ИСПОЛЬЗОВАНИЕ ПИРИДОКСИНА ДЛЯ ПОВЫШЕНИЯ ПРОТИВООПУХОЛЕВОЙ АКТИВНОСТИ МЕТИОНИН-ГАММАЛИАЗЫ НА МОДЕЛЯХ ПЕРЕВИВАЕМЫХ ОПУХОЛЕЙ МЫШЕЙ

We presented results of monotherapy and combination therapy of transplantable murine tumor models using methionine-gamma-lyase (MGL) and pyridoxine hydrochloride. We studied MGL from Clostridium sporogenes and Citrobacter freundii. We used Lewis lung carcinoma (LLC), melanoma B16, leukemias P388 and L1210 and Fisher lymphadenosis L5178y. Neither monotherapy with MGL nor combination of MGL and pyridoxine demonstrated antitumor activity against P388 and L5178y. In the murine L1210 leukemia model, MGL C. sporogenes injected intraperitoneally in the dose of 2000 U/kg, 11 times with a 12-hour interval increased the life span of mice (ILS=22 %, р=0.035). In the LLC model, the combination of MGL C. sporogenes at a dose of 400 U/kg, i.p., 4 times with a 48-hour interval and pyridoxine at a dose of 250 mg/kg led to tumor growth inhibition (TGI=55 %, р<0.001) on the first day after the completion of treatment. Monotherapy with MGL or pyridoxine in the same regimens resulted in a 24 % TGI (р=0.263) or 21 % TGI (р=0.410), respectively. In a pair-wise comparison of treatments, MGL + pyridoxine was more effective compared to MGL used alone (р=0.061) and MGL + pyridoxine was more effective then pyridoxine alone (р=0.031). MGL from C. freundii at a dose of 200 U/kg, 4 times with a 48-hour interval plus pyridoxine at a dose of 500 mg/kg injected on day 9 after the completion of treatment led to 50 % TGI, whereas MGL monotherapy at a single dose of 400 U/kg or pyridoxine monotherapy in the same regimen showed 5 % TGI (р=0.991) and 4 % TGI (р=0.998), respectively. The pair-wise comparison showed that MGL (200 U/kg) + pyridoxine was more effective than MGL (400 U/ kg) alone (р<0.001) and pyridoxine alone (р=0.003). In the B16 model, the combination of MGL injected i.p at a dose of 2000 U/kg and pyridoxine at a dose of 300 mg/kg showed 56 % TGI on day 1after the completion of treatment (р=0.045) and 35 % TGI on day 3 (р=0.038). Pyridoxine significantly increased the anticancer effect of MGL: MGL 1000 U/kg i.p and MGL 1000 U/kg i.p. + pyridoxine 300 mg/kg led to TGI=45 % (р=0.034) on day 3 after the completion of treatment. Single maximum tolerated dose after multiple i.p. administration was defined as 2000 U/kg, simultaneous administration of pyridoxine did not increase the toxicity of MGL. In conclusion, LLC and B16 are sensitive to MGL treatment, and pyridoxine may increase the efficacy of MGL.Приведены экспериментальные данные монотерапии и комбинированной терапии моделей перевиваемых опухолей мышей препаратами метионин-γ-лиазы (МГЛ) и пиридоксина гидрохлорида. Изучены МГЛ Clostridium sporogenes и Citrobacter freundii. Использованы перевиваемые модели опухолей мышей: карцинома легкого Льюис (LLC), меланома В16, лимфолейкоз P388, лимфолейкоз L1210, лимфаденоз Фишера L5178y. На моделях P388, L5178y МГЛ не показала противоопухолевой активности ни в монорежиме, ни в сочетании с пиридоксином. На модели L1210 было получено пограничное увеличение продолжительности жизни (УПЖ) 22 %, р=0,035 при применении МГЛ C. sporogenes в дозе 2000 Е/кг 11-кратно внутрибрюшинно с интервалом 12 ч. На LLC показано, что на 1-е сут после окончания лечения одновременное внутрибрюшное (в/б) введение МГЛ C. sporogenes 400 Е/кг 4-кратно с интервалом 48 ч и пиридоксина в дозе 250 мг/кг вызывало ТРО=55 % (р<0,001), монотерапия МГЛ или пиридоксином в аналогичных дозах и режимах применения вызывала ТРО=24 % (р=0,263) и 21 % (р=0,410) соотетственно. При попарном сравнении: комбинированная терапия МГЛ + пиридоксин против монотерапии МГЛ в аналогичном режиме р=0,061, против монотерапии пиридоксином р=0,031. На LLC МГЛ C. freundii 200 Е/кг 4-кратно с интервалом 48 ч и пиридоксина в дозе 500 мг/кг одновременно на 9-е сут после окончания лечения вызывало ТРО=50 % (р=0,001), при этом монотерапия МГЛ в разовой дозе 400 Е/кг или пиридоксином в аналогичном режиме применения вызывала ТРО=+5 % (р=0,991) и 4 % (р=0,998) соответственно. При попарном сравнении: комбинированная терапия МГЛ 200 Е/кг + пиридоксин против монотерапии МГЛ 400 Е/кг в аналогичном режиме р<0,001, против монотерапии пиридоксином р=0,003. На модели меланома B16 МГЛ 2000 в/б + пиридоксин 300 мг/кг вызывает ТРО 56 % на 1-е сут (р=0,045) и 35 % на 3-и сут (р=0,038). При комбинированной терапии МГЛ + пиридоксин последний значимо повышал противоопухолевую активность МГЛ в сочетаниях: МГЛ 1000 Е/кг в/б и МГЛ 1000 Е/кг в/б + пиридоксин 300 мг/кг ТРО=45 % (р=0,034) на 3-и сут после окончания лечения. При внутривенном введении МГЛ 500 Е/кг и МГЛ 500 Е/кг + пиридоксин последний повышал эффективность лечения: максимальное ТРО 50 % на 1-е сут после окончания лечения (р=0,085, различие не достоверно) и 21 % на 3-и сут после окончания лечения ТРО 22 % (р=0,965, различие не достоверно).Разовая максимальная переносимая доза при многократном в/б введении составила 2000 Е/кг, одновременное применение пиридоксина не усугубляло токсичности МГЛ. Таким образом, LLC и меланома В16 обладают чувствительностью к терапии МГЛ. Одновременное введение пиридоксина на модели LLC и В16 достоверно повышает её эффективность

