Purification and partial characterization of megamodulin, a heat-stable protein factor from E. coli and its stimulatory effect on RNA polymerase holoenzyme

Megamodulin, a heat-stable protein from Escherichia coli was isolated and purified near homogeneity as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis. It had a molecular weight of 71,000 and pl between 3.5 and 4.0. This factor stimulated E. coli RNA polymerase 71-fold in the presence of a synthetic template such as poly (rA).p(dT). When TATAAA sequence was used as template, the RNA polymerase activity was increased 68 times by this factor. The possible mechanism by which this protein factor may regulate the RNA polymerase activity has been described.     

Modeling of habitat suitability of Asiatic black bear (Ursus thibetanus gedrosianus) in Iran in future

Future changes in climate are imminent and they threat endangered and rare species due to habitat destruction. The Asiatic black bear (Ursus thibetanus gedrosianus) is a rare and vulnerable species whose habitat fragmentation and habitat loss decreased the size of its population significantly. Climate change is another threat to this species that is investigated in this research work. Aiming at this goal, ten species distribution models (SDMs) were applied as helpful tools for evaluating the potential effectiveness of climate change in habitat suitability of Asiatic black bear in Iran. Potential dispersal of Asiatic black bear was modeled as a function of 32 environmental variables for the current time and 2070 for 44 climate change scenarios (CC scenario) of future climate. Our results showed that modeling result depended on type of model. Our results confirmed that one of the greatest threats in the near future for Asiatic black bear was the change of suitable habitat due to climate change. All the CC scenarios showed that migration of this species would be to the north and west areas with higher elevation and that an increase in area would be more than a decrease in area in all scenarios. Recognizing and protecting potential future habitats are of the important activities to conserve this species and identify areas with conservation priority.     

Evaluation of the role of the national parks for Persian leopard (<Emphasis Type="Italic">Panther pardus saxicolor</Emphasis>, Pocock 1927) habitat conservation (case study: Tandooreh National Park,Iran)

Having knowledge of the habitat requirements of top predators is important for their conservation and also for the stability of wildlife communities since they are keystone species of ecosystems. This study aims to predict suitable habitats for the Persian leopard (Panther pardus saxicolor, Pocock 1927) in Tandooreh National Park, Iran. This species is the largest leopard subspecies in the Middle East and is at high risk of extinction. Presence points for the species have been obtained from field data, literature review, atlas data, and IUCN records. Variables related to human disturbance, terrain, land use/land cover, and the leopards’ prey were used to predict the habitat selection of the Persian leopard by employing binary logistic regression. Our model predicted that the presence of Persian leopards is restricted to the southern, north-western, and western areas of the park which are under severe fragmentation threat. Moreover, leopards avoided areas near human development such as roads. We have also found that the availability of prey such as Capra aegagrus, Ovis vignei, and Ovis orientalis is one of the important parameters to predict habitat suitability for leopards. Our model provides a suitable tool for national park managers to identify biodiversity hotspots since leopards are often associated with high biodiversity.     

Metabolomics and lipidomics reveal perturbation of sphingolipid metabolism by a novel anti-trypanosomal 3-(oxazolo[4,5-<Emphasis Type="Italic">b</Emphasis>]pyridine-2-yl)anilide

Introduction

Trypanosoma brucei is the causative agent of human African trypanosomiasis, which is responsible for thousands of deaths every year. Current therapies are limited and there is an urgent need to develop new drugs. The anti-trypanosomal compound, 3-(oxazolo[4,5-b]pyridine-2-yl)anilide (OXPA), was initially identified in a phenotypic screen and subsequently optimized by structure–activity directed medicinal chemistry. It has been shown to be non-toxic and to be active against a number of trypanosomatid parasites. However, nothing is known about its mechanism of action.

Objective

Here, we have utilized an untargeted metabolomics approach to investigate the biochemical effects and potential mode of action of this compound in T. brucei.

Methods

Total metabolite extracts were analysed by HILIC-chromatography coupled to high resolution mass spectrometry.

