Influence of antioxidant effect of stobadine derivative in condition of kidney ischemia-reperfusion in a pre-clinical experiment (effect in therapy)

The goal of the study was to monitor the antioxidative effect of stobadine derivative under conditions of ischemia-reperfusion of laboratory rat kidney tissue. 40 animals were subjected to kidney tissue ischemia (60 min) followed by reperfusion (10 min). After that, the animals were divided by random selection into 4 groups (n = 10). The treated groups were given stobadine derivative in peroral doses of 5, 10 and 20 mg/kg in 0.5% solution of Avicel once a day, the placebo group was given only the solution of Avicel. One group (n = 10) was an intact group (without ischemia-reperfusion and without treatment), for comparison. Once a week, selected laboratory parameters were determined in all animals. On the 15th day the animals were exsanquined and organs were recovered for histopathological examination. We discovered a statistically significant changes of the superoxiddismutase and glutathione peroxidase catalytic activity; changes of total antioxidative capacity and malondialdehyde in the treated groups compared to the groups of placebo and intact. Other examined laboratory parameters (creatinine, urea and uric acid in blood; creatinine, urea, total protein in urine; diuresis) exhibited significant changes too. The results of biochemical examination show a protective antioxidative effect of the compound studied. The results of histopathological examination support this assumption.     

Flexibility and structural conservation in a c-KIT G-quadruplex

A quadruplex sequence from the promoter region of the c-KIT gene forms a stable quadruplex, as characterized by crystallographic and NMR methods. Two new crystal structures are reported here, together with molecular dynamics simulation studies on these quadruplex crystal structures and an NMR structure. The new crystal structures, each in a distinct space group and lattice packing arrangement, together with the existing structures, demonstrate that the c-KIT quadruplex fold does not change with differing environments, suggesting that quadruplex topological dynamism is not a general phenomenon. The single and dinucleotide loops in these structures show a high degree of conformational flexibility within the three crystal forms and the NMR ensemble, with no evidence of clustering to particular conformers. This is in accord with the findings of high loop flexibility from the molecular dynamics studies. It is suggested that intramolecular quadruplexes can be grouped into two broad classes (i) those with at least one single-nucleotide loop, often showing singular topologies even though loops are highly flexible, and (ii) with all loops comprising at least two nucleotides, leading to topological dynamism. The loops can have more stable and less dynamic base-stacked secondary structures.     

The plant alkaloid and anti-leukemia drug homoharringtonine sensitizes resistant human colorectal carcinoma cells to TRAIL-induced apoptosis via multiple mechanisms

TNF-related apoptosis-inducing ligand (TRAIL) is a pro-apoptotic ligand from the TNF-alpha family that is under consideration, along with agonistic anti-TRAIL receptor antibodies, as a potential anti-tumor agent. However, most primary human tumors are resistant to monotherapy with TRAIL apoptogens, and thus the potential applicability of TRAIL in anti-tumor therapy ultimately depends on its rational combination with drugs targeting these resistances. In our high-throughput screening for novel agents/drugs that could sensitize TRAIL-resistant colorectal cancer cells to TRAIL-induced apoptosis, we found homoharringtonine (HHT), a cephalotaxus alkaloid and tested anti-leukemia drug, to be a very effective, low nanomolar enhancer of TRAIL-mediated apoptosis/growth suppression of these resistant cells. Co-treatment of TRAIL-resistant RKO or HT-29 cells with HHT and TRAIL led to the effective induction of apoptosis and the complete elimination of the treated cells. HHT suppressed the expression of the anti-apoptotic proteins Mcl-1 and cFLIP and enhanced the TRAIL-triggered activation of JNK and p38 kinases. The shRNA-mediated down-regulation of cFLIP or Mcl-1 in HT-29 or RKO cells variably enhanced their TRAIL-induced apoptosis but it did not markedly sensitize them to TRAIL-mediated growth suppression. However, with the notable exception of RKO/sh cFLIP cells, the downregulation of cFLIP or Mcl-1 significantly lowered the effective concentration of HHT in HHT + TRAIL co-treatment. Combined HHT + TRAIL therapy also led to the strong suppression of HT-29 tumors implanted into immunodeficient mice. Thus, HHT represents a very efficient enhancer of TRAIL-induced apoptosis with potential application in TRAIL-based, anti-cancer combination therapy.     