Sunlight-Exposed Biofilm Microbial Communities Are Naturally Resistant to Chernobyl Ionizing-Radiation Levels

BACKGROUND: The Chernobyl accident represents a long-term experiment on the effects of exposure to ionizing radiation at the ecosystem level. Though studies of these effects on plants and animals are abundant, the study of how Chernobyl radiation levels affect prokaryotic and eukaryotic microbial communities is practically non-existent, except for a few reports on human pathogens or soil microorganisms. Environments enduring extreme desiccation and UV radiation, such as sunlight exposed biofilms could in principle select for organisms highly resistant to ionizing radiation as well. METHODOLOGY/PRINCIPAL FINDINGS: To test this hypothesis, we explored the diversity of microorganisms belonging to the three domains of life by cultivation-independent approaches in biofilms developing on concrete walls or pillars in the Chernobyl area exposed to different levels of radiation, and we compared them with a similar biofilm from a non-irradiated site in Northern Ireland. Actinobacteria, Alphaproteobacteria, Bacteroidetes, Acidobacteria and Deinococcales were the most consistently detected bacterial groups, whereas green algae (Chlorophyta) and ascomycete fungi (Ascomycota) dominated within the eukaryotes. Close relatives to the most radio-resistant organisms known, including Rubrobacter species, Deinococcales and melanized ascomycete fungi were always detected. The diversity of bacteria and eukaryotes found in the most highly irradiated samples was comparable to that of less irradiated Chernobyl sites and Northern Ireland. However, the study of mutation frequencies in non-coding ITS regions versus SSU rRNA genes in members of a same actinobacterial operational taxonomic unit (OTU) present in Chernobyl samples and Northern Ireland showed a positive correlation between increased radiation and mutation rates. CONCLUSIONS/SIGNIFICANCE: Our results show that biofilm microbial communities in the most irradiated samples are comparable to non-irradiated samples in terms of general diversity patterns, despite increased mutation levels at the single-OTU level. Therefore, biofilm communities growing in sunlight exposed substrates are capable of coping with increased mutation rates and appear pre-adapted to levels of ionizing radiation in Chernobyl due to their natural adaptation to periodical desiccation and ambient UV radiation

