Three‐step procedure for preparation of pure <i>Bacillus altitudinis</i> ribonuclease

Dudkina, Elena; Ulyanova, Vera; Mahmud, Rashed; Khodzhaeva, Vera; Dao, Linh; Vershinina, Valentina; Kolpakov, A. I.; Ilinskaya, О. N.

doi:10.1002/2211-5463.12023

Cited by 33 publications

(20 citation statements)

References 36 publications

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“…The guanyl-preferring RNase from B. pumilus , binase (12.2 kDa, 109 amino acid residues, pI 9.5), was isolated from the culture fluid of native binase producer as homogenous protein using the three-step procedure described earlier ( Dudkina et al, 2016 ). The binase catalytic activity (1.4 × 10 6 units/mg) was determined by measurement of high-polymeric yeast RNA hydrolysis products according to modified method of Kolpakov and Il’inskaia (1999) .…”

Section: Methodsmentioning

confidence: 99%

Binase Immobilized on Halloysite Nanotubes Exerts Enhanced Cytotoxicity toward Human Colon Adenocarcinoma Cells

et al. 2017

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Many ribonucleases (RNases) are considered as promising tools for antitumor therapy because of their selective cytotoxicity toward cancer cells. Binase, the RNase from Bacillus pumilus, triggers apoptotic response in cancer cells expressing RAS oncogene which is mutated in a large percentage of prevalent and deadly malignancies including colorectal cancer. The specific antitumor effect of binase toward RAS-transformed cells is due to its direct binding of RAS protein and inhibition of downstream signaling. However, the delivery of proteins to the intestine is complicated by their degradation in the digestive tract and subsequent loss of therapeutic activity. Therefore, the search of new systems for effective delivery of therapeutic proteins is an actual task. This study is aimed to the investigation of antitumor effect of binase immobilized on natural halloysite nanotubes (HNTs). Here, we have developed the method of binase immobilization on HNTs and optimized the conditions for the enzyme loading and release (i); we have found the non-toxic concentration of pure HNTs which allows to distinguish HNTs- and binase-induced cytotoxic effects (ii); using dark-field and fluorescent microscopy we have proved the absorption of binase-loaded HNTs on the cell surface (iii) and demonstrated that binase-halloysite nanoformulations possessed twice enhanced cytotoxicity toward tumor colon cells as compared to the cytotoxicity of binase itself (iv). The enhanced antitumor activity of biocompatible binase-HNTs complex confirms the advisability of its future development for clinical practice.

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Section: Methodsmentioning

confidence: 99%

Binase Immobilized on Halloysite Nanotubes Exerts Enhanced Cytotoxicity toward Human Colon Adenocarcinoma Cells

et al. 2017

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“…The protein samples were frozen overnight at −20 ∘ C, followed by freezing for 15 min at −80 ∘ C and then lyophilized for 48 h at −50 ∘ C under vacuum using a Labconco FreeZone 2.5 Liter Freeze Dry System (Labconco, USA). Homogeneity and authenticity of the purified enzyme were confirmed by PAAG electrophoresis, Western blotting, and MALDI TOF/TOF mass spectrometry on UltrafleXtreme (Bruker Corporation, Germany) as described in [12].…”

Section: Bacteria Growth Condition and Binasementioning

confidence: 99%

Ribonuclease fromBacillusActs as an Antiviral Agent against Negative- and Positive-Sense Single Stranded Human Respiratory RNA Viruses

Mahmud

Müller

Romanova

et al. 2017

BioMed Research International

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Bacillus pumilus ribonuclease (binase) was shown to be a promising antiviral agent in animal models and cell cultures. However, the mode of its antiviral action remains unknown. To assess the binase effect on intracellular viral RNA we have selected single stranded negative- and positive-sense RNA viruses, influenza virus, and rhinovirus, respectively, which annually cause respiratory illnesses and are characterized by high contagious nature, mutation rate, and antigen variability. We have shown that binase exerts an antiviral effect on both viruses at the same concentration, which does not alter the spectrum of A549 cellular proteins and expression of housekeeping genes. The titers of influenza A (H1N1pdm) virus and human rhinovirus serotype 1A were reduced by 40% and 65%, respectively. A preincubation of influenza virus with binase before infection significantly reduced viral titer after single-cycle replication of the virus. Using influenza A virus mini genome system we showed that binase reduced GFP reporter signaling indicating a binase action on the expression of viral mRNA. Binase reduced the level of H1N1pdm viral NP mRNA accumulation in A549 cells by 20%. Since the viral mRNA is a possible target for binase this agent could be potentially applied in the antiviral therapy against both negative- and positive-sense RNA viruses.

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“…Биназа была изолирована как гомогенный белок из культуральной жидкости продуцента по методике, описанной Дудкиной с соавт. [12]. Каталитическая активность биназы охарактеризована ранее по отношению к синтетическим субстратам [13] и высокополимерной дрожжевой РНК [10].…”

Section: материалы и методыunclassified

Binase-Induced Changes of Tumor Cell Membranes

Zelenikhin

Горбунова

Beuerlein

et al. 2017

Moscow Univ. Biol.Sci. Bull.

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Экзогенные рибонуклеазы бацилл могут селективно индуцировать апоптоз злокачественных клеток. В работе проведен анализ способности рибонуклеазы Bacillus pumilusбиназы-индуцировать процессы, приводящие к динамическому нарушению целостности мембран клеток аденокарциномы легких человека линии А549. Охарактеризовано влияние фермента в различных концентрациях на состояние цитоплазматической мембраны клеток и мембран митохондрий. При помощи методов проточной цитофлуориметрии и флуоресцентной микроскопии установлено, что воздействие биназы приводит к нарушению нормального функционирования мембран обоих типов, причем вначале нарушения затрагивают мембраны митохондрий. Проведенное исследование позволило выявить и визуализировать этапы влияния биназы на мембранные структуры клеток-мишеней и подтвердить, что бактериальная РНКаза индуцирует апоптоз клеток-мишеней преимущественно по "внутреннему" (митохондриальному) пути.

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Three‐step procedure for preparation of pure Bacillus altitudinis ribonuclease

Cited by 33 publications

References 36 publications

Binase Immobilized on Halloysite Nanotubes Exerts Enhanced Cytotoxicity toward Human Colon Adenocarcinoma Cells

Binase Immobilized on Halloysite Nanotubes Exerts Enhanced Cytotoxicity toward Human Colon Adenocarcinoma Cells

Ribonuclease fromBacillusActs as an Antiviral Agent against Negative- and Positive-Sense Single Stranded Human Respiratory RNA Viruses

Binase-Induced Changes of Tumor Cell Membranes

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