonas aureofaciens, Pseudomonas putida, Methylovorus mays have been studied. These microorganisms were shown to be in stable associations with plants. The colonized plants were characterized by more rapid growth, a higher yield, and better adaptation to in vivo conditions. The colonized plants were more resistant to bacterial phytopathogens Erwinia carotovora and Pseudomonas syringae. Plants colonized by naphthalene-resistant bacteria can grow steadily on a medium containing this compound. The results demonstrate the prospects of the use of beneficial associative microorganisms in the development of technologies for plant protection against biotic and abiotic stressors.

Marker-free transgenic Camelina sativa (L.) plants carrying a synthetic gene for cecropin P1, an antimicrobial peptide, under the control of the cauliflower mosaic virus 35S RNA promoter have been obtained and analyzed. The plants were transformed with an agrobacterial binary vector free of selective genes of antibiotic and herbicide resistance. The marker-free transformants were screened via measurement of the antibacterial activity of cecropin P1 and enzyme immunoassay. The obtained plants exhibited an increased resistance to infection with the bacteria Erwinia carotovora, the fungi Fusarium graminearum, and oxidative stress during infection. Analysis of the fatty acid composition of seed oil showed an increased amount of α-linolenic acid in the transgenic Camelina lines as compared to unmodified plants. The results indicate that the cecropin P1 gene can be included in an integral antistress plant-protective system.

A study of the plants rapeseed (Brassica napus L.) with artificial gene antimicrobial peptide cecropin P1 has shown enhanced resistance of transgenic plants to phytopathogenic microorganisms Erwinia carotovora, Fusarium oxysporum, Sclerotinia sclerotiorum and oxidative stress, as compared to the nontransformed plants.

Transgenic tobacco (Nicotiana tabacum L.) plants with an artificial gene from the antimicrobial peptide bombinin (bom) have been obtained and studied. The presence of the bom gene in the genome of kanamycin-resistant plants was shown by PCR. Expression of the bom gene was confirmed by antimicrobial activity measurements in leaf extracts. The obtained plants were morphogenetically resistant to Erwinia carotovora bacteria and Rhizoctonia solani fungi phytopathogens. In addition, the protective oxidative reaction to the infection, i.e., the SOD activity and proline content, were lower in transgenic plants than in the infected nontransgenic plants. Plants with the expression of the antimicrobial bombinin peptide gene are promising for use in agricultural biotechnology as plant protectors.

Получены безмаркерные растения камелины (Camelina sativa (L.)), с геном антимикробного пептида цекропина Р1

The method of producing of water extract from transgenic Kalanchoe pinnata L. plants expressing cecropin P1 gene has been developed and adapted for pharmaceutical production. Chlorophorm has been shown to be an efficient preservative for this extract. The antibiotic activity of the extract has kept well even after 1-h boiling. In addition, the prolonged antimicrobial activity of the extract has been confirmed by the “accelerated aging” method. Being added to culture medium, the leaf extract from transgenic K. pinnata plants has provided more intensive callus growth and improved rhizogenesis in ice plant ( Mesembryanthemum crystallinum ). The obtained results demonstrate an increased biological activity of the extract from kalanchoe plants producing antimicrobial cecropin P1 peptide and good prospects for its use in pharmacology.

Management of purulent wounds is a problem that requires particular attention: wounds are a common injury type for which suppurative complications are frequent, mortality rates are high and antimicrobial therapy may be ineffective due to the presence of drug-resistant bacteria in the wound. In this work we have studied the effectiveness of wound treatment with the leaf extract of transgenic Kalanchoe pinnata modified to produce antimicrobial peptide cecropin P1. Purulent wounds infected with Staphylococcus aureus were modeled in Wistar rats. Four groups of animals were formed, with 10 animals in each group. In all groups, the wounds were cleansed with 3 % hydrogen peroxide solution once a day; all groups except the controls received additional treatment. Group 2 received 10 % cefazolin solution, group 3 received kalanchoe juice, group 4 received the juice of cecropin P1-producing kalanchoe. Histologic stains of biopsy samples were performed after rats were sacrificed by anesthetic overdose on days 3, 10 and 14 after treatment onset. On day 3, wound dynamics was the same in all groups. On day 10 exudate was still observed in the controls; in group two exudation was almost finished and regeneration was about to begin; in groups 3 and 4 the wound defect was filled with granulation tissue. In spite of epidermal repair along the wound edges in groups 2 and 3, there still was some sloughing and granulation tissue was less mature than in group 4. We recommend conducting more extensive clinical research of the leaf extract of cecropin P1-containing transgenic Kalanchoe pinnata.