A Gene Encoding l-Methionine γ-Lyase Is Present in Enterobacteriaceae Family Genomes: Identification and Characterization of Citrobacter freundii l-Methionine γ-Lyase

Citrobacter freundii cells produce l-methionine γ-lyase when grown on a medium containing l-methionine. The nucleotide sequence of the hybrid plasmid with a C. freundii EcoRI insert of about 3.0 kbp contained two open reading frames, consisting of 1,194 nucleotides and 1,296 nucleotides, respectively. The first one (denoted megL) encoded l-methionine γ-lyase. The enzyme was overexpressed in Escherichia coli and purified. The second frame encoded a protein belonging to the family of permeases. Regions of high sequence identity with the 3′-terminal part of the C. freundii megL gene located in the same regions of Salmonella enterica serovar Typhimurium, Shigella flexneri, E. coli, and Citrobacter rodentium genomes were found

USE OF PYRIDOXINE TO INCREASE ANTICACNER ACTIVITY OF METHIONINE-GAMMA-LYASE IN MURINE CANCER MODELS

We presented results of monotherapy and combination therapy of transplantable murine tumor models using methionine-gamma-lyase (MGL) and pyridoxine hydrochloride. We studied MGL from Clostridium sporogenes and Citrobacter freundii. We used Lewis lung carcinoma (LLC), melanoma B16, leukemias P388 and L1210 and Fisher lymphadenosis L5178y. Neither monotherapy with MGL nor combination of MGL and pyridoxine demonstrated antitumor activity against P388 and L5178y. In the murine L1210 leukemia model, MGL C. sporogenes injected intraperitoneally in the dose of 2000 U/kg, 11 times with a 12-hour interval increased the life span of mice (ILS=22 %, р=0.035). In the LLC model, the combination of MGL C. sporogenes at a dose of 400 U/kg, i.p., 4 times with a 48-hour interval and pyridoxine at a dose of 250 mg/kg led to tumor growth inhibition (TGI=55 %, р<0.001) on the first day after the completion of treatment. Monotherapy with MGL or pyridoxine in the same regimens resulted in a 24 % TGI (р=0.263) or 21 % TGI (р=0.410), respectively. In a pair-wise comparison of treatments, MGL + pyridoxine was more effective compared to MGL used alone (р=0.061) and MGL + pyridoxine was more effective then pyridoxine alone (р=0.031). MGL from C. freundii at a dose of 200 U/kg, 4 times with a 48-hour interval plus pyridoxine at a dose of 500 mg/kg injected on day 9 after the completion of treatment led to 50 % TGI, whereas MGL monotherapy at a single dose of 400 U/kg or pyridoxine monotherapy in the same regimen showed 5 % TGI (р=0.991) and 4 % TGI (р=0.998), respectively. The pair-wise comparison showed that MGL (200 U/kg) + pyridoxine was more effective than MGL (400 U/ kg) alone (р<0.001) and pyridoxine alone (р=0.003). In the B16 model, the combination of MGL injected i.p at a dose of 2000 U/kg and pyridoxine at a dose of 300 mg/kg showed 56 % TGI on day 1after the completion of treatment (р=0.045) and 35 % TGI on day 3 (р=0.038). Pyridoxine significantly increased the anticancer effect of MGL: MGL 1000 U/kg i.p and MGL 1000 U/kg i.p. + pyridoxine 300 mg/kg led to TGI=45 % (р=0.034) on day 3 after the completion of treatment. Single maximum tolerated dose after multiple i.p. administration was defined as 2000 U/kg, simultaneous administration of pyridoxine did not increase the toxicity of MGL. In conclusion, LLC and B16 are sensitive to MGL treatment, and pyridoxine may increase the efficacy of MGL

Comparative Analysis of the lux Operons in Aliivibrio logei KCh1 (a Kamchatka Isolate) and Aliivibrio salmonicida ▿

Here we provide a molecular description of a new psychrophilic strain, KCh11, of marine luminescent bacteria classified as Aliivibrio logei. We sequenced the entire lux operon of A. logei KCh1 and showed that it is substantially similar to the lux operon of Aliivibrio salmonicida. It was demonstrated that the reduced production of bioluminescence in A. salmonicida is most likely defined by a specific defect in its luxD gene