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.     

A histological and immunohistochemical study of the effects of N-acetyl cysteine on retinopathy of prematurity by modifying insulin-like growth factor-1

Retinopathy of prematurity (ROP) is a vasoproliferative disorder that occurs in premature infants and may lead to permanent visual impairment. We investigated both the possible protective role of N-acetyl cysteine (NAC) for preventing ROP and the role of IGF-1 in the disorder. Forty-five newborn rats were divided into three groups. Group 1 was raised in room air as controls. Group 2 was exposed to 60% oxygen for 14 days after birth, then transferred to room air. Group 3 was exposed to the same conditions as group 2, but received intraperitoneal injections of NAC on postnatal days 7–17. After 35 days, both eyes of all rats were processed for histology. Some sections were stained with hematoxylin and eosin to assess structural changes and other sections were immunostained to determine the location of IGF-1. Frozen sections also were prepared and stained for adenosine triphosphatase to detect retinal blood vessels. Compared to the controls, more blood vessels, many of which were abnormal, and increased IGF-1 expression were observed in group 2. In group 3, abnormal blood vessels and IGF-1 expression were less evident. NAC appeared to be an effective vascular-protective agent for ROP by decreasing IGF-1 expression.     

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.     

Selection of thermotolerant bradyrhizobial strains for nodulation of soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L.) in semi-arid regions of Iran

Nodulation of soybeans grown in semi-arid region of southern parts of Iran is poor due to high air and soil temperatures. Here we identified thermotolerant isolates of soybean bradyrhizobia and evaluated the nitrogen fixation efficiency of the isolates under heat stress conditions in greenhouse and field experiments. The ability of fifty-six bradyrhizobial isolates to grow on solid or in liquid yeast extract mannitol medium at 38 and 41°C was evaluated. We identified 19 isolates, which were able to grow at 38°C and 10 isolates able to grow at 41°C. Greenhouse experiments were carried out at 28 and 38°C to study the nitrogen-fixing capacity of the isolates under optimal and high temperature conditions. Ten isolates had a symbiotic index of effectiveness of 80% or greater compared with nitrogen-fertilized treatments in greenhouse experiments at 28°C. Some thermotolerant isolates demonstrated good nitrogen-fixing performance at 38°C. Eight isolates were selected for use in a field trial in the natural high temperature environment of the Dezful region in Iran. Our results demonstrate that geographical origin can have a great influence on the successful selection of thermotolerant bradyrhizobia. Our thermotolerant isolates were mainly obtained from high-temperature regions, and improved shoot dry matter, nitrogen-uptake and seed yield of the plants.     

Conserved sequences in enzymes of the UDP-GlcNAc/MurNAc family are essential in hamster UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase

The UDP-GlcNAc/MurNAc family of eukaryotic and prokaryotic enzymes use UDP-GlcNAc or UDP-MurNAc-pentapeptide as donors, dolichol-P or polyprenol-P as acceptors, and generate sugar-P-P-polyisoprenols. A series of six conserved sequences, designated A through F and ranging from 5 to 13 amino acid residues, has been identified in this family. To determine whether these conserved sequences are required for enzyme function, various mutations were examined in hamster UDP- GlcNAc:dolichol-P GlcNAc-1-P transferase (GPT). Scramble mutations of sequences B-F, generated by scrambling the residues within each sequence, demonstrated that each is important in GPT. While E and F scrambles appeared to prevent stable expression of GPT, scrambling of B- D resulted in GPT mutants that could be stably expressed and bound tunicamycin, but lacked enzymatic activity. Further, the C and D scramble mutants had an unexpected sorting defect. Replacement of sequences B-F with prokaryotic counterparts from either the B.subtilis mraY or E.coli rfe genes also affected GPT by preventing expression of the mutant protein (B, F) or inhibiting its enzymatic activity (C-E). For the C-E replacements, no acquisition of acceptor activity for polyprenol-P, the fully unsaturated natural bacterial acceptor, was detected. These studies show that the conserved sequences of the UDP- GlcNAc/MurNAc family are important, and that the eukaryotic and prokaryotic counterparts are not freely interchangeable. Since several mutants were efficiently expressed and bound tunicamycin, yet lacked enzymatic activity, the data are consistent with these sequences having a direct role in product formation.