Results

Significant accumulation of ceramides was observed in OXPA-treated T. brucei. To further understand drug-induced changes in lipid metabolism, a lipidomics method was developed which enables the measurement of hundreds of lipids with high throughput and precision. The application of this LC–MS based approach to cultured bloodstream-form T. brucei putatively identified over 500 lipids in the parasite including glycerophospholipids, sphingolipids and fatty acyls, and confirmed the OXPA-induced accumulation of ceramides. Labelling with BODIPY-ceramide further confirmed the ceramide accumulation following drug treatment.

Conclusion

These findings clearly demonstrate perturbation of ceramide metabolism by OXPA and indicate that the sphingolipid pathway is a promising drug target in T. brucei.

     

Thawed human hepatocytes in primary culture.

In drug metabolism studies, isolated and cultured human hepatocytes provide a useful model for overcoming the difficulty of extrapolating from animal data. In vitro studies with human hepatocytes are scarce because of the lack of livers and suitable methods of storage. After developing a new method for cryopreservation of human hepatocytes, we evaluated the effects of deep freezing storage on their viability, morphology, and functional and toxicological capabilities in classical culture conditions. Freshly isolated human hepatocytes were cryopreserved in medium containing 10% Me2SO and 20% fetal calf serum, using a Nicool ST20 programmable freezer (-1.9 degrees C/min for 18 min and -30 degrees C/min for 4 min). Cells were stored in liquid nitrogen. Viability of thawed human hepatocytes was 50-65% as assessed by erythrosin exclusion test prior to purification on a Percoll density gradient. Morphological criteria showed that thawed human hepatocytes require an adaptation period to the medium after seeding. Functional assessments showed that human hepatocytes which survive freezing and thawing preserve their protein synthesis capabilities and are able to secrete a specific protein, anionic peptidic fraction, which is involved in the hepatic uptake of bile-destined cholesterol. We then studied Midazolam biotransformation to test metabolic functions, and erythromycin toxicity by Neutral Red test (cell viability) and 3-(4,5-dimethylthiazol-2-yl)-diphenyl tetrazolium bromide test (cell metabolism). All of these experiments indicated that thawed human hepatocytes should be used 38 h after seeding for optimum recovery of their functions: membrane integrity, protein synthesis, and stabilization of drug metabolism enzymes.     

Influence of the chlorine concentration on the radiation efficiency of a XeCl exciplex lamp

The influence of the chlorine concentration on the radiation efficiency of coaxial exciplex lamps (excilamps) excited by a dielectric barrier discharge (DBD) in binary Xe-Cl₂ mixtures at pressures of 240–250 Torr is investigated experimentally and theoretically. The experiments were carried out at Cl₂ concentrations in the range of 0.01–1%. The DBD characteristics were calculated in the framework of a one-dimensional hydrodynamic model at Cl₂ concentrations in the range of 0.1–5%. It is found that the radiation intensities of the emission bands of Xe*₂(172 nm) and XeCl* (308 nm) are comparable when the chlorine concentration in the mixture is in the range of 0.01–0.1%. In this case, in the mixture, the radiation intensity of the Xe*₂ molecule rapidly decreases with increasing Cl₂ concentration and, at a chlorine concentration of ≥0.2%, the radiation of the B → X band of XeCl* molecules with a peak at 308 nm dominates in the discharge radiation. The radiation efficiency of this band reaches its maximum value at chlorine concentrations in the range of 0.4–0.5%. The calculated efficiencies of DBD radiation exceed those obtained experimentally. This is due to limitations of the one-dimensional model, which assumes the discharge to be uniform in the transverse direction, whereas the actual excilamp discharge is highly inhomogeneous. The influence of the chlorine concentration on the properties of the DBD plasma in binary Xe-Cl₂ mixtures is studied numerically. It is shown that an increase in the Cl₂ concentration in the mixture leads to the attachment of electrons to chlorine atoms and a decrease in the electron density and discharge conductivity. As a result, the electric field and the voltage drop across the discharge gap increase, which, in turn, leads to an increase in the average electron energy and the probability of dissociation of Cl₂ molecules and ionization of Xe atoms and Cl₂ molecules. The total energy deposited in the discharge rises with increasing chlorine concentration due to an increase in the power spent on the heating of positive and negative ions. The power dissipated by electrons decreases with increasing chlorine concentration in the working mixture. Recommendations on the choice of the chlorine content in the mixture for reducing the intensity of VUV radiation of the second continuum of the Xe*₂ excimer without a substantial decrease in the excilamp efficiency are formulated.