Previously transgenic Kalanchoe pinnata plants producing an antimicrobial peptide cecropin P1 (CecP1) have been reported. Now we report biological testing K. pinnata extracts containing CecP1 as a candidate drug for treatment of wounds infected with Candida albicans. The drug constitutes the whole juice from K. pinnata leaves (not ethanol extract) sterilized with nanofiltration. A microbicide activity of CecP1 against an animal fungal pathogen in vivo was demonstrated for the first time. However, a favorable therapeutic effect of the transgenic K. pinnata extract was attributed to a synergism between the fungicide activity of CecP1 and wound healing (antiscar), revascularizing, and immunomodulating effect of natural biologically active components of K. pinnata. A commercial fungicide preparation clotrimazole eliminated C. albicans cells within infected wounds in rats with efficiency comparable to CecP1-enriched K. pinnata extract. But in contrast to K. pinnata extract, clotrimazole did not exhibit neither wound healing activity nor remodeling of the scar matrix. Taken together, our results allow assumption that CecP1-enriched K. pinnata extracts should be considered as a candidate drug for treatment of dermatomycoses, wounds infected with fungi, and bedsores.

Procedure of manufacturing K. pinnata water extracts containing cecropin P1 (CecP1) from the formerly described transgenic plants is established. It included incubation of leaves at +4°C for 7 days, mechanical homogenization of leaves using water as extraction solvent, and heating at +70°C for inactivating plant enzymes. Yield of CecP1 (after heating and sterilizing filtration) was 0.3% of total protein in the extract. The water extract of K. pinnata + CecP1 exhibits favorable effect on healing of wounds infected with S. aureus (equal to Cefazolin) and with a combination of S. aureus with P. aeruginosa (better than Cefazolin). Wild-type K. pinnata extract exhibited evident microbicide activity against S. aureus with P. aeruginosa but it was substantially strengthened in K. pinnata + CecP1 extract. K. pinnata extracts (both wild-type and transgenic) did not exhibit general toxicity and accelerated wound recovery. Due to immunomodulating activity, wild-type K. pinnata extract accelerated granulation of the wound bed and marginal epithelialization even better than K. pinnata + CecP1 extract. Immunomodulating and microbicide activity of K. pinnata synergizes with microbicide activity of CecP1 accelerating elimination of bacteria.

Проблема лечения гнойных ран актуальна в хирургии в связи с распространенностью ран различной этиологии, частотой гнойных осложнений, высокой летальностью, появлением антибиотикорезистентных штаммов бактерий. В работе исследована эффективность фармакотерапии раневого процесса экстрактом листьев трансгенного каланхоэ перистого с антимикробным пептидом цекропином Р1. Гнойную рану моделировали на крысах линии Wistar с внесением в рану культуры Staphylococcus aureus. Сформировали 4 группы по 10 животных в каждой. Во всех группах раны обрабатывали ежедневно однократно 3 % раствором перекиси водорода и дополнительным препаратом, кроме группы 1 (контрольной). В группе 2 использовали 10 % раствор цефазолина, в группе 3 сок каланхоэ, в группе 4 сок каланхоэ с цекропином Р1. Гистологическое исследование раневых биоптатов производили на 3, 10 и 14 сутки с начала лечения после выведения крыс из эксперимента путем передозировки наркоза. Результаты лечения через 3 сут были схожими во всех группах. Через 10 сут для ран крыс контрольной группы была отмечена незавершенность фазы экссудации, группы 2 переход фазы экссудации в фазу регенерации, а групп 3 и 4 покрытие грануляционной тканью. Несмотря на восстановление эпидермиса по краям ран в группах 2 и 3, кое-где сохранялся струп, а грануляционная ткань была менее зрелой, чем в группе 4. Результаты позволяют рекомендовать экстракт листьев трансгенного каланхоэ перистого с цекропином Р1 для широкого клинического изучения.

Kalanchoe pinnata L. plants bearing an artificial CP1 gene encoding the cecropin P1 antimicrobial peptide have been obtained. The presence of the CP1 gene in the plant genome has been confirmed by PCR. Cecropin P1 synthesis in transgenic plants has been shown by MALDI mass spectrometry and Western blotting. The obtained plants have been highly resistant to bacterial and fungal phytopathogens, and their extracts have demonstrated antimicrobial activity towards human and animal pathogens. It has been shown that transgenic plants bearing the CP1 gene can be colonized by the beneficial associative microorganisms Methylovorus mays.

Transgenic kalanchoe plants (Kalanchoe pinnata L.) expressing the antimicrobial peptide cecropin P1 gene (cecP1) under the control of the 35S cauliflower mosaic virus 35S RNA promoter and the selective neomycin phosphotransferase II (nptII) gene under the control of the nopaline synthase gene promoter were studied. The 35S promoter methylation and the cecropin P1 biosynthesis levels were compared in plants growing on media with and without kanamycin. The low level of active 35S promoter methylation further decreases upon cultivation on kanamycin-containing medium, while cecropin P1 synthesis increases.

Kalanchoe pinnate (Kalanchöe pinnata L. ) plants with synthetic gene of antimicrobial peptide cecropin P1 (CP1) under the control of promoter 35S RNA of cauliflower mosaic virus (CaMV 35S) were produced. For transformation, a modified binary vector not containing selective genes of tolerance against antibiotics and herbicides was used. Screening of the marker-free transformed plants was conducted on the medium without selective antibiotics by revealing antibacterial activity of plant extracts and cecropin P1. The marker-free plants produced displayed increased resistance against bacterial and fungus phytopathogens, while their extracts were characterized by antimicrobial activity for human and animal pathogens. These plants meet the requirements of biosafety and may be used as producers of cecropin P1 in pharmaceutics.