Raw 1H NMR spectra of Fusarium graminearum hyphae can be found at the website of the pesticide metabolomics group (PMG) of the Agricultural University of Athens at the address: http://www.aua.gr/pesticide-metabolomicsgroup/Resources/Fusarium_graminearum_NMR_spectra.html, accession number PMG-01–17. The data set support the research article "Implication of Fusarium graminearum Primary Metabolism in its Resistance to Benzimidazole Fungicides as revealed by 1H NMR Metabolomics" [1].
Цель исследования: изучение взаимодействия комплекса протеолитических ферментов препарата лонголитин из анаморфного гриба Arthrobotrys longa с антикоагулянтным и антитромботическим препаратом пиявитом. Материалы и методы. Препарат пиявит смешивали в разных разведениях с различными концентрациями лонголитина и определяли фибринолитическую активность (ФА) смеси. В другой серии опытов пиявит добавляли в погруженную культуру гриба-продуцента. По окончании эксперимента в пробах культуральной жидкости (КЖ) определяли ФА. Антикоагулянтные свойства КЖ измеряли методом рекальцификации и записи тромбоэластограммы при добавлении КЖ к плазме крови здоровых интактных крыс. Результаты. В опытах in vitro пиявит дозозависимо угнетал ФА лонголитина до полного ее подавления при концентрации 16 мг/мл. Добавление пиявита к культуре гриба-продуцента вызывало усиление биосинтеза препарата с увеличением ФА. КЖ при этом обладала высокой антикоагулянтной активностью. Время рекальцификации и показатель R тромбоэластограммы в КЖ были статистически достоверно удлинены, увеличивалась и антиполимеризационная активность КЖ. Заключение. Совместное применение двух антитромботических средства (лонголитина и пиявита) в исследовании in vitro привело к уменьшению фибринолитических, а значит, и тромболитических свойств лонголитина. Таким образом, одновременное использование этих препаратов требует осторожности. Aim: to study the interaction of Longolitin (complex of proteolytic enzymes from anamorphic fungus Arthrobotrys longa) with anticoagulative and antithrombotic drug Piyavit. Materials and methods. We mixed diff erent dilutions of Piyavit with various concentrations of Longolitin and determined fibrinolytic activity (FA) of the mixture. In other experimental series we added Piyavit to submerged culture of the fungus-producer. At the end of the experiment we determined FA in culture liquid samples (CL). Anticoagulative properties of CL were measured by recalcification method and thromboelastograms recording with CL addition to blood plasma of healthy intact rats. Results. In in vitro experiments Piyavit dose-dependently inhibited FA of Longolitin until it’s completely suppression at concentration of 16 mg/ml. Piyavit addition to the culture of fungus-producer caused the enhancement of drug biosynthesis with FA increasing. At the same time CL had a high anticoagulative activity. Recalcifi cation time and parameter R of thromboelastogram in CL were statistically signifi cant elongated, also CL anti-polymerization activity increased. Conclusion. Combined application of two antithrombotic agents (Longolitin and Piyavit) in in vitro study led to decreasing of fibrinolytic, and so thrombolytic properties of Longolitine. Thus, the simultaneous use of these drugs requires caution. Adding piyavit to the culture of the producer fungus led to an increase in the biosynthesis of the preparation. Thus, caution is needed at simultaneous using of these drugs.
The cultivation conditions (cultivation duration, nitrogen sources, and initial pH of the medium) of the strain Tolypocladium inflatum k1, a producer of extracellular proteases, has been optimized to achieve maximal plasminogen activator activity. It has been shown that Tolypocladium inflatum k1 forms at least two proteinases with activator activity towards plasminogen. One of them is thiol-dependent, EDTA-sensitive serine proteinase, which activates plasminogen and does not hydrolyze fibrin, indicating its higher specificity and prospects for the development of new thrombolytic agents.
Production of proteinases with plasmin-like and plasminogen-activating activities by a micromycete Arthrobotrys longa 1 was studied. Polycyclic growth of the producer in submerged cultures was observed, with an endogenous rhythm of periods of intense microconidia formation alternating with the phases of drastic increase in the content of producing mycelium. The highest plasmin-like and plasminogen-activating activities (up to 1000 and 500 cond. U/mL, respectively) were found to correlate with the polycyclic growth of the producer, coinciding with the stage of microconidia germination. Enhanced secretion of proteinases with plasmin-like and plasminogen-activating activity was found to be associated with increased specific growth rate of А. longa 1.
Проведен скрининг продуцентов протеиназ с фибринолитической (плазминоподобной и активаторной к плазминогену) и коллагенолитической активностями среди 83 штаммов микроскопических грибов разных экологических групп. Энтомопатогенные микромицеты, по сравнению с сапротрофами, потенциальными фитопатогенами и эпифитами секретировали протеиназы с более высокой фибринолитической и коллагенолитической активностью. Выявлены штаммы микромицетов, протеолитические ферменты которых обладали коллагеназной активностью и штаммы-продуценты протеиназ с плазминоподобной активностью. Среди исследованных микромицетов не было обнаружено штаммов, способных секретировать протеиназы только с активаторной к плазминогену активностью. Найден продуцент внеклеточных протеиназ с высокой активаторной к плазминогену, плазминоподобной и коллагенолитической активностями Tolypocladium inflatum k1. Показано, что удельная активаторная к плазминогену активность T. inflatum k1 на 20% выше плазминоподобной активности.
Screening for producers of proteinases with fibrinolytic (plasmin-like and plasminogen-activating) and collagenolytic activities was carried out among 83 strains of microscopic fungi belonging to various ecological groups. Entomopathogenic micromycetes secreted proteinases with higher fibrinolytic and collagenolytic activity than saprotrophic, potentially phytopathogenic, and epiphytic strains. Micromycete strains possessing proteolytic enzymes with collagenase activity were revealed, as were the strains producing proteinases with plasminlike activity. None of the strains studied secreted proteinases possessing only plasminogen-activating activity. Tolypocladium inflatum k1 was found to be a producer of extracellular proteinases with high plasminogen-activating, plasminlike, and collagenolytic activities. The specific plasminogen-activating activity of T. inflatum k1 was shown to be 20% higher than its plasminlike activity.