In Vitro Stability of Phytase from Recombinant Bacteria E. Coli BL21 (DE3) EAS1-AMP

The objective of the research was to inquire the Km, Vm, activity, intracellular phytase stability exposed to pH variation, temperature variation and protease (pepsin and pancreas) in vitro. The phytase was produced from recombinant bacteria E. coli BL21(DE3) EAS1-AMP using 1.5 mM IPTG as inducer. Intracellular enzyme was extracted via freeze shock and centrifugation. Pure enzyme was acquired through NI-NTA agarose column. The enzyme was then tested for Km, Vm, phytase activity and stability against pH, temperature and protease. Treatment levels for stability against protease were P0: without protease, P1: addition of pepsin, P2: addition of pepsin and pancreas, and the data were statistically analyzed using analysis of variance of one-way Completely Randomized Design. Crude intracellular phytase had Vm 6.39 υM/sec, Km 34.82 υM, and 277 units activity. Intracellular phytas was stable at pH 4–6 and 0–550 C. Protease level influenced the activity of intracellular phytase (P<0.05). Intracellular phytase was stable against pepsin but not pancreas. Keywords: Km, Vm, activity, intracellular phytase, pH, temperature, protease

Quantitative RNA-seq Analysis Unveils Osmotic and Thermal Adaptation Mechanisms Relevant for Ectoine Production in Chromohalobacter salexigens

Quantitative RNA sequencing (RNA-seq) and the complementary phenotypic assays were implemented to investigate the transcriptional responses of Chromohalobacter salexigens to osmotic and heat stress. These conditions trigger the synthesis of ectoine and hydroxyectoine, two compatible solutes of biotechnological interest. Our findings revealed that both stresses make a significant impact on C. salexigens global physiology. Apart from compatible solute metabolism, the most relevant adaptation mechanisms were related to “oxidative- and protein-folding- stress responses,” “modulation of respiratory chain and related components,” and “ion homeostasis.” A general salt-dependent induction of genes related to the metabolism of ectoines, as well as repression of ectoine degradation genes by temperature, was observed. Different oxidative stress response mechanisms, secondary or primary, were induced at low and high salinity, respectively, and repressed by temperature. A higher sensitivity to H2O2 was observed at high salinity, regardless of temperature. Low salinity induced genes involved in “protein-folding-stress response,” suggesting disturbance of protein homeostasis. Transcriptional shift of genes encoding three types of respiratory NADH dehydrogenases, ATP synthase, quinone pool, Na+/H+ antiporters, and sodium-solute symporters, was observed depending on salinity and temperature, suggesting modulation of the components of the respiratory chain and additional systems involved in the generation of H+ and/or Na+ gradients. Remarkably, the Na+ intracellular content remained constant regardless of salinity and temperature. Disturbance of Na+- and H+-gradients with specific ionophores suggested that both gradients influence ectoine production, but with differences depending on the solute, salinity, and temperature conditions. Flagellum genes were strongly induced by salinity, and further induced by temperature. However, salt-induced cell motility was reduced at high temperature, possibly caused by an alteration of Na+ permeability by temperature, as dependence of motility on Na+-gradient was observed. The transcriptional induction of genes related to the synthesis and transport of siderophores correlated with a higher siderophore production and intracellular iron content only at low salinity. An excess of iron increased hydroxyectoine accumulation by 20% at high salinity. Conversely, it reduced the intracellular content of ectoines by 50% at high salinity plus high temperature. These findings support the relevance of iron homeostasis for osmoadaptation, thermoadaptation and accumulation of ectoines, in C. salexigens.España Ministerio de Economía y Competitividad BIO2015-63949-RJunta de Andalucía P11-CVI-729

Duramycin-induced calcium release in cancer cells

Introduction: Duramycin through binding with phosphatidylethanolamine (PE) has shown potential to be an effective anti-tumour agent. However its mode of action in relation to tumour cells is not fully understood. Methods: PE expression on the surface of a panel of cancer cell lines was analysed using duramycin and subsequent antibody labelling then analysed by flow cytometry. Cell viability was also assessed via flow cytometry using annexin V and propidium iodide (PI). Calcium ion (Ca²⁺) release by tumour cells in response to duramycin was determined by spectrofluorometry following incubation with Fluo-3, AM. Confocal microscopy was performed on the cancer cell line AsPC-1 to assess real time cell response to duramycin treatment. Results: Duramycin was able to detect cell surface PE expression on all 15 cancer cell lines screened, which was shown to be duramycin concentration dependent. However higher concentrations induced necrotic cell death. Duramycin induced calcium ion (Ca²⁺) release from the cancer cell lines also in a concentration and time dependent manner. Confocal microscopy showed an influx of PI into the cells over time and induced morphological changes. Conclusion: Duramycin induces Ca²⁺ release from cancer cell lines in a time and concentration dependent relationship

Application of magnetically induced hyperthermia on the model protozoan Crithidia fasciculata as a potential therapy against parasitic infections