Bioluminescent bioreporter Pseudomonas putida TVA8 as a detector of water pollution. Operational conditions and selectivity of free cells sensor

Conditions influencing bioluminescence output from Pseudomonas putida TVA8 harboring chromosomal tod-luxCDABE fusion were followed. In complex media, cell growth was not influenced by the presence of toluene at 53 mg/L. Bioluminescence induction was tested in minimal medium. At 15 °C the highest bioluminescence induced with toluene (1.325 mg/L) was reached after 6 h. At 25 °C the bioluminescence maximum was approximately 20% lower but this was reached after 3.5 h, and at temperatures of 7 °C, 28 °C, 30 °C and 34 °C, bioluminescence peaked at ≤60% of the maximum. Time courses of bioluminescence were dependent on cell concentrations. The heights of bioluminescence maxima were proportional to toluene concentration in the range 0–26 mg/L. Twenty-three organic pollutants (10³× diluted saturated solutions) were tested as bioluminescent inducers. The bioluminescence maximum decreased in the order: ethylbenzene > toluene > phenol > benzene > 4-ethyltoluene > 4-fluorotoluene > cumene > isobutylbenzene > styrene > trichloroethylene > o-, p-xylene > cresol > m-xylene > 2-methylnaphthalene > benzylchloride > naphthalene > salicylic acid > hexachlorobenzene > 2-chloronaphthalene > biphenyl > 2-bromonaphthalene > 1,3,5-triethylbenzene. Bioluminescence was also induced with ethanol and methanol and the presence of these alcohols in concentrations of ≤1% increased bioluminescence of toluene. The induction of bioluminescence from samples of wastewater and groundwater contaminated with BTEX (benzene, toluene, ethylbenzene and xylene) from 0.5 to 120 mg/L was demonstrated.     

Sample preparation in separation of the extracellular chitinolytic enzymes of the human intestinal bacterium Clostridium paraputrificum J4 from the culture fluids

Membrane ultrafiltration (UF) was used in sample preparation of the culture fluids of the human intestinal bacterium Clostridium paraputrificum strain J4 containing seven extracellular chitinolytic isoenzymes (38-90 kDa). The subsequent filtration of the bacteria-free supernatants was carried out through Millipore membranes with cut-off 100 and 30 kDa for separation of undigested components of the culture medium and bacterial metabolites with molecular weight higher and lower than that of the target enzymes. The chitinolytic enzymes, which were the minor components in the culture fluids, were concentrated at UF as well. The aim of the research consisted in evaluation of the effect of component composition of bacteria-free supernatants and the chemical nature of membrane active layer on partial fractionation of the chitinolytic enzymes, their recovery in retentates and purification degree. On the basis of the obtained experimental results, the sample preparation procedure of the culture fluids of C. paraputrificum J4 was established to be used further in chromatographic separations of the chitinolytic enzymes.     

Synergistic cytotoxicity of perifosine and ABT‐737 to colon cancer cells

An acidic environment and hypoxia within the tumour are hallmarks of cancer that contribute to cell resistance to therapy. Deregulation of the PI3K/Akt pathway is common in colon cancer. Numerous Akt‐targeted therapies are being developed, the activity of Akt‐inhibitors is, however, strongly pH‐dependent. Combination therapy thus represents an opportunity to increase their efficacy. In this study, the cytotoxicity of the Akt inhibitor perifosine and the Bcl‐2/Bcl‐xL inhibitor ABT‐737 was tested in colon cancer HT‐29 and HCT‐116 cells cultured in monolayer or in the form of spheroids. The efficacy of single drugs and their combination was analysed in different tumour‐specific environments including acidosis and hypoxia using a series of viability assays. Changes in protein content and distribution were determined by immunoblotting and a “peeling analysis” of immunohistochemical signals. While the cytotoxicity of single agents was influenced by the tumour‐specific microenvironment, perifosine and ABT‐737 in combination synergistically induced apoptosis in cells cultured in both 2D and 3D independently on pH and oxygen level. Thus, the combined therapy of perifosine and ABT‐737 could be considered as a potential treatment strategy for colon cancer.     

Serotonin localization and its functional significance during mouse preimplantation embryo development

Serotonin is a neurotransmitter functioning also as a hormone and growth factor. To further investigate the biological role of serotonin during embryo development, we analysed serotonin localization as well as the expression of specific serotonin 5-HT1D receptor mRNA in mouse oocytes and preimplantation embryos. The functional significance of serotonin during the preimplantation period was examined by studying the effects of serotonin on mouse embryo development. Embryo exposure to serotonin (1 microM) highly significantly reduced the mean cell number, whereas lower concentrations of serotonin (0.1 microM and 0.01 microM) had no significant effects on embryo cell numbers. In all serotonin-treated groups a significant increase in the number of embryos with apoptotic and secondary necrotic nuclei was observed. Expression of serotonin 5-HT1D receptor mRNA in mouse oocytes and preimplantation embryos was confirmed by in situ hybridization showing a clearly distinct punctate signal. Immunocytochemistry results revealed the localization of serotonin in oocytes and embryos to the blastocyst stage as diffuse punctate cytoplasmic labelling. It appears that endogenous and/or exogenous serotonin in preimplantation embryos could be involved in complex autocrine/paracrine regulations of embryo development and embryo-maternal interactions.