Magnetic hyperthermia is currently an EU-approved clinical therapy against tumor cells that uses magnetic nanoparticles under a time varying magnetic field (TVMF). The same basic principle seems promising against trypanosomatids causing Chagas disease and sleeping sickness, since therapeutic drugs available display severe side effects and drug-resistant strains. However, no applications of this strategy against protozoan-induced diseases have been reported so far. In the present study, Crithidia fasciculata, a widely used model for therapeutic strategies against pathogenic trypanosomatids, was targeted with Fe_{3}O_{4} magnetic nanoparticles (MNPs) in order to remotely provoke cell death using TVMFs. The MNPs with average sizes of d approx. 30 nm were synthesized using a precipitation of FeSO_{4}4 in basic medium. The MNPs were added to Crithidia fasciculata choanomastigotes in exponential phase and incubated overnight. The amount of uploaded MNPs per cell was determined by magnetic measurements. Cell viability using the MTT colorimetric assay and flow cytometry showed that the MNPs were incorporated by the cells with no noticeable cell-toxicity effects. When a TVMF (f = 249 kHz, H = 13 kA/m) was applied to MNP-bearing cells, massive cell death was induced via a non-apoptotic mechanism. No effects were observed by applying a TVMF on control (without loaded MNPs) cells. No macroscopic rise in temperature was observed in the extracellular medium during the experiments. Scanning Electron Microscopy showed morphological changes after TVMF experiments. These data indicate (as a proof of principle) that intracellular hyperthermia is a suitable technology to induce the specific death of protozoan parasites bearing MNPs. These findings expand the possibilities for new therapeutic strategies that combat parasitic infections.Comment: 9 pages, four supplementary video file

Elevated intracellular cAMP exacerbates vulnerability to oxidative stress in optic nerve head astrocytes.

Glaucoma is characterized by a progressive loss of retinal ganglion cells and their axons, but the underlying biological basis for the accompanying neurodegeneration is not known. Accumulating evidence indicates that structural and functional abnormalities of astrocytes within the optic nerve head (ONH) have a role. However, whether the activation of cyclic adenosine 3',5'-monophosphate (cAMP) signaling pathway is associated with astrocyte dysfunction in the ONH remains unknown. We report here that the cAMP/protein kinase A (PKA) pathway is critical to ONH astrocyte dysfunction, leading to caspase-3 activation and cell death via the AKT/Bim/Bax signaling pathway. Furthermore, elevated intracellular cAMP exacerbates vulnerability to oxidative stress in ONH astrocytes, and this may contribute to axonal damage in glaucomatous neurodegeneration. Inhibition of intracellular cAMP/PKA signaling activation protects ONH astrocytes by increasing AKT phosphorylation against oxidative stress. These results strongly indicate that activation of cAMP/PKA pathway has an important role in astrocyte dysfunction, and suggest that modulating cAMP/PKA pathway has therapeutic potential for glaucomatous ONH degeneration

Insertion of T4-lysozyme (T4L) can be a useful tool for studying olfactory-related GPCRs.

The detergents used to solubilize GPCRs can make crystal growth the rate-limiting step in determining their structure. The Kobilka laboratory showed that insertion of T4-lysozyme (T4L) in the 3rd intracellular loop is a promising strategy towards increasing the solvent-exposed receptor area, and hence the number of possible lattice-forming contacts. The potential to use T4L with the olfactory-related receptors hOR17-4 and hVN1R1 was thus tested. The structure and function of native and T4L-variants were compared. Both receptors localized to the cell membrane, and could initiate ligand-activated signaling. Purified receptors not only had the predicted alpha-helical structures, but also bound their ligands canthoxal (MW = 178.23) and myrtenal (MW = 150.22). Interestingly, the T4L variants had higher percentages of soluble monomers compared to protein aggregates, effectively increasing the protein yield that could be used for structural and function studies. They also bound their ligands for longer times, suggesting higher receptor stability. Our results indicate that a T4L insertion may be a general method for obtaining GPCRs suitable for structural studies

His+ reversions Caused in Salmonella typhimurium by different types of ionizing radiation

The yield of his+ reversions in the Ames Salmonella tester strain TA2638 has been determined for 60Co γ rays, 140 kV X rays, 5.4 keV characteristic X rays, 2.2 MeV protons, 3.1 MeV α particles, and 18 MeV/U Fe ions. Inactivation studies were performed with the same radiations. For both mutation and inactivation, the maximum effectiveness per unit absorbed dose was obtained for the characteristic X rays, which have a dose averaged linear energy transfer (LET) of roughly 10 keV/μm. The ratio of the effectiveness of this radiation to γ rays was 2 for inactivation and about 1.4 for the his+ reversion. For both end points the effectiveness decreases substantially at high LET, i.e., for the α particles and the Fe ions. The composition of the bottom and the top agar was the one recommended by Maron and Ames [Mutat. Res. 113, 173-215 (1983)] for application in chemical mutagenicity tests. The experiments with the less penetrating radiations differed from the usual protocol by utilization of a technique of plating the bacteria on the surface of the top agar. As in an earlier study [Roos et al., Radiat. Res. 104, 102-108 (1985)] greatly enhanced yields of mutations, relative to the spontaneous reversion rate, were obtained in these experiments by performing the irradiations 6 h after plating, which differs from the conventional procedure to irradiate the bacteria shortly